WO2023051685A1 - Method for amplifying erythroid progenitors or erythroblasts, and application thereof - Google Patents

Method for amplifying erythroid progenitors or erythroblasts, and application thereof Download PDF

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WO2023051685A1
WO2023051685A1 PCT/CN2022/122562 CN2022122562W WO2023051685A1 WO 2023051685 A1 WO2023051685 A1 WO 2023051685A1 CN 2022122562 W CN2022122562 W CN 2022122562W WO 2023051685 A1 WO2023051685 A1 WO 2023051685A1
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alkyl
aryl
heterocyclyl
alkenyl
alkynyl
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PCT/CN2022/122562
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French (fr)
Chinese (zh)
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李湘盈
吴顺康
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北京大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • the invention provides a method for expanding erythroid precursor cells or erythroblasts from various cell sources, promoting erythroid development, and finally producing mature denucleated erythrocytes and its application.
  • Blood transfusion is the area of greatest need.
  • autologous blood transfusion is a potential method to alleviate and restore blood loss during and after surgery. It's an important way to get enough red blood cells for those with rare blood types who don't have a readily available blood supply.
  • various diseases can be treated by the efficient expansion of red blood cells in vivo.
  • patients with bone marrow failure such as those with congenital pure red developmental aplastic anemia and Diamond-Blackfan anemia (DBA)
  • DBA Diamond-Blackfan anemia
  • the disease is currently Apart from high-dose hormones, blood transfusion, and bone marrow transplantation, there is no effective treatment.
  • EPO erythropoietin
  • CFU-E colony-forming unit erythroid progenitors
  • Some patients with chronic kidney disease will be unable to respond to conventional anemia treatments due to EPO resistance. So once a treatment that reduces EPO resistance is discovered, it could not only improve kidney inflammation in patients, but also alleviate symptoms of anemia and potentially improve patient outcomes and survival.
  • erythrocytes can serve as long-lived circulating delivery vehicles in vivo. Therefore, erythroid precursor cells can be genetically engineered so that they carry specific enzymes on their surface or inside them in genetically engineered red blood cell therapy. These engineered expanded erythrocytes can be or are designed as carriers of small molecule chemical drugs, or proteins (including various enzymes) to achieve various medical purposes of clinical treatment, for example, erythrocyte engineering can be used clinically to treat innate metabolic disorders Enzyme deficiency and other diseases that require long-term treatment in order to achieve long-term and sustained-release therapeutic effects.
  • BRAF kinase inhibitors are able to act through paradoxical activation of the MAPK pathway during erythroid development (i.e. BRAF inhibitors in cells with BRAF wild-type and upstream RAS protein activation, by binding to the wild-type BRAF ATP pocket, Keep BRAF in the activated conformation, greatly promote and stabilize the dimerization of BRAF and CRAF, so that CRAF can maintain the activated conformation, and then activate the downstream MAPK/ERK pathway, thereby promoting cell proliferation, differentiation and survival), and effectively expand the erythroid Precursor cells or erythroid cells (erythroid progenitors, EPs or erythroblasts, EBs), which can culture EPs in any erythroid medium, and the morphology, function and erythroid surface markers of EPs or EBs remain basically unchanged after treatment Change.
  • BRAF inhibitors in cells with BRAF wild-type and upstream RAS protein activation by binding to the wild-type BRAF ATP pocket, Keep BRAF in the activated conformation
  • BRAF inhibitors can greatly promote erythroid development and proliferation in conditions of cytokine deficiency such as pathological states. Moreover, when combined with existing erythropoietic agents such as TGF- ⁇ inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or erythropoietin and/or stem cell factor, BRAF Kinase inhibitors produce stronger amplification.
  • the present invention provides a medicament for the treatment of primary or secondary anemia, comprising a BRAF kinase inhibitor and one or more pharmaceutically acceptable excipients.
  • the medicament further comprises TGF- ⁇ inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or erythropoietin and/or stem cell factor.
  • the invention provides a kit comprising: a) a BRAF kinase inhibitor, and optionally b) erythropoietin.
  • the kit further includes: c) TGF- ⁇ inhibitor and/or SMAD2/3 inhibitor and/or glucocorticoid and/or erythropoietin and/or stem cell factor.
  • the invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF- ⁇ inhibitor, or a BRAF kinase inhibitor and SMAD2 /3 inhibitors, or BRAF kinase inhibitors, TGF- ⁇ inhibitors and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of a medicament for expanding erythroid precursor cells or erythroblasts .
  • the invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF- ⁇ inhibitor, or a BRAF kinase inhibitor and SMAD2 /3 inhibitors, or BRAF kinase inhibitors, TGF- ⁇ inhibitors and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of medicines for the treatment of primary or secondary anemia .
  • the invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF- ⁇ inhibitor, or a BRAF kinase inhibitor and SMAD2 /3 inhibitors, or BRAF kinase inhibitors, TGF- ⁇ inhibitors and SMAD2/3 inhibitors, and their use in combination with glucocorticoids in expanding erythroid precursor cells or erythroblasts.
  • the invention provides a method of expanding erythroid precursor cells or erythroblasts comprising administering to a subject a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or Or stem cell factor, or BRAF kinase inhibitor and TGF- ⁇ inhibitor, or BRAF kinase inhibitor and SMAD2/3 inhibitor, or BRAF kinase inhibitor, TGF- ⁇ inhibitor and SMAD2/3 inhibitor, and their combination with sugar A combination of corticosteroids.
  • the invention provides a method of expanding erythroid precursor cells or erythroblasts comprising administering to a subject a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin, or a BRAF kinase inhibitor agent and stem cell factor, or a combination of BRAF kinase inhibitor, erythropoietin, and stem cell factor.
  • the invention provides methods of expanding erythroid precursor cells or erythroblasts.
  • methods of expanding a population of erythroid precursor cells are also provided.
  • the EPs of the present invention have a small, round and non-adhesive uniform morphology.
  • the present invention also provides a method for preventing or treating related diseases, comprising importing a sufficient number of EPs amplified by the method, and carrying drugs to treat the diseases.
  • the present invention also provides a sufficient number of EPs or genetically modified EPs required for medicines for treating or preventing related diseases.
  • the present invention can be combined with the method WO2014183071 of the inventor's previous application for genetic modification of human EPs, which can be further differentiated into mature red blood cells with invertase or chemical drugs.
  • human EPs are expected to improve therapeutic efficacy and could be used clinically to treat genetic diseases such as phenylketonuria and even cancer.
  • Human EPs can also be engineered to produce the required enzymes on the surface of red blood cells for the development of new treatments for cancer and other diseases.
  • MEP megakaryocyte-erythroid progenitor
  • blood transfusions have become a very common therapy, and modified red blood cells have been used as natural drug delivery vehicles in the body, their clinical application is limited.
  • PBMC peripheral blood mononuclear cells
  • EPO resistance may be one of the causes of chronic inflammatory anemia. There are currently no existing treatments for EPO resistance.
  • the method provided by the present invention solves the above-mentioned problems.
  • the inventive method has at least the following advantages:
  • EPs in human PBMCs are amplified in large quantities, and the development and expression of red blood cells can be regulated by genetically modifying EPs;
  • Human EPs amplified in vitro can be used clinically, including but not limited to blood transfusion in various situations.
  • human EPs expanded in vitro can be used to treat anemia or excessive blood loss;
  • EPO concentration or EPO resistance
  • other cytokines necessary for erythroid growth it can promote the differentiation, maturation and denucleation of erythroid precursor cells or erythroblasts, and relieve EPO resistance or Symptoms of anemia caused by cytokine deficiency.
  • FIG. 1A Schematic representation of high-throughput screening (HTS) of small molecule expansion of erythroid precursor cells.
  • Figure 1B Results of high-throughput screening.
  • FIG. 1A Exploring the optimal concentration of GDC-0879 treatment.
  • Figure 2B Expansion effect of various BRAF inhibitors on erythroid precursor cells over a wide concentration range (one administration only).
  • Figure 2C Expansion effect of selected BRAF inhibitors on erythroid precursor cells over a broad concentration range (three doses).
  • Figure 3A Growth curves of three small molecules at optimal concentrations in erythroid differentiation system.
  • Figure 3B Growth curves of erythroblasts after small molecule treatment.
  • FIGS 4A-4E FACS results of GDC-0879 in differentiation medium.
  • Figures 5A-5D Colony formation results.
  • Figure 5A is the statistics of the number of erythrocyte colonies (middle) and the area of a single erythrocyte colony (right) in different Methocult H4435 administration groups and the control group
  • Figure 5B shows the results of erythrocyte-specific colony formation on the 14th day
  • Figure 5C shows The results of erythrocyte-specific colony formation on day 14 were detected under a bright-field microscope using a 5 ⁇ objective lens.
  • Figure 5D shows that BRAF inhibitors promote erythropoiesis and lineage development of cord blood-derived CD34+ cells (myeloid medium).
  • Figures 6A-6E Effects of small molecules targeting BRAF on promoting proliferation of erythroid precursor cells.
  • Figure 6A shows the results of detection of other small molecules targeting BRAF with the same target (the concentration of small molecules not specifically marked is 1 ⁇ M, and the treatment is 9 days), and
  • Figure 6B shows that other inhibitors of BRAF are effective in formazan containing only EPO.
  • Figure 6C shows the erythrocyte-specific colony formation on day 14 of other combinations containing BRAF inhibitors in methylcellulose medium containing EPO only
  • Fig. 6D shows the colony formation results under the light field microscope
  • Fig. 6E shows the colony forming unit counting results of each well of Fig. 6B and 6C.
  • Figures 7A-7E Effective dosing cycle assay.
  • Figure 7A shows the test results of GDC-0879 administered in different cycles from the 0th day to the 12th day, and the final cell number statistics were performed on the 15th day
  • Figure 7B shows the 0-3 day treatment group and the control group Comparison of the number of cells on the 13th day
  • Figure 7C shows the FACS results of the experimental group and the control group on the 13th day in the differentiation medium
  • Figure 7D shows that the statistics of the enucleation rate of each group administered for 3 days on the 13th to 16th day ( Treatment group, 1 ⁇ M GDC-0879 treatment group)
  • Figure 7E shows statistics on the enucleation rate of each group in the two administration periods (6 consecutive days) from day 13 to 16 (treatment group: 1 ⁇ M GDC-0879 treatment).
  • Figures 8A-8K In vitro expansion of erythroblasts in PBMCs.
  • Figure 8A shows the cells of the treatment group and the control group on the 9th day
  • Figure 8B shows the change in the number of erythroblasts in the PBMC of healthy donors (HD1-HD5) in vitro
  • Figure 8C shows the changes in the number of erythroblasts in the healthy donors (HD1-HD5) on the 9th day (HD1-HD5)
  • PBMCs were cultured in serum-free erythrocyte expansion medium on the 9th day of FACS results
  • Figure 8D shows the statistics of the FACS results of healthy donors (HD1-HD5) PBMCs on the 10th day cell group ratio
  • Figure 8E shows the statistics of the ratio of cell groups on the 10th day of FACS
  • Figure 8F shows the FACS results of PBMCs from healthy donors (Donor1-Donor5) cultured in serum-free erythrocyte expansion medium
  • Figure 8G shows the results of healthy donors (Donor1
  • Figures 9A-9L Therapeutic effects of drugs under low EPO conditions.
  • Figure 9A shows the effect of GDC-0879 in different concentrations of erythropoietin (EPO) differentiation medium
  • Figure 9B shows the FACS results on day 14 after 5-10 days of culture without EPO
  • Figure 9C shows the 1/3 concentration FACS results on the 15th day after cultured under EPO conditions for 1-15 days
  • Figure 9D shows the erythrocyte denucleation rate under the conditions of various EPO concentrations from 13 to 19 days
  • Figure 9E shows the concentration of erythrocytes with 0% SCF or 5% EPO at low cytokine concentrations
  • Figure 9F shows 0% SCF (left) and 5% EPO ( Right)
  • Figure 9G and 9H
  • FIGS. 9I and 9J are the erythrocyte surface markers ( The expression of CD117, CD71 and CD235),
  • Figure 9K is a typical erythrocyte photo of samples cultured under different stress conditions,
  • Figure 9L representative Giemsa-benzidine stained erythrocyte images under different pressure culture conditions.
  • Figures 10A-10F Effects of in vitro treatment of anemia caused by defective pure red blood cell regeneration after hematopoietic stem cell transplantation.
  • Figure 10A shows that GDC-0879 may promote the development and self-renewal of erythroid progenitor cells in erythrocyte development failure or pure erythrocyte aplasia after hematopoietic stem cell transplantation
  • Figure 10B shows the bone marrow single Growth curves of nucleated cells (BMMNCs)
  • Figure 10C shows the erythrocyte surface markers ( CD71, CD235 and Hoechst 33342)
  • Figure 10D is the representative Giemsa-benzidine stained erythrocytes obtained from EFAHSCT BMMNCs after erythroid culture
  • Figure 10E is 50000 EFAHSCT BMMNCs in H4435 myeloid methylcellulose medium
  • Representative images in Figure 10F show the number of colonies of different lineages from 50,000 EFAHSCT-derived BMMNCs cultured
  • FIG. 11 SB-590885 potently expands erythroid precursors in peripheral mononuclear cells from DBA anemia patients.
  • Figure 12 Statistics of SB-590885 effectively expanding erythroid precursor cells in peripheral mononuclear cells of DBA anemia patients.
  • Figures 13A-13E BRAF inhibitors effectively promote erythroid differentiation and erythroid proliferation in MDS patient cells.
  • Figure 14 Expansion of hematopoietic stem progenitor cells (HSPC) by BRAF inhibitors.
  • FIG. 15 BRAF inhibitors promote hematopoiesis in mice (normal conditions).
  • FIGS 16A-16E BRAF inhibitors promote hematopoiesis in mice (acute hemolysis model).
  • FIGS 17A-17B BRAF inhibitors promote hematopoiesis in mice (acute hemolysis model).
  • FIG. 18 BRAF inhibitors induce paradoxical activation of the MAPK pathway during erythroid differentiation.
  • Figure 20 Paradoxical activation of MAPK/ERK pathway by BRAF inhibitors is dependent on both upstream cytokine signaling and downstream signaling.
  • alkyl refers to a saturated linear or branched chain hydrocarbon group of 1 to 18 carbon atoms, wherein said alkyl group may be optionally substituted independently by one or more substituents described below.
  • the alkyl group may be monovalent, and examples thereof include C 1 -C 12 alkyl, C 1 -C 8 alkyl, C 1 -C 6 alkyl, such as methyl (Me, -CH 3 ), ethyl ( Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, - CH 2 CH(CH 3 ) 2 ), 2-butyl (s
  • the alkyl group may have 2 monovalent radical centers resulting from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Examples include, but are not limited to , methylene ( -CH2- ), 1,2-ethyl (-CH2CH2-) , 1,3-propyl ( -CH2CH2CH2- ) , 1, 4-Butyl (-CH 2 CH 2 CH 2 CH 2 -) etc.
  • alkenyl refers to a straight or branched chain hydrocarbon radical of 2 to 18 carbon atoms, having at least one site of unsaturation, i.e. a carbon-carbon sp double bond, wherein said alkenyl radical may optionally be replaced by one or more
  • the substituents are independently substituted and include groups with cis and trans orientations, or "E" and "Z" orientations.
  • alkyl and alkenyl groups alone or as part of a larger group such as an alkoxy group (i.e. -OR group where R is the alkyl and alkenyl group) or as part of a larger group such as an alkylthio group moieties (ie -SR groups, where R is the alkyl and alkenyl groups described).
  • an alkoxy group i.e. -OR group where R is the alkyl and alkenyl group
  • alkylthio group moieties ie -SR groups, where R is the alkyl and alkenyl groups described.
  • alkynyl refers to a straight or branched chain hydrocarbon group of 2 to 18 carbon atoms, having at least one site of unsaturation, i.e. a carbon-carbon sp triple bond, wherein said alkynyl group may optionally be replaced by one or more The above substituents are independently substituted.
  • the alkynyl group may be monovalent, examples of which include C 2 -C 12 alkynyl, C 2 -C 8 alkynyl, C 2 -C 6 alkynyl, including but not limited to, ethynyl (-C ⁇ CH) and propynyl (propargyl, -CH 2 C ⁇ CH) and the like.
  • the alkynyl group may have two monovalent radical centers resulting from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. Examples include, but are not limited to, ethynylene (-C ⁇ C-), propargylene ( -CH2C ⁇ C- ) , and 4-pentynylene ( -CH2CH2CH2C ⁇ C- ) wait.
  • aryl refers to a monovalent aromatic hydrocarbon radical having 6 to 20 carbon atoms, resulting from the removal of a hydrogen atom from a carbon atom of a parent aromatic ring system.
  • aryl groups include C 6-14 aryl, C 6-10 aryl, and some aryl groups in the exemplary structures are represented by "Ar".
  • Aryl includes bicyclic rings containing an aromatic ring fused to a non-aromatic or partially saturated ring.
  • Typical aryl groups include, but are not limited to, groups derived from benzene, substituted benzenes, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3, 4-tetrahydronaphthyl, etc.
  • Aryl groups are optionally substituted independently with one or more substituents described herein.
  • heterocycle refers to ring atoms having 3 to 25 ring atoms (containing 2 to 20 carbon atoms and 1 to 5 ring atoms selected from N, O, P and heteroatoms of S), saturated, partially saturated (i.e., having one or more double and/or triple bonds within the ring) or aromatic carbocyclic groups in which at least one ring atom is independently selected from nitrogen, oxygen and sulfur and the remaining ring atoms are carbon, wherein one or more ring atoms are optionally independently substituted with one or more substituents described herein.
  • the heterocycle can be a monocyclic ring having 3 to 7 ring atoms (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S) or a ring having 7 to 10 ring atoms (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example: bicyclic [4,5], [5,5], [5,6] or [6,6 ]system.
  • Heterocycles are described in: Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W.A.
  • a heterocyclyl group can be a carbon-bonded group or a heteroatom-bonded group.
  • heterocycle includes heterocycloalkoxy.
  • Heterocyclyl also includes radicals in which heterocyclyl is fused to a carbocyclic, heterocyclic, aromatic or heteroaromatic ring.
  • heterocyclic groups include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidine Base, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrroliny
  • Spiro groups are also included within the scope of this invention.
  • the heterocyclic groups herein are optionally substituted independently with one or more substituents described herein.
  • heteroaryl includes 1) monocyclic aromatic 5-, 6- and 7-membered rings containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur; and 2) 8 to 20 atom Fused ring systems wherein at least one aromatic ring contains one or more heteroatoms independently selected from nitrogen, oxygen and sulfur.
  • heteroaryl groups are pyridyl (including, for example, 2-hydroxypyridyl), imidazolyl, imidazopyridyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, thiazolyl, pyrazinyl, tetra Azolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolyl, indolyl, benzimidazolyl, benzofuryl , cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl , thiadiazoly
  • Heterocycles can be carbon-attached or nitrogen-attached (if applicable). For example, but not limited to: carbon-bonded heterocyclic ring binding position at 2, 3, 4, 5 or 6 positions of pyridine; 3, 4, 5 or 6 positions of pyridazine; 2, 4, 5 or 6 positions of pyrimidine ; 2, 3, 5 or 6 positions of pyrazine; 2, 3, 4 or 5 positions of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole; 2, 4 or 5 of oxazole, imidazole or thiazole position; 3, 4 or 5 positions of isoxazole, pyrazole or isothiazole; 2 or 3 positions of aziridine; 2, 3 or 4 positions of azetidine; 2, 3, 4 positions of quinoline , 5, 6, 7 or 8 positions; or 1, 3, 4, 5, 6, 7 or 8 positions of isoquinoline.
  • nitrogen-bonded heterocyclic rings are bound at aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline , 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1-position of 1H-indazole; isoindole or 2-position for isoindoline; 4-position for morpholine; and 9-position for carbazole or ⁇ -carboline.
  • Carbocycle refers to a monocyclic non-aromatic, saturated or unsaturated ring having 3 to 12 carbon atoms, or a ring having 7 to A bicyclic non-aromatic, saturated or unsaturated ring of 12 carbon atoms.
  • Monocyclic carbocycles have 3 to 7, or 3 to 6, more usually 5 or 6 ring atoms.
  • Bicyclic carbocycles have 7 to 12 ring atoms, for example arranged in a bicyclic [4,5], [5,5], [5,6] or [6,6] system; or have 9 or 10 ring atoms, arranged Form bicyclo[5,6] or [6,6] systems, or become bridged ring systems, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane.
  • Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclo Hexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, Cyclodecyl, Cycloundecyl and Cyclododecyl.
  • protecting group refers to a substituent, typically used to block or protect a particular functional group while reacting other functional groups of a compound.
  • an "amino-protecting group” is a substituent attached to an amino group that blocks or protects the amino function in a compound.
  • Suitable amino protecting groups include acetyl, trifluoroacetyl, phthalimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc ).
  • hydroxyl protecting group refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality.
  • Suitable hydroxy protecting groups include acetyl, trialkylsilyl, dialkylphenylsilyl, benzoyl, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl group and tetrahydropyranyl group.
  • Carboxy protecting group refers to a carboxyl substituent that blocks or protects the carboxyl functionality.
  • Common carboxyl protecting groups include -CH2CH2SO2Ph , cyanoethyl , 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2 -(p-tosyl)ethyl, 2-(p-nitrophenylsulfinyl)ethyl, 2-(diphenylphosphino)-ethyl, nitroethyl and the like.
  • protecting groups and their use see TW Greene and P. Wuts, Protective Groups in Organic Synthesis, 3rd ed., John Wiley & Sons, New York, 1999; and P. Kocienski, Protecting Groups, 3rd ed., Verlag, 2003.
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and is capable of being activated enzymatically or hydrolytically or by Conversion to the more active parent form. See, eg, Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp.375-382, 615th Meeting Harbor (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery” Directed Drug Delivery , Borchardt et al., (ed.), pp. 247-267, Humana Press (1985).
  • Prodrugs of the present invention include, but are not limited to, phosphate-containing prodrugs, phosphorothioate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine prodrugs and others that can be Conversion of 5-fluorouridine prodrugs to more active non-cytotoxic drugs.
  • Examples of cytotoxic drugs that can be derivatized as prodrugs for use in the present invention include, but are not limited to, those chemotherapeutic drugs described above.
  • alkyl, alkenyl, cycloalkyl, and cycloalkenyl groups described herein can be optionally substituted with the following substituents: aryl, heteroaryl, heterocyclyl, C 1-6 alkoxy, C 1-6 alkylthio, aryl C 1-6 alkoxy, aryl C 1-6 alkylthio, amino, mono- or di-C 1-6 alkylamino, aminosulfonyl, cycloalkyl, Cycloalkenyl, carboxyl and its ester, amide, ureido, guanidino, C 1-6 alkyl guanidino, amidino, C 1-6 alkyl amidino, C 1-6 acyloxy, hydroxyl and halogen or Any of their combined substituents.
  • Another substituent may also be a cyano group.
  • Preferred optional substituents include water-soluble groups; those of ordinary skill in the art are familiar with suitable water-soluble moieties, including hydroxyl, amino groups. More preferred optional substituents include amino, mono- or di-C 1-6 alkyl, amino, amino-containing heterocyclyl or hydroxy or any combination thereof.
  • the aryl, heterocyclyl, and heteroaryl groups described herein can be optionally substituted with the following substituents: halogen, hydroxy, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1-6 alkyl , halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, nitro, cyano, azido , amino, mono- and di-NC 1-6 alkylamino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, carbamoyl, mono- and di-NC 1-6 alkyl Carbamoyl, C 1-6 alkoxycarbonyl, aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, urea, carbamic acid Est
  • heteroC 1-6 alkyl- refers to a C 1-6 carbon chain in which the terminal carbon atoms of the chain are replaced by heteroatoms selected from N, O or S, for example, C 1-6 alkylamino, C 1 -6 alkoxy, C 1-6 alkylthio.
  • C 1-6 alkylheteroC 1-6 alkyl refers to a C 3-13 alkyl chain in which one carbon atom is replaced by a heteroatom selected from N, O or S, for example, C 1-6 Alkylamino C 1-6 alkyl or C 1-6 alkylaminodiC 1-6 alkyl, C 1-6 alkoxy C 1-6 alkyl-, C 1-6 alkylthio C 1- 6 alkyl-, or C 1-6 alkylthio diC 1-6 alkyl.
  • stereoisomer refers to compounds that have identical chemical constitution but differ in the arrangement of the atoms or groups in space.
  • tautomer or “tautomeric form” refers to structural isomers of different energies, which are interconvertible via a low energy barrier.
  • proton tautomers also known as prototropic tautomers
  • Valence tautomers involve interconversion by recombination of some of the valence electrons.
  • the term "pharmaceutically acceptable” means that a substance or composition must be chemically and/or toxicologically compatible with the other ingredients making up the formulation and/or the mammal receiving its treatment.
  • the term "pharmaceutically acceptable salt” refers to those carboxylate, amino acid addition salts of the compounds of the present invention, which are suitable within the scope of sound medical judgment for use in contact with patient tissues without undue toxicity, irritation Effects, allergic reactions, etc., commensurate with a reasonable benefit/risk ratio, are valid for their intended application, including, where possible, zwitterionic forms of the compounds of the invention.
  • isotopic derivative refers to a compound in which one or more atoms are replaced by an atom having an atomic mass or mass number different from that normally found in nature. All isotopes of any particular atom or element specified and their use are intended to be within the scope of the compounds of the present invention. Exemplary isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 32 P, 33 P, 35 S, 18 F, 36 Cl, 123 I and 125 I.
  • solvate refers to a form of a compound, or a salt thereof, which is associated with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding.
  • solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like.
  • Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid.
  • “Solvate” includes both solution state solvates and isolatable solvates. Representative solvates include hydrates, ethanolates and methanolates.
  • hydrate refers to a compound that combines with water. Generally, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined.
  • a hydrate of a compound can be represented, for example, by the general formula RxxH2O , where R is the compound, and x is a number greater than zero.
  • a given compound may form more than one hydrate type, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1, for example, hemihydrates (R ⁇ 0.5H2 O)) and polyhydrates (x is a number greater than 1, eg, dihydrate (R ⁇ 2H 2 O) and hexahydrate (R ⁇ 6H 2 O)).
  • the compounds of the invention may be in amorphous or crystalline form (polymorphs). Furthermore, the compounds of the invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the invention.
  • polymorph refers to a crystalline form of a compound (or a salt, hydrate or solvate thereof) in a particular crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms generally have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can cause one crystalline form to predominate. Various polymorphs of a compound can be prepared by crystallization under different conditions.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • More expansion of erythroid progenitors and erythroblasts in CD34+ derived cells obtained from bone marrow, umbilical cord blood or iPSC/ESC is also of scientific interest, especially the expansion of erythroid precursors allows us to obtain more erythroid lineage stem/progenitor cells, thereby enriching our understanding of the mechanisms underlying erythroid development.
  • EPs or EBs are derived from stem cells in donor PBMCs.
  • the stem cells are genetically modified prior to or during culture.
  • the stem cells are hematopoietic stem cells.
  • the hematopoietic stem cells are derived from bone marrow or umbilical cord blood.
  • the subject of the experiment is a mammal. In another example, the subject is a human, mouse or rat.
  • erythroid precursor cells or erythroblasts can be cultured in vitro in culture medium.
  • Media include erythroid differentiation medium or serum-free erythroid differentiation medium and expansion medium.
  • the erythrocyte differentiation medium comprises IMDM, 10% FBS, 5% human serum, 300 ⁇ g/ml holo-transferrin, 2 IU/ml heparin, 10 ⁇ g/ml insulin, 2 mM L-glutamine; then supplemented with 3 IU /ml erythropoietin, 50ng/ml human stem cell factor and 10ng/ml interleukin (IL)-3.
  • erythrocyte differentiation and expansion in serum-free medium includes SFEM II supplemented with 3 IU/mL erythropoietin, 50 ng/ml human stem cell factor, 10 ng/ml interleukin (IL)-3 and Insulin-like growth factor (IGF)-1.
  • SFEM II supplemented with 3 IU/mL erythropoietin, 50 ng/ml human stem cell factor, 10 ng/ml interleukin (IL)-3 and Insulin-like growth factor (IGF)-1.
  • preferred BRAF kinase inhibitors are compounds of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • the BRAF kinase inhibitor induces paradoxical activation of MAPK;
  • the BRAF kinase inhibitor promotes RAF protein dimerization;
  • the BRAF kinase inhibitor induces ⁇ C-helix and DFG domain in BRAF kinase IN conformation of at least one of them, and the IN conformation of R506;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof:
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • TGF- ⁇ inhibitors currently on the market and in clinical practice are well known to those skilled in the art, and all of them can be used for the purpose of the present invention.
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof:
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • the SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof:
  • Glucocorticoid is a kind of adrenocortical hormone, which is a steroid hormone secreted by the zona fascicularis in the middle layer of the adrenal cortex, and can also be synthesized artificially in vitro by chemical methods. It plays an important role in regulating the body's development, growth, metabolism, and immune function. It is the most important regulatory hormone for the body's stress response, and it is also the most widely used and effective anti-inflammatory and immunosuppressant clinically. In urgent or critical situations, glucocorticoids are often the drug of choice.
  • Glucocorticoid drugs mainly act on the downstream pathway of intracellular glucocorticoid receptors, increase the production of red blood cells and hemoglobin, increase platelets and fibrinogen, increase the amount of monocytes and neutrophils entering the blood circulation, and lymphocytes and decreased eosinophils and basophils.
  • Glucocorticoids are clinical drugs currently used to treat anemia, especially refractory anemia, bone marrow failure anemia (such as DBA anemia), which can expand erythroid precursor cells (BFU-E and CFU-E) in vitro , can also be used in vivo to treat anemia and promote the production of red blood cells in the body.
  • Anemia diseases are mainly classified into anemia caused by decreased or defective red blood cell production, anemia caused by destruction of red blood cells, and anemia caused by excessive blood loss.
  • the anemia caused by reduced or defective erythropoiesis may be primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome, dyskeratosis congenita, Fanconi anemia, Anemia due to congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, myelodysplastic syndrome.
  • DBA Diamond-Blackfan anemia
  • CDA congenital dyserythropoietic anemia
  • thalassemia thalassemia
  • sickle cell anemia myelodysplastic syndrome.
  • the anemia caused by the reduction or defect of red blood cell production can also be secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency anemia, iron deficiency anemia, chronic Inflammatory anemia, anemia of endocrine disease, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary aplastic anemia is caused by: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO incompatibility, stem cell transplantation, pyoderma gangrenosum), lymphoproliferative diseases (such as Chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin lymphoma, etc.), other hematological malignancies (such as
  • the anemia caused by the destruction of red blood cells may be primary, including hereditary spherocytosis, hereditary ellipsocytosis, abeta lipoproteinemia, anemia caused by enzyme deficiency (such as pyruvate kinase and hexose Kinase deficiency leads to glycolysis deficient anemia, glucose 6-phosphate dehydrogenase deficiency and glutathione synthetase deficiency, anemia due to increased oxidative stress).
  • enzyme deficiency such as pyruvate kinase and hexose Kinase deficiency leads to glycolysis deficient anemia, glucose 6-phosphate dehydrogenase deficiency and glutathione synthetase deficiency, anemia due to increased oxidative stress.
  • the anemia caused by the destruction of red blood cells can also be secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic diseases (such as hemolytic disease of newborns), red blood cell Mechanical injury (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), infection-induced hemolytic anemia (eg, malaria infection).
  • antibody-mediated mild autoimmune hemolytic anemia e.g, cold agglutinin hemolytic anemia, hemolytic diseases (such as hemolytic disease of newborns), red blood cell Mechanical injury (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), infection-induced hemolytic anemia (eg, malaria infection).
  • the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood sampling in laboratory tests, and optionally combined with insufficient erythropoiesis), external/internal injuries (acute blood loss caused by various traumas or operations), gastrointestinal lesions Acute bleeding (eg, varicose lesions, peptic ulcers) or chronic bleeding (eg, angiodysplasia), chronic bleeding due to gynecological disorders, acute or chronic bleeding due to cancer (including colorectal and bladder cancers, especially in late stage), blood-feeding intestinal nematode infections (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws and medical procedures.
  • intestinal nematode infections such as hookworm and whipworm causing hemorrhagic anemia
  • iatrogenic anemia blood loss from repeated blood draws and medical procedures.
  • anemic disease may be resistant to erythropoietin or glucocorticoids or other drugs for the treatment of anemia.
  • the present invention provides a medicament for treating primary or secondary anemia, which comprises a BRAF kinase inhibitor and one or more pharmaceutically acceptable excipients.
  • the drug further comprises a TGF- ⁇ inhibitor and/or a SMAD2/3 inhibitor and/or a glucocorticoid.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase
  • the IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives:
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof:
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof:
  • anemia is anemia caused by decreased or defective erythropoiesis.
  • anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome , dyskeratosis congenita, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, anemia caused by myelodysplastic syndrome.
  • DBA Diamond-Blackfan anemia
  • CDA congenital dyserythropoietic anemia
  • thalassemia thalassemia
  • sickle cell anemia anemia caused by myelodysplastic syndrome.
  • anemia caused by reduced or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency type anemia, iron deficiency anemia, chronic inflammatory anemia, endocrine disease anemia, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary regeneration Obstacle anemia is caused by the following reasons: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, gangrenous pyoderma, etc.) disease), lymphoproliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin
  • anemia is anemia due to destruction of red blood cells.
  • anemia due to destruction of red blood cells is primary, including hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, enzyme deficiency Anemias resulting from (eg, glycolysis deficient anemia due to pyruvate kinase and hexokinase deficiencies, glucose 6-phosphate dehydrogenase and glutathione synthase deficiencies, anemia due to increased oxidative stress).
  • anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • antibody-mediated mild autoimmune hemolytic anemia eg, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • anemia is anemia caused by excessive blood loss.
  • the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood collection as detected by the laboratory, and optionally combined with insufficient erythropoiesis), external/internal injuries (various trauma or Acute blood loss due to surgery), acute bleeding due to gastrointestinal lesions (eg, variceal lesions, peptic ulcer) or chronic blood loss (eg, angiodysplasia), chronic blood loss due to gynecological diseases, cancer (including colorectal cancer and bladder cancer) Acute or chronic blood loss from cancer, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
  • the disease is resistant to erythropoietin or glucocorticoids or other currently available drugs for the treatment of anemia.
  • the invention provides a kit comprising:
  • TGF- ⁇ inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or erythropoietin and/or stem cell factor are examples of TGF- ⁇ inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or erythropoietin and/or stem cell factor.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BA
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
  • the present invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF- ⁇ inhibitor, or a BRAF kinase inhibitor and Use of SMAD2/3 inhibitors, or BRAF kinase inhibitors, TGF- ⁇ inhibitors and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of a medicament for expanding erythroid precursor cells or erythroblasts use.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BA
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives: Luspatercept, Sotatercept, SIS3 HCl , Alantolactone, Halofuginone, and AUDA.
  • the present invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and a TGF-beta inhibitor, or a BRAF kinase inhibitor and a SMAD2/3 inhibitor, or a BRAF kinase inhibitor, a TGF-beta inhibitor Agents and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of medicines for the treatment of primary or secondary anemia.
  • anemia is anemia caused by decreased or defective erythropoiesis.
  • anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome , dyskeratosis congenita, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, anemia caused by myelodysplastic syndrome.
  • DBA Diamond-Blackfan anemia
  • CDA congenital dyserythropoietic anemia
  • thalassemia thalassemia
  • sickle cell anemia anemia caused by myelodysplastic syndrome.
  • anemia caused by reduced or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency type anemia, iron deficiency anemia, chronic inflammatory anemia, endocrine disease anemia, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary regeneration Obstacle anemia is caused by the following reasons: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, gangrenous pyoderma, etc.) disease), lymphoproliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin
  • anemia is anemia due to destruction of red blood cells.
  • anemia due to destruction of red blood cells is primary, including hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, enzyme deficiency Anemias resulting from (eg, glycolysis deficient anemia due to pyruvate kinase and hexokinase deficiencies, glucose 6-phosphate dehydrogenase and glutathione synthase deficiencies, anemia due to increased oxidative stress).
  • anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • antibody-mediated mild autoimmune hemolytic anemia eg, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • anemia is anemia caused by excessive blood loss.
  • the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood collection as detected by the laboratory, and optionally combined with insufficient erythropoiesis), external/internal injuries (various trauma or Acute blood loss due to surgery), acute bleeding due to gastrointestinal lesions (eg, variceal lesions, peptic ulcer) or chronic blood loss (eg, angiodysplasia), chronic blood loss due to gynecological diseases, cancer (including colorectal cancer and bladder cancer) Acute or chronic blood loss from cancer, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
  • the disease is resistant to erythropoietin or glucocorticoids or other drugs for the treatment of anemia.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BA
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, Regorafenib, or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
  • the present invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF- ⁇ inhibitor, or a BRAF kinase inhibitor and SMAD2/3 inhibitors, or BRAF kinase inhibitors, TGF-beta inhibitors and SMAD2/3 inhibitors, and their use in combination with glucocorticoids for expanding erythroid precursor cells or erythroblasts.
  • said BRAF kinase inhibitor delays the progression of terminal phase differentiation of erythrocytes.
  • said erythroid precursor cells are selected from the group consisting of erythroid colony forming units (CFU-E), burst forming unit erythroid progenitors (BFU-E) and proerythroblasts.
  • the erythroid precursor cells are genetically modified erythroid precursor cells or drug-loadable erythroid precursor cells.
  • said genetically modified erythroid precursor cells are capable of treating genetic diseases, such as phenylketonuria and cancer.
  • said erythroid precursor cells or erythroblasts are derived from blood sources, including blood and blood products of mammals such as mice, rats, humans, and the like.
  • said erythroid precursor cells or erythroblasts are CD34+ derived cells obtained from bone marrow, umbilical cord blood or embryonic/induced pluripotent stem cells (iPSC/ESC).
  • erythroid precursor cells or erythroblasts are derived from in vitro differentiated and expanded human erythroid precursor cells and stem cells from peripheral blood mononuclear cells (PBMC), or from PBMC of stem cells.
  • PBMC peripheral blood mononuclear cells
  • stem cells are hematopoietic stem cells.
  • hematopoietic stem cells are derived from bone marrow or umbilical cord blood.
  • stem cells are genetically modified before or during culturing.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BA
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, Regorafenib, or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
  • said BRAF kinase inhibitor is used at a concentration of 1 nM to 100 ⁇ M, preferably 100 nM to 10 ⁇ M, more preferably 1 ⁇ M to 10 ⁇ M.
  • erythropoietin is also used concomitantly.
  • the invention provides a method of expanding erythroid precursor cells or erythroblasts comprising administering to a subject a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and /or stem cell factor, or BRAF kinase inhibitor and TGF- ⁇ inhibitor, or BRAF kinase inhibitor and SMAD2/3 inhibitor, or BRAF kinase inhibitor, TGF- ⁇ inhibitor and SMAD2/3 inhibitor, and their combination Combinations of glucocorticoids.
  • said erythroid precursor cells are selected from the group consisting of erythroid colony forming units (CFU-E), burst forming unit erythroid progenitors (BFU-E) and proerythroblasts.
  • the subject is a mammal, such as a mouse, a rat, a human, or the like.
  • anemia is anemia caused by decreased or defective erythropoiesis.
  • anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome , dyskeratosis congenita, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, anemia caused by myelodysplastic syndrome.
  • DBA Diamond-Blackfan anemia
  • CDA congenital dyserythropoietic anemia
  • thalassemia thalassemia
  • sickle cell anemia anemia caused by myelodysplastic syndrome.
  • anemia caused by reduced or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency type anemia, iron deficiency anemia, chronic inflammatory anemia, endocrine disease anemia, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary regeneration Obstacle anemia is caused by the following reasons: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, gangrenous pyoderma, etc.) disease), lymphoproliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin
  • anemia is anemia due to destruction of red blood cells.
  • anemia due to destruction of red blood cells is primary, including hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, enzyme deficiency Anemias resulting from (eg, glycolysis deficient anemia due to pyruvate kinase and hexokinase deficiencies, glucose 6-phosphate dehydrogenase and glutathione synthase deficiencies, anemia due to increased oxidative stress).
  • anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • antibody-mediated mild autoimmune hemolytic anemia eg, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • anemia is anemia caused by excessive blood loss.
  • the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood collection as detected by the laboratory, and optionally combined with insufficient erythropoiesis), external/internal injuries (various trauma or Acute blood loss due to surgery), acute bleeding due to gastrointestinal lesions (eg, variceal lesions, peptic ulcer) or chronic blood loss (eg, angiodysplasia), chronic blood loss due to gynecological diseases, cancer (including colorectal cancer and bladder cancer) Acute or chronic blood loss from cancer, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
  • the disease is resistant to erythropoietin or glucocorticoids or other drugs for the treatment of anemia.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BA
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
  • said BRAF kinase inhibitor is used at a concentration of 1 nM to 100 ⁇ M, preferably 100 nM to 10 ⁇ M, more preferably 1 ⁇ M to 10 ⁇ M.
  • erythropoietin and/or stem cell factor are also used concomitantly.
  • the differentiation medium includes: IMDM, 10% FBS, 5% human serum, 300 ⁇ g/ml holo-transferrin (Sigma-Aldrich, St.Louis, MO, USA, Cat T0665), 2IU/ml heparin (Sigma-Aldrich, St.
  • Serum-free differentiation medium includes: SFEM II (StemCell Technologies, Vancouver, BC, Canada, Cat 09655), supplemented with 3IU/mL erythropoietin (Amgen, Thousand Oaks, CA, Cat 55513-144-10), 50ng/ml Human stem cell factor (StemCell, Vancouver, BC, CA, Cat 78062), 10ng/ml interleukin (IL)-3 (StemCell, Vancouver, BC, CA, Cat 78042) and insulin-like growth factor (IGF)-1 ( 40ng/ml, PeproTech, Rocky Hill, NJ, USA, Cat AF-100-11).
  • SFEM II SteCell Technologies, Vancouver, BC, Canada, Cat 09655
  • 3IU/mL erythropoietin Amgen, Thousand Oaks, CA, Cat 55513-144-10
  • 50ng/ml Human stem cell factor StemCell, Vancouver, BC, CA, Cat 78062
  • 10ng/ml interleukin (IL)-3 Stem
  • Hematopoietic stem and progenitor cell serum-free medium includes: SFEM II (StemCell Technologies, Vancouver, BC, Canada, Cat 09655), supplemented with 100 ng/ml human stem cell factor (StemCell, Vancouver, BC, CA, Cat 78062), 100 ng/ml FLT3-L (StemCell, Vancouver, BC, CA, Cat 78137), 50ng/ml thrombopoietin (TPO) (StemCell, Vancouver, BC, CA, Cat 78210), 10 ⁇ g/ml human low density lipoprotein (Low density lipoprotein) ) (StemCell, Vancouver, BC, CA, Cat 02698).
  • SFEM II SteCell Technologies, Vancouver, BC, Canada, Cat 09655
  • TPO thrombopoietin
  • Fresh umbilical cord blood was mixed with phosphate buffered solution (DPBS) at a ratio of 1:1, and then carefully poured into a 50mL SepMate separation tube filled with lymphocyte separation agent (Lymphoprep) in the lower layer with a pipette, and centrifuged in a precooled to 4°C Centrifuge at 1200g for 10min. Then the supernatant serum and peripheral mononuclear cells were transferred to a new 50mL tube and centrifuged at 2000rpm for 10min.
  • DPBS phosphate buffered solution
  • BRAF inhibitor solution Dissolve SB-590885 in 2% Cremophor EL, 2% N,N-dimethylacetamide, pH 5.0, to 30mg/ml.
  • GDC-0879 was dissolved in 50% PGE 300 and 50% PBS to 50mg/ml.
  • Encorafenib was dissolved in 50% PGE 300 and 50% PBS to 30mg/ml.
  • SB-590885 is administered by intraperitoneal injection, once a day unless otherwise mentioned.
  • GDC-0879 and Encorafenib were administered by gavage.
  • mice To induce acute hemolytic anemia: Dissolve phenylhydrazine in PBS and inject 60 mg/kg intraperitoneally into mice.
  • Example 1 High-throughput screening of small molecule expansion of erythroid precursor cells
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 02690) for 3 days, and then screened the compound library.
  • SFEM II StemCell Technologies, Vancouver, BC, CA, Cat 09655
  • CC100 StemCell Technologies, Vancouver, BC, CA, Cat 02690
  • GDC-0879 is a potent BRAF inhibitor with IC 50 of 0.13nM.
  • FIG. 1A it is a schematic diagram of high-throughput screening (HTS).
  • HTS high-throughput screening
  • Example 2-1 Explore the optimal concentration of GDC-0879 treatment
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cell density was kept below 100,000/ml every time the medium was changed.
  • Add 20% volume of Cell to each well on the ninth day Cell Viability reagent after incubating at 37°C in the dark for 2 hours, detect the absorbance at 560nM and 590nM on the BioTek multi-functional microplate detector, calculate the ratio of 560/590 in each well, subtract the background ratio and calculate for each experimental group Ratio relative to the mean of the control group.
  • the experiments used concentrations of 0.1 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M and 20 ⁇ M GDC-0879 and concentrations of 0.1 ⁇ M, 1 ⁇ M Galunisertib (LY2157299) and combinations of 0.1 ⁇ M GDC-0879 and 1 ⁇ M Galunisertib.
  • GDC-0879 has a wide range of effective concentrations. In differentiation medium, GDC-0879 at 100nM to 10 ⁇ M is most effective for the expansion of erythroid precursor cells. The optimal effective concentration of GDC-0879 is in the range of 1-10 ⁇ M, and the erythroid expansion of cord blood-derived CD34+ cells can reach more than 5 times.
  • TGF- ⁇ inhibitors were previously reported as potential erythroblast stimulators.
  • Galunisertib (LY2157299) is an oral, selective TGF- ⁇ receptor type I (TGF- ⁇ RI) kinase inhibitor with IC50 of 56nM.
  • GDC-0879 combined with Galunisertib showed about 6-fold amplification on the 9th day. Medium and small molecules were changed every 3 days.
  • GDC GDC-0879
  • Gal Galunisertib
  • Dex dexamethasone.
  • Example 2-2 Explore the optimal concentration of BRAF inhibitor treatment
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
  • Figure 2B shows the effect of a single initial dose of BRAF inhibitor on the expansion of erythroid precursors only.
  • Figure 2B shows that after 3 days of expansion culture, 200 ⁇ L of differentiation medium containing 300 CD34+ cells was added to a 96-well plate, and then small molecule compounds were added to each well. Three biological replicates were prepared for each small molecule in this screening, and culture was continued for 7 days without changing the medium thereafter.
  • Figure 2B shows the expansion effect of various BRAF inhibitors on erythroid precursor cells in a wide range of concentrations (administered only once).
  • Fig. 2B shows the results obtained by the 96-well plate assay that different concentrations of BRAF inhibitors stimulate the proliferation of erythroid progenitor cells on Day 7.
  • Vemu Vemurafenib; Enco: Encorafenib; Dabra: Dabrafenib; SB: SB-590885; PLX: PLX-8394; GDC: GDC-0879.
  • Statistical data histograms are mean ⁇ SD, **: P ⁇ 0.01, ***: P ⁇ 0.001.
  • SB and GDC are BRAF inhibitors that can better promote the proliferation of early erythroid stem progenitor cells.
  • Figure 2C shows the expansion effect of selected BRAF inhibitors on erythroid precursor cells over a wide range of concentrations (three doses).
  • GDC-0879 has a wide effective concentration range. GDC-0879 at 100 nM to 10 ⁇ M in differentiation medium is most effective for the expansion of erythroid precursor cells. The optimal effective concentration of GDC-0879 is in the range of 300nM-10 ⁇ M, and the erythroid expansion of cord blood-derived CD34+ cells can reach 5-10 times.
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
  • the cells were counted at the beginning of Day 0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day 0, and the cell density was kept below 200,000/ml when changing the medium.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed.
  • Figure 3A shows the effect of BRAF inhibitors on the expansion of erythroblasts.
  • Figure 3A is the growth curves of three small molecules in the erythroid differentiation system at the optimal concentration. Three biological replicates were set for each condition, and statistical data are mean ⁇ SD, ***: P ⁇ 0.001, ****: P ⁇ 0.0001.
  • Fig. 3B shows the synergistic effect of BRAF inhibitors and TGF- ⁇ inhibitors on the expansion of erythroblasts.
  • Fig. 3B The growth curve of erythroblasts after small molecule treatment in differentiation medium is shown in Fig. 3B.
  • Fig. 3B The growth curve of erythroblasts after small molecule treatment in differentiation medium is shown in Fig. 3B.
  • red blood cells expanded about 10 times.
  • Administration of GDC-0879 in combination with Galunisertib resulted in approximately 12-fold expansion of red blood cells at day 12.
  • GDC GDC-0879
  • Gal Galunisertib
  • LY LY2109761.
  • Data are presented as mean ⁇ SD.
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. A total of 9 days were administered, and no treatment was administered after the 9th day.
  • the FACS results in Figure 4A show the effects of different concentrations of GDC-0879 on the development of erythrocytes and the expression of the erythroid surface marker CD235a (Glycophorin A) in the differentiation medium on day 6 (small molecule treatment for 6 days).
  • the FACS results in Figure 4B show the effects of different concentrations of GDC-0879 on the development of erythrocytes and the expression of the erythroid surface marker CD235a (Glycophorin A) in the differentiation medium on day 6 (small molecule treatment for 6 days).
  • CD235a erythroid surface marker CD235a
  • the main cell group in the 1 ⁇ M GDC-0879 treatment group was still CD235a-negative, while the main cell group in the control group was CD235a-positive, that is, GDC-0879 delayed the terminal differentiation process of erythrocytes, allowing more self-renewal of erythroblasts.
  • Figure 4C shows the effects of different concentrations of GDC-0879 in the differentiation medium on day 12 on the development of erythrocytes and the expression of the erythroid surface marker CD235a (Glycophorin A) (small molecule treatment for 6 days).
  • CD71 expression decreased on day 12 until erythrocytes matured.
  • the treatment group still had high CD71 expression cell population, indicating that the red blood cell differentiation in the GDC-0879 treatment group was delayed.
  • Figure 5A shows that BRAF inhibitors promote PBMC-derived colony formation.
  • PBMC Resuscitate PBMC from a healthy donor one day before the experiment, and overnight in the differentiation medium.
  • 100,000 PBMC cells per well (35 mm) are seeded in methylcellulose containing cytokines necessary for the development of myeloid cells
  • Methocult H4435 StemCell, Vancouver, BC, CA, Cat H4435
  • the medicines of the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed, counted and area counted using a Leica inverted phase-contrast microscope at 14 days.
  • Figure 5A is the statistics of the number of erythrocyte colonies (middle) and the area of a single erythrocyte colony (right) in different administration groups of Methocult H4435 and the control group.
  • GDC GDC-0879;
  • Dex Dexamethasone. Three biological repetitions were set for each condition, and statistical data are mean ⁇ SD; ns: no significant difference, P>0.05; ****: P ⁇ 0.0001.
  • Figure 5B shows that BRAF inhibitors promote erythropoiesis of CD34+ cells derived from umbilical cord blood (EPO only medium).
  • the CD34+ cells were revived one day before the experiment, and overnight in the differentiation medium. On the first day, 600 CD34+ cells per well (35 mm) were inoculated in the methylcellulose medium Methocult H4330 containing only EPO (StemCell, Vancouver, BC, CA, Cat H4330), the drugs in the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed at 14 days using a Leica inverted phase contrast microscope.
  • Figure 5B shows the results of erythrocyte-specific colony formation at day 14.
  • 600 CD34+ cells were pre-cultured overnight in erythroid differentiation medium containing only EPO, and cultured in 35 mm wells in methylcellulose medium containing only EPO the next day, and on the 14th day of culture Take photos of the medium.
  • no large BFU-E colonies could be seen with naked eyes and pictures, but large BFU-E colonies could be seen with the naked eye in the GDC-0879 treatment group.
  • Figure 5C shows the results of red blood cell-specific colony formation on day 14 detected under a bright-field microscope using a 5 ⁇ objective lens. There were significant differences in the colony area and the number of cells per colony between the treatment group and the control group. Scale bar, 500 ⁇ m.
  • Figure 5D shows that BRAF inhibitors promote erythropoiesis and lineage development of cord blood-derived CD34+ cells (myeloid medium).
  • the CD34+ cells were revived one day before the experiment, and kept overnight in the hematopoietic stem cell maintenance medium.
  • 200 CD34+ cells per well were inoculated in methylcellulose containing cytokines required for each lineage of myeloid development
  • Methocult H4435 StemCell, Vancouver, BC, CA, Cat H4435
  • the medicines of the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed at 14 days using a Leica inverted phase contrast microscope.
  • BRAF inhibitors did not result in a significant hematopoietic lineage shift (Panel D).
  • A At the beginning (Day 0), 100 CD34+ cells were inoculated on the culture medium of the treatment group and the control group, and then eluted with PBS on the fourteenth day and counted for each well.
  • B Number of red blood cells (Non CD11b+) in each well.
  • C The proportion of granulocytes (CD11b+) in each well.
  • D On Methocult H4435, UCB-hCD34+ was administered, and the number of colonies grown from GDC and the control group was counted.
  • Example 6 The effect of small molecules targeting BRAF on promoting the proliferation of erythroid precursor cells
  • Example 6-1 Small molecule test results for the same target
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. A total of 9 days were administered, and no treatment was administered after the 9th day.
  • Figure 6A shows the results of detection of other small molecules targeting BRAF with the same target (the concentration of small molecules not specifically marked is 1 ⁇ M, treated for 9 days).
  • the combined treatment group of 1 ⁇ M Encorafenib and Dabrafenib had more than 3 times the number of red blood cell expansion on the 10th day.
  • Example 6-2 The effect of small molecules targeting BRAF on promoting the proliferation of erythroid precursor cells
  • the CD34+ cells were revived one day before the experiment, and overnight in the differentiation medium.
  • 300 CD34+ cells per well (22mm) were inoculated in EPO-only methylcellulose medium Methocult H4330 (StemCell Technologies, Vancouver) , BC, CA, Cat H4330), the drugs in the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed and counted using a Leica inverted phase-contrast microscope at 14 days.
  • Figure 6B shows the results of erythrocyte-specific colony formation on day 14 in EPO-only methylcellulose medium with other inhibitors of BRAF.
  • 300 CD34+ cells were pre-cultured overnight in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100, and then placed in 35mm wells the next day, and cultured on the 14th day base photo. 2 replicates per treatment group.
  • Gal treated with 2 ⁇ M Galunisertib
  • Enco treated with 5 ⁇ M Encorafenib
  • Dabr treated with 5 ⁇ M Dabrafenib
  • GDC treated with 5 ⁇ M GDC-0879
  • SB treated with 5 ⁇ M SB-590885
  • Ctrl control (DMSO).
  • Figure 6C shows the results of erythrocyte-specific colony formation at day 14 for other combinations containing BRAF inhibitors in EPO-only methylcellulose media.
  • 300 CD34+ cells were pre-cultured overnight in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100, and then placed in 35mm wells the next day, and cultured on the 14th day base photo. 2/3 replicates per treatment group.
  • GDC Gal treated with 2 ⁇ M galunisertib and 5 ⁇ M GDC-0879
  • SB Gal treated with 2 ⁇ M galunisertib and 5 ⁇ M SB-590885
  • Ctrl control.
  • Figure 6D shows the colony formation results under the light field microscope, and the erythrocyte-specific colony formation results on day 14 were detected under the 5 ⁇ objective lens. Under the microscope, the largest colony of erythrocytes in Figures 6B and 6C is shown in Figure D. The colony area and the number of cells per colony were significantly different between the treatment group and the control group.
  • Gal treated with 2 ⁇ M Galunisertib
  • Enco treated with 5 ⁇ M Encorafenib
  • Dabr treated with 5 ⁇ M Dabrafenib
  • GDC treated with 5 ⁇ M GDC-0879
  • SB treated with 5 ⁇ M SB-590885
  • +SB 2 ⁇ M Galunisertib and 5 ⁇ M SB-590885 treatment
  • Ctrl control.
  • Scale bar 500 ⁇ m.
  • Gal treated with 2 ⁇ M Galunisertib; Enco: treated with 5 ⁇ M Encorafenib; Dabr: treated with 5 ⁇ M Dabrafenib;
  • GDC treated with 5 ⁇ M GDC-0879; SB: treated with 5 ⁇ M SB-590885;
  • Gal+GDC treated with 2 ⁇ M Galunisertib and 5 ⁇ M GDC-0879; : 2 ⁇ M Galunisertib and 5 ⁇ M SB-590885 treatment;
  • Ctrl control (DMSO).
  • BRAF inhibitors generally have paradoxical activation effects, and have the potential to promote erythroid proliferation synergistically with other small molecules in the colony formation ability.
  • Embodiment 7 effective dosing cycle test
  • Embodiment 7-1 Effective administration cycle test result 1
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed.
  • Different administration time groups were administered for 0-3 days, 3-6 days, 6-9 days and 9-12 days respectively.
  • Figure 7A shows the test results of GDC-0879 administered in different cycles from the 0th day to the 12th day, and the final cell count was counted on the 15th day, with 3 days as the administration period, to explore the drug dependence of continuous administration effect.
  • Figure 7B shows the comparison of the number of cells in the 0-3 day treatment group and the control group on day 13. In the different cycles of red blood cells treated in the differentiation medium, the expansion ability of the 6-9 day treatment group was the strongest. This figure demonstrates the persistent effect of GDC-0879 expansion. The number of cells in the single-dose treatment group also increased significantly, indicating that GDC-0879 expanded erythroid precursor cells at all stages.
  • Embodiment 7-2 Effective administration cycle test result 2
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. Dosing for 3 days, after the 3rd day, no more dosing treatment.
  • Fig. 7C shows the FACS results of the experimental group and the control group on day 13 in the differentiation medium.
  • the treatment group was administered from the 0th to the 3rd day. It can be seen from the FACS graph that the differentiation rate of the 1 ⁇ M GDC-0879 treatment group for 0-3 days was almost the same as that of the control group on the 13th day, and even had a higher enucleation rate.
  • 1 ⁇ M GDC-0879 was administered only from day 1 to day 3. This indicated that early drug administration still promoted proliferation, but did not affect differentiation at all, and even promoted terminal maturation and denucleation.
  • Embodiment 7-3 Effective administration cycle test result 3
  • the cells were counted at the beginning of Day 0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day 0, and the cell density was kept below 100,000/ml when changing the medium.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed.
  • Different administration time groups were administered for 0-3 days, 3-6 days, 0-6 days, 3-9 days and 6-9 days respectively.
  • Figure 7D shows statistics on the enucleation rate of each group administered for 3 days from day 13 to day 16 (treatment group, 1 ⁇ M GDC-0879 treatment group). It can be seen from the figure that GDC-0879 does not directly affect the process of denucleation and terminal maturation of erythrocytes.
  • the vertical axis is the percentage of cells.
  • Figure 7E shows statistics on the enucleation rate of each group in the two administration periods (6 consecutive days) from day 13 to day 16 (treatment group: 1 ⁇ M GDC-0879 treatment). It can be seen from the figure that GDC-0879 does not directly affect the process of denucleation and terminal maturation of erythrocytes.
  • the vertical axis is the percentage of cells.
  • Example 8 Expansion of erythroblasts in PBMCs in vitro
  • Example 8-1 In vitro expansion of erythroblasts from healthy donor PBMCs in serum-free differentiation medium
  • the cells were counted at the start of Day 0, and 500,000 PBMC cells from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) were inoculated in each well of serum-free differentiation medium, and at Day 0, according to the corresponding concentration for administration.
  • the medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced. After the ninth day of administration, the different administration groups were centrifuged and the cell pellets were photographed.
  • Figure 8A shows the cells of the treated and control groups at day 9.
  • PBMCs from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) were cultured in serum-free erythrocyte expansion medium (SFEM II (StemCell Technologies, Vancouver, BC, Canada, Cat 09655)) for 9 days, and 3 IU/ ml erythropoietin (Amgen, Thousand Oaks, CA, Cat 55513-144-10), 50ng/ml human stem cell factor (StemCell, Vancouver, BC, CA, Cat 78062), 10ng/ml interleukin (IL)- 3 (StemCell, Vancouver BC CA Cat 78042) and insulin-like growth factor (IGF)-1 (40ng/mL, PeproTech, Rocky Hill, NJ, USA, Cat AF-100-11)).
  • SFEM II serum-free erythrocyte expansion medium
  • IGF insulin-like growth factor
  • Example 8-2 In vitro expansion of erythroblasts in PBMC Result 1
  • the cells were counted at the start of Day 0, and 500,000 PBMC cells from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) were inoculated in each well of serum-free differentiation medium, and at Day 0, according to the corresponding concentration for administration.
  • the culture medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the number of cells was counted on the ninth day of administration.
  • Figure 8B shows the change in the number of erythroblasts in the PBMCs of healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) expanded in vitro on day 9.
  • GDC GDC-0879; SB: SB-590885. Data are presented as mean ⁇ SD.
  • BRAF inhibitors can efficiently expand the erythroid precursor cells therein, and a large number of erythrocytes can be obtained without any separation means for erythroid precursor cells.
  • Example 8-3 In vitro expansion of erythroblasts in PBMC results 2
  • the cells were counted at the beginning of Day 0, and 500,000 PBMC cells from healthy donors were inoculated in each well of serum-free differentiation medium, and administered at the corresponding concentration on Day 0, and the cell density was kept at Below 1000000/ml.
  • the medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cells were detected by flow cytometry on the ninth day of administration.
  • Figure 8C shows the FACS results of PBMCs from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) cultured in serum-free erythrocyte expansion medium on day 9.
  • Treatment group 1 ⁇ M SB-590885.
  • Figure 8D shows the statistics of the FACS results of the PBMC of healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) on day 10 of the cell population ratio.
  • the vertical axis is the percentage of cells. Double negative: CD71 and CD235a double negative; Double positive: CD71 and CD235a double positive; SB: SB-590885; GDC: GDC-0879.
  • Example 8-4 In vitro expansion of erythroblasts in PBMC results 3
  • the medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cells were detected by flow cytometry on the ninth day of administration.
  • Figure 8F shows the FACS results of PBMC from healthy donors (Donor1-Donor5) cultured in serum-free erythrocyte expansion medium. Treatment group: 1 ⁇ M SB-590885.
  • Figure 8G shows the FACS results of PBMC from healthy donors (Donor1-Donor5) cultured in serum-free erythrocyte expansion medium. Treatment group: 1 ⁇ M SB-590885.
  • BRAF inhibitors can greatly expand the proportion of erythroid precursor cells in PBMC, and better promote the proliferation of erythroid precursor cells in PBMC.
  • Example 8-5 In vitro expansion of erythroblasts in PBMC Result 4
  • Dex glucocorticoid dexamethasone
  • Example 8-6 In vitro expansion of erythroblasts in PBMC Result 5 (SB-590885)
  • Figure 8I Whole-body clonogenic images and representative erythroid monoclonal colony images of SB and controls administered to PBMCs on Methocult H4435.
  • Figure 8K Statistics of the number of erythrocyte colonies (left) and the area of individual erythrocyte colonies in different groups (right) in different Methocult H4435 administration groups and control groups.
  • Embodiment 9 Therapeutic effect of medicine under low EPO condition
  • Embodiment 9-1 Therapeutic effect of medicine under low EPO condition 1
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • Figure 9A shows the effect of GDC-0879 in different concentrations of erythropoietin (EPO) differentiation medium.
  • EPO erythropoietin
  • Embodiment 9-2 Therapeutic effect of medicine under low EPO condition 2
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, for a total of six days, and no further administration for 4 days after six days.
  • Flow cytometry detection was performed on the 14th day of culture.
  • Figure 9B shows the FACS results on day 14 after 5-10 days of culture in the absence of EPO.
  • Left 1 ⁇ M GDC-0879 treatment from day 5 to day 10; right: control.
  • the treatment group used complete erythropoietin differentiation medium (3IU/ml erythropoietin, EPO) for the first 4 days, and used erythropoietin (EPO)-free differentiation medium for 5 to 10 days.
  • EPO erythropoietin
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. Culture in only 1/3 EPO concentration (i.e. 1IU EPO). The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the treatment group was only administered for 9 days. A total of 15 days were cultured in this 1/3 EPO concentration differentiation medium.
  • Figure 9C shows the FACS results on the 15th day after culturing for 1-15 days under the condition of 1/3 concentration of EPO. From day 1 to day 14, 1/3 EPO differentiation medium (1IU/ml EPO) was used. In the treatment group, 1 ⁇ M GDC-0879 was added to the culture medium from day 1 to day 9.
  • Embodiment 9-4 Therapeutic effect of medicine under low EPO condition 4
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the culture medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the administration group was administered for 12 days.
  • a total of 19 days were cultured in the differentiation medium of these two EPO concentrations.
  • Fig. 9D shows the statistics of the denucleation rate of red blood cells under the conditions of various EPO concentrations from day 13 to day 19. Most of the cells in the control group were apoptotic by day 15, therefore no further data were available after day 15.
  • the treatment group was treated with 1 ⁇ M GDC-0879 from day 1 to day 12.
  • 3 IU/ml erythropoietin was added to the differentiation medium; in the 1/3 EPO group, 1.5 IU/ml EPO was added to the differentiation medium.
  • BRAF inhibitors can promote normal erythroid development, promote erythroid proliferation, and maintain erythroid denucleation ratio.
  • Example 9-5 Effect of BRAF Inhibitor Treatment and Promotion of Erythroid Differentiation under Low Cytokine Concentration Conditions
  • Example 9-5a Therapeutic effect of drugs under low EPO and SCF conditions 1
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • Differentiation medium with EPO concentration and SCF concentration were changed in each group (3 IU for Full Ctrl, 0.15 IU for 5% EPO, no SCF for NO SCF).
  • Figure 9E is a growth curve of UCB CD34+ cells treated with 2 ⁇ M GDC-0879 administration in erythroid medium with low cytokine concentrations of 0% SCF or 5% EPO. In all conditions, the number of cells in the administration group was an order of magnitude higher than that in the control group.
  • Figure 9F is the flow cytometry of 0% SCF (left) and 5% EPO (right) low concentrations of cytokines differentiated (UCB CD34+ cells) on day 9 in the control group (top) and GDC-0879 treatment group (bottom) Representative images of cell surface markers. Compared with the control group, the erythroid development process of the administration group was normal, and the proportion of the undeveloped group was lower.
  • Example 9-7 Effect of BRAF Inhibitor Treatment and Promotion of Erythroid Differentiation under Low Cytokine Concentration-2
  • Figures 9G and 9H Expression of typical erythrocyte surface markers (CD117, CD71 and CD235) of erythroblasts in different erythropoietic conditioned media of GDC treatment group and control group on day 6.
  • Figures 9I and 9J Expression of erythrocyte surface markers (CD117, CD71 and CD235) in different erythropoietic conditioned media in the GDC treatment group and control group on day 14.
  • Figure 9J shows the difference in the expression of CD235 between GDC and the control group under different stress conditions.
  • Figure 9K 5% E.: Typical photographs of red blood cells of samples cultured under different stress conditions after centrifugation of 5% EPO-conditioned medium for 14 days (the number of cells in each EP tube is equal); S.Free: cultured without SCF base. Apart from the cytokine differences mentioned, the other components in the conditioned medium were the same as in the erythropoietic medium.
  • Example 10 In vitro therapeutic effect of anemia caused by pure red blood cell regeneration defects after hematopoietic stem cell transplantation
  • the cells were counted at the start of Day0, and 500,000 PBMC cells from patients with erythroid dysplasia after hematopoietic stem cell transplantation were inoculated in serum-free differentiation medium per well, and administered at the corresponding concentration on Day0. When changing the medium, keep the cell density below 1,000,000/ml. The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cells were detected by flow cytometry and cell number on the 10th day of administration.
  • FIG. 10A shows that GDC-0879 may promote the development and self-renewal of erythroid progenitors in erythroid failure or pure erythroid aplasia after hematopoietic stem cell transplantation.
  • the patient had a history of leukemia and had a low red blood cell count after a hematopoietic stem cell transplant.
  • After 10 days of treatment of this patient's PBMC with GDC-0879 in differentiation medium not only the cell number expanded to about 6-fold (Fig. 10A), but also FACS showed a low proportion of CD71/CD235a negative cell population and good erythroid development outcome.
  • FIG. 10B Media and small molecules were changed every 3 days. Data are expressed as mean ⁇ SD, and statistical results are calculated from three independent biological replicates.
  • FIG. 10B Growth curves of bone marrow mononuclear cells (BMMNCs) from patients with defective erythroid regeneration after hematopoietic stem cell transplantation (EFAHSCT). *Represents pairwise comparisons between different treatment and control groups.
  • GDC GDC-0879; SB: SB-590885; ***: P ⁇ 0.001; ****: P ⁇ 0.0001.
  • Figure 10C Red blood cell surface markers (CD71, CD235, and Hoechst 33342) in SB-treated (bottom) and control (top) erythrocyte development of BMMNCs from EFAHSCT at days 6 (left) and 16 (right).
  • Figure 10F The number of colonies of different lineages cultured from 50,000 EFAHSCT BMMNCs in H4435 myeloid methylcellulose medium.
  • ns no significant difference; ***: p ⁇ 0.001. Data are shown as mean ⁇ SD. Statistical significance of two-group comparisons was assessed using an unpaired Student's t-test.
  • BRAF inhibitors can rescue erythroid developmental failure in patients with EFAHSCT, greatly promote the differentiation and development of erythroid, and expand the number of cells at the same time.
  • Example 11 SB-590885 effectively expands erythroid precursor cells in peripheral mononuclear cells of patients with DBA anemia
  • PBMC cells derived from DBA patients were revived one day before the experiment, and overnight in the differentiation medium.
  • 300,000 PBMC cells per well were inoculated in the methyl group containing EPO.
  • Cellulose medium Methocult H4330 (StemCell, Vancouver, BC, CA, Cat H4330), supplemented to 10ng/ml IL3 (StemCell, Vancouver, BC, CA, Cat 78042) and 50ng/ml hSCF (StemCell, Vancouver, BC, CA, Cat 78062), the medicines of the administration group were premixed evenly, and cultivated in the incubator for 14 days. At 14 days, the colonies were photographed using a Leica inverted phase-contrast microscope and the number of erythroid colonies in each well was counted.
  • Figure 11 shows that in the methylcellulose medium containing EPO, IL3, and SCF, 1 ⁇ M SB-590885 treated peripheral mononuclear cells of different types of DBA patients, and inoculated 300,000 PBMCs per well; the figure shows the results of erythrocyte colony formation at 14 days. Erythrocyte colony formation results of SB-590885 cultured in methylcellulose medium for erythroid cell culture for 14 days. The results of erythrocyte-specific colony formation on day 14 were detected with a 5 ⁇ objective lens. Under the microscope, typical erythrocyte colonies formed by in vitro culture of PBMC from each DBA patient. The colony area of the treatment group was significantly different from that of the control group.
  • RPL11Mut refers to DBA patients with RPL11 mutation, and there are two patient samples of DBA1 and DBA2 in this batch;
  • RPL5Mut refers to DBA patients with RPL5 mutation.
  • Ctrl treated without administration; Treated: only initially administered 1 ⁇ M SB-590885.
  • Example 12 Statistics of SB-590885 effectively amplifying erythroid precursor cells in peripheral mononuclear cells of patients with DBA anemia
  • Figure 12 shows the statistics for the results of Figure 11. Take photos of the erythroid colonies grown on methylcellulose medium in Figure 11, and use FIJI software to count the area of a single erythroid colony, and calculate the individual and average erythroid colonies in two replicate wells area. Statistical data were subjected to unpaired Student's t-test and statistical significance was calculated. Data are presented as mean ⁇ SD; *: p-value ⁇ 0.05; **: p-value ⁇ 0.01.
  • Example 13 BRAF inhibitors effectively promote erythroid differentiation and erythroid proliferation of MDS patient cells
  • MDS Myelodysplastic Syndrome. It is mainly due to the abnormal development (cancerous) of the blood progenitor cells of the patient, or the blood progenitor cells die in the bone marrow for various reasons, or after entering the circulation. In turn, the number of immature or defective cells is lower than that of healthy cells, and the number of healthy red blood cells, healthy white blood cells, and platelets is too small, but the number of pre-cancerous blood progenitor cells is abnormally increased.
  • Figure 13C Area statistics of the erythroid colonies in Figure 13A.
  • Figure 13E Erythrocyte surface markers (CD71, CD235 and Hoechst 33342) in the erythroid development process of BMMNC of MDS patients treated with GDC (lower) and control group (upper) on day 6 (left) and day 9 (right) .
  • Example 13 proves that BRAF inhibitors can rescue erythroid dysplasia in MDS patients, greatly promote the differentiation and development of erythroid, and simultaneously expand the number of cells.
  • BRAF inhibitors have an expansion effect on early erythroid precursor cells
  • the source of BRAF on erythroid progenitor cells that is, the drug's expansion effect on CD34+ cells
  • a and B are not from the same batch of CD34+.
  • Example 15 BRAF inhibitors promote hematopoiesis in mice 1 (normal conditions)
  • A Schematic representation of in vivo administration of BRAF inhibitors.
  • RBC red blood cells
  • HGB hemoglobin
  • MCV mean corpuscular volume
  • Example 16 BRAF inhibitors promote hematopoiesis in mice 2 (acute hemolysis model 1)
  • PHZ phenylhydrazine
  • PHZ can induce hemolytic anemia of red blood cells.
  • acute hemolytic anemia can be induced in mice, which can be used as an acute anemia model to verify the stress hematopoiesis promotion model of BRAF inhibitors.
  • FIG 16A Schematic representation of administration of BRAF inhibitors in the phenylhydrazine (PHZ)-induced hemolytic anemia model.
  • C57BL/6N mice were pretreated with vehicle (control group), GDC-0879 (30 mg/kg) or Encorafenib (25 mg/kg) for 10 days at 6–8 weeks prior to injection of phenylhydrazine (PHZ) on day 0 ( P.O., B.D., Mon-Fri 3 weeks). Blood samples were collected and examined on Day 0, Days 2-5, and Days 7-11. Mice were sacrificed on day 12, when bone marrow and spleen samples were collected. Red arrows indicate the days on which blood samples were collected. Yellow arrows indicate days on which spleen samples were collected.
  • Figure 16B Determination of red blood cells (RBC) (left panel) and red blood cell distribution width CV (RDW-CV) (middle panel) on day 0, days 2-5, and days 7-11. Spleens were weighed on day 12 (right panel). Error bars represent mean ⁇ standard deviation of 4 or 5 biological replicates. The two groups (ns: P>0.05; *: P ⁇ 0.05, **: P ⁇ 0.01) were statistically analyzed by unpaired two-tailed Student's t test.
  • FIG. 16C Hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), platelet count (PLT), white blood cell count (WBC) and PHZ-induced hemolytic anemia in the in vivo experiment in Example 16 Reticulocyte ratio.
  • HGB Hemoglobin
  • HCT hematocrit
  • MCH mean corpuscular hemoglobin
  • PHT platelet count
  • WBC white blood cell count
  • PHZ-induced hemolytic anemia in the in vivo experiment in Example 16 Reticulocyte ratio.
  • Figure 16D Non-erythroid (CD71 and Terl 19 double negative) ratio in the spleen at day 12 after PHZ induction in the in vivo experiment of Example 16.
  • FIG. 16E CD117+ progenitor cell ratio in bone marrow on day 12 after PHZ induction in Example 16.
  • BRAF inhibitors can promote stress hematopoiesis in vivo, especially extramedullary hematopoiesis (splenic hematopoiesis) in an acute hemolysis model.
  • Example 17 BRAF inhibitors promote hematopoiesis in mice 3 (acute hemolysis model 2)
  • Figure 17A 21 doses of GDC-0879 (50 mg/kg, P.O.) and SB-590885 (10 mg/kg, IP) were administered in the PHZ-induced hemolytic anemia model (Monday to Wednesday Q.D. and Thursday to Friday B.I.D. 2 week) diagram.
  • Figure 17B Measurement of red blood cell (RBC), HGB, HCT and red blood cell distribution width CV (RDW-CV) on days 0-4 and 7-11. 3 mice per group.
  • RBC red blood cell
  • HGB red blood cell
  • RDW-CV red blood cell distribution width CV
  • Example 18 BRAF inhibitors induce paradoxical activation of the MAPK pathway during erythroid differentiation
  • BRAF inhibitors are ATP-competitive inhibitors that competitively bind to the ATP-binding pocket in the BRAF mutant protein, and then it locks the R-Spine ( ⁇ C-helix) and DFG domain residue positions, thereby blocking the R-Spine And the formation of the DFG domain and the active conformation of the BRAF protein, thus preventing the activation of the mutant BRAF protein and the activation of downstream MAPKs.
  • BRAFV600E mutation In melanoma with BRAFV600E mutation, BRAFV600E mutation directly mediates downstream ERK activation after activating MEK, and finally leads to tumorigenesis.
  • BRAF inhibitors In normal BRAF (wild type), the addition of BRAF inhibitors will cause BRAF to be in an activated conformation similar to ATP binding. At this time, BRAF loses its kinase activity, but the conformation becomes activated, which makes it easy to align with the R-Spine of CRAF.
  • the formation of the R506 salt bridge greatly promotes the efficiency of BRAF and CRAF to form heterodimers.
  • the formation of BRAF-CRAF dimers is the key to RAS-RAF-MEK signal transduction in the MAPK pathway.
  • SCF Stem Cell Factor
  • EPO Erythropoietin
  • BRAF inhibitors SB-590885, GDC-0879 and Encorafenib could all activate pMEK and pERK to achieve paradoxical activation of the MAPKs pathway (Figure 18). It is worth noting that SB-590885 and GDC-0879 have stronger contradictory activation effects, not only in the stronger phosphorylation of pMEK and pERK, but also in the activation of pCRAF S338. Stronger MAPKs paradoxically activate small molecules. In summary, it can be basically concluded that BRAF inhibitors activate the MAPK/ERK pathway through paradox.
  • Figure 18 Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot.
  • Example 19 BRAF inhibitors paradoxically activate MAPK/ERK pathway and have concentration-dependent effects
  • Figure 19 Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot.
  • Example 20 Paradoxical activation of MAPK/ERK pathway by BRAF inhibitors depends on both upstream cytokine signaling and downstream signal transduction
  • hematopoietic stem cells first need to be activated from a quiescent state to a dividing state, similar to a transition from a long-term hematopoietic stem cell to a short-term hematopoietic stem cell, which is accompanied by cell division and strong activation of proliferation, a process that relies on extracellular signals to stimulate its entry into division.
  • the development of blood cells from hematopoietic stem cells to terminal lineages also requires the stimulation of various lineage-related cytokines.
  • One of the more common cytokines is stem cell factor (SCF).
  • SCF stem cell factor receptor
  • KIT stem cell factor receptor
  • both SCF and EPO activate the RAS/MAPK pathway to varying degrees to promote the proliferation, survival and development of erythroid progenitor cells. Therefore, the activated RAS brought about by cytokines is the result of normal hematopoiesis and erythroid development, and this result is the basis of paradoxical activation.
  • BRAF inhibitors are drugs that potentially promote cell proliferation, so it is necessary to consider whether it will bring about a broad spectrum of abnormal proliferation or potential carcinogenesis.
  • Pharmacological inhibition of BRAF downstream MEK protein phosphorylation is a common approach for tumor therapy.
  • this project needs to prove that BRAF inhibitors will not cause RAS-independent proliferation and will not have significant carcinogenic effects; on the other hand, this project also needs to prove whether the direct inhibition of BRAF downstream MEK/ERK can directly Block paradox activation.
  • Figure 20 Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot.
  • C From left to right, the drug was administered under the condition of containing SCF and EPO, containing only SCF, and containing only EPO.
  • Example 21 Paradoxical activation of MAPK/ERK pathway by BRAF inhibitors is mainly dependent on activation of CRAF
  • Figure 21 Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot.
  • A Protein expression of each knockdown group.
  • B Comparison of the doubling of the number of cells in the BRAF inhibitor administration group on Day 7 compared with the DMSO treatment group in the control group and the knockdown group. Three biological replicates were set for each condition, and the statistical data were mean ⁇ SD, ns: no significant difference, P>0.05; **: P ⁇ 0.01.
  • Example 22 BRAF inhibitors delay but only limitedly perturb transcriptional profiles of erythroid development
  • BRAF inhibitors are known to significantly delay the expression of surface differentiation marker proteins during erythroid differentiation; on the growth curve, after 6 days of exponential expansion of erythroid precursor cells, the cell number increased significantly slowing, which is characteristic of erythroid terminal differentiation, i.e., the balance of proliferation and differentiation is skewed toward differentiation during the final stages.
  • the administration of BRAF inhibitors did delay the time of slowing down the number of cell proliferation, so it can be confirmed that BRAF inhibitors lead to a significant delay in erythroid development in terms of cell surface markers and cell proliferation. After 72 hours of BRAF inhibitor treatment, it can also be determined that BRAF inhibitor can also delay the differentiation of erythrocytes at the transcriptional level.
  • Figure 22 Cells of erythroid precursor cells (CD71+, CD235-) were sorted, and transcriptional profiling was performed after 2 ⁇ M administration of GDC-0879 for 72 hours.
  • A Differences in the expression of specific genes between the treatment group (GDC) and the control group (DMSO).
  • B Volcano map of differential genes between the control group and the BRAF inhibitor-administered group (Foldchange>1.5; FDR ⁇ 0.1).
  • C GSEA enrichment analysis of downregulated genes (FDR ⁇ 0.1).
  • This example demonstrates that BRAF inhibitor treatment limitedly affected the transcriptional profile, delayed erythroid differentiation, and exhibited more early stem-progenitor characteristics. And it illustrates the synergistic effect of BRAF inhibitors and glucocorticoids from the perspective of transcriptional profiling.

Abstract

A method for amplifying erythroid progenitors or erythroblasts and ultimately producing mature denucleated red blood cells, and an application thereof, specifically comprising using a BRAF kinase inhibitor, and a combination thereof with a TGF-β inhibitor and/or an SMAD2/3 inhibitor and/or a glucocorticoid and/or erythropoietin and/or stem cell factor.

Description

一种对红系前体细胞或成红细胞进行扩增的方法及其应用A method for expanding erythroid precursor cells or erythroblasts and its application
本申请要求2021年9月30日提交的中国申请202111161019.0的优先权。中国申请202111161019.0的全部内容并入本文作为参考。This application claims priority from Chinese application 202111161019.0 filed on September 30, 2021. The entire contents of Chinese application 202111161019.0 are incorporated herein by reference.
发明领域field of invention
本发明提供了对各种细胞来源中的红系前体细胞或成红细胞进行扩增，促进红系发育过程，并最终产生成熟脱核的红细胞的方法及其应用。The invention provides a method for expanding erythroid precursor cells or erythroblasts from various cell sources, promoting erythroid development, and finally producing mature denucleated erythrocytes and its application.
背景技术Background technique
来源安全的血液可以在各种情况下挽救生命，尤其是在各类紧急情况和目前的全球性流行病的治疗中。对血液巨大的需求、血液的安全性和可持续性一直以来都是全球各国颇为关注的问题。但是目前血液的来源基本依靠自愿捐献，截至目前，全球每年总计献血1.185亿次，输血8500万单位。如果可以实现体内或实验室内对红细胞的有效扩增，则会很大程度上提供一个全新的血液来源，有效的缓解目前面临的血液来源短缺压力。Safely sourced blood saves lives in a variety of situations, especially in emergencies and the current global pandemic. The huge demand for blood, the safety and sustainability of blood have always been issues of great concern to countries all over the world. However, the current source of blood basically relies on voluntary donations. Up to now, the world has donated a total of 118.5 million times of blood and transfused 85 million units of blood every year. If the effective expansion of red blood cells in vivo or in the laboratory can be achieved, it will provide a new blood source to a large extent, and effectively alleviate the current shortage of blood sources.
因此，对红系前体细胞或成红细胞的有效扩增可以很容易地应用于广泛的临床和基础科学研究中。其中输血是需求最大的领域。例如，自体输血是一种缓解和恢复手术期间和手术后失血的潜在方法。对于那些具有稀有血型但没有现成血液供应的人来说，这是获得足够红细胞的重要途径。Therefore, the efficient expansion of erythroid precursor cells or erythroblasts can be easily applied in a wide range of clinical and basic scientific research. Blood transfusion is the area of greatest need. For example, autologous blood transfusion is a potential method to alleviate and restore blood loss during and after surgery. It's an important way to get enough red blood cells for those with rare blood types who don't have a readily available blood supply.
此外，通过在体内对红细胞的有效扩增可以治疗多种疾病。对于骨髓衰竭患者，例如患有先天性的纯红发育再障疾病及Diamond-Blackfan贫血患者(DBA)，患者会出现低效率的成红细胞和低红细胞数量(并最终贫血)，但目前这种疾病除了高剂量激素、输血、骨髓移植外，无有效的治疗方法。治疗骨髓衰竭和***(EPO)耐药性贫血需要一种在红细胞生成中比EPO更早发挥作用的药物，并能促进集落形成单位红系祖细胞(CFU-E)的形成。部分慢性肾病患者会因为EPO耐药性使得常规的贫血治疗方式难以起效。所以一旦发现降低EPO抵抗的治疗手段，它不仅可以改善患者的肾脏炎症，还可以缓解贫血症状，并有可能改善患者的预后和生存率。In addition, various diseases can be treated by the efficient expansion of red blood cells in vivo. In patients with bone marrow failure, such as those with congenital pure red developmental aplastic anemia and Diamond-Blackfan anemia (DBA), patients develop inefficient erythroblasts and low red blood cell counts (and eventually anemia), but the disease is currently Apart from high-dose hormones, blood transfusion, and bone marrow transplantation, there is no effective treatment. Treatment of bone marrow failure and erythropoietin (EPO)-resistant anemia requires a drug that acts earlier than EPO in erythropoiesis and promotes the formation of colony-forming unit erythroid progenitors (CFU-E). Some patients with chronic kidney disease will be unable to respond to conventional anemia treatments due to EPO resistance. So once a treatment that reduces EPO resistance is discovered, it could not only improve kidney inflammation in patients, but also alleviate symptoms of anemia and potentially improve patient outcomes and survival.
此外，红细胞可以作为体内长寿命的循环传递载体。因此，可以通过对红系前体细胞进行基因改造，以便在基因工程红细胞疗法中，它们可在其表面或内部携带特定的酶。这些工程扩增的红细胞可以或被设计为小分子化学药物、或蛋白质(包括各种酶)的载体，以达到各种临床治疗的医疗目的，例如临床上可将红细胞工程用于治疗先天性代谢酶缺乏症和其他需要长期治疗的疾病，以期实现长期及缓释的治疗效果。In addition, erythrocytes can serve as long-lived circulating delivery vehicles in vivo. Therefore, erythroid precursor cells can be genetically engineered so that they carry specific enzymes on their surface or inside them in genetically engineered red blood cell therapy. These engineered expanded erythrocytes can be or are designed as carriers of small molecule chemical drugs, or proteins (including various enzymes) to achieve various medical purposes of clinical treatment, for example, erythrocyte engineering can be used clinically to treat innate metabolic disorders Enzyme deficiency and other diseases that require long-term treatment in order to achieve long-term and sustained-release therapeutic effects.
相比基因编辑手段，通过药物手段例如使用小分子化合物更容易为临床接受，所以使用小分子化合物扩增红系前体细胞或成红细胞是一种可行且富有临床转化潜力的方法。目前对于能够实现扩增红系前体细胞或成红细胞的小分子存在需求。Compared with gene editing methods, pharmaceutical means such as the use of small molecule compounds are more likely to be clinically accepted, so the use of small molecule compounds to expand erythroid precursor cells or erythroblasts is a feasible and clinically transformative method. There is currently a need for small molecules that enable the expansion of erythroid precursor cells, or erythroblasts.
发明概述Summary of the invention
本发明人发现，BRAF激酶抑制剂能够在红系发育过程中通过MAPK通路的矛盾激活(即BRAF抑制剂在BRAF野生型且上游RAS蛋白激活的细胞中，通过结合在野生型BRAF ATP口袋中，使得BRAF保持激活构象，极大促进和稳定BRAF和CRAF的二聚化，从而使得CRAF保持激活构象，进而激活下游的MAPK/ERK通路，从而促进细胞增殖、分化和存活)，有效扩增红系前体细胞或成红细胞(erythroid progenitors,EPs或erythroblasts,EBs)，其能够在任何红系培养基中培养EPs，并且EPs或EBs在经处理后，形态、功能和红系表面标记物基本保持不变。给药BRAF抑制剂可以在病理状态 等细胞因子不足的情况下极大促进红系发育和增殖。而且，当与TGF-β抑制剂和/或SMAD2/3抑制剂和/或糖皮质激素和/或***和/或干细胞因子等现有的促进红系生成的药物结合使用时，BRAF激酶抑制剂能够产生更强的扩增效果。The present inventors have found that BRAF kinase inhibitors are able to act through paradoxical activation of the MAPK pathway during erythroid development (i.e. BRAF inhibitors in cells with BRAF wild-type and upstream RAS protein activation, by binding to the wild-type BRAF ATP pocket, Keep BRAF in the activated conformation, greatly promote and stabilize the dimerization of BRAF and CRAF, so that CRAF can maintain the activated conformation, and then activate the downstream MAPK/ERK pathway, thereby promoting cell proliferation, differentiation and survival), and effectively expand the erythroid Precursor cells or erythroid cells (erythroid progenitors, EPs or erythroblasts, EBs), which can culture EPs in any erythroid medium, and the morphology, function and erythroid surface markers of EPs or EBs remain basically unchanged after treatment Change. Administration of BRAF inhibitors can greatly promote erythroid development and proliferation in conditions of cytokine deficiency such as pathological states. Moreover, when combined with existing erythropoietic agents such as TGF-β inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or erythropoietin and/or stem cell factor, BRAF Kinase inhibitors produce stronger amplification.
因此，在一个方面中，本发明提供了一种用于治疗原发性或继发性贫血的药物，其包含BRAF激酶抑制剂和一种或多种药学上可接受的赋形剂。在优选方面中，所述药物还包含TGF-β抑制剂和/或SMAD2/3抑制剂和/或糖皮质激素和/或***和/或干细胞因子。Accordingly, in one aspect, the present invention provides a medicament for the treatment of primary or secondary anemia, comprising a BRAF kinase inhibitor and one or more pharmaceutically acceptable excipients. In a preferred aspect, the medicament further comprises TGF-β inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or erythropoietin and/or stem cell factor.
在另一个方面中，本发明提供了一种试剂盒，其包括：a)BRAF激酶抑制剂，和任选地，b)***。在优选方面中，所述试剂盒还包括：c)TGF-β抑制剂和/或SMAD2/3抑制剂和/或糖皮质激素和/或***和/或干细胞因子。In another aspect, the invention provides a kit comprising: a) a BRAF kinase inhibitor, and optionally b) erythropoietin. In a preferred aspect, the kit further includes: c) TGF-β inhibitor and/or SMAD2/3 inhibitor and/or glucocorticoid and/or erythropoietin and/or stem cell factor.
在另一个方面中，本发明提供了BRAF激酶抑制剂，或者BRAF激酶抑制剂和***和/或干细胞因子，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合在制备用于扩增红系前体细胞或成红细胞的药物中的用途。In another aspect, the invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF-β inhibitor, or a BRAF kinase inhibitor and SMAD2 /3 inhibitors, or BRAF kinase inhibitors, TGF-β inhibitors and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of a medicament for expanding erythroid precursor cells or erythroblasts .
在另一个方面中，本发明提供了BRAF激酶抑制剂，或者BRAF激酶抑制剂和***和/或干细胞因子，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合在制备用于治疗治疗原发性或继发性贫血的药物中的用途。In another aspect, the invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF-β inhibitor, or a BRAF kinase inhibitor and SMAD2 /3 inhibitors, or BRAF kinase inhibitors, TGF-β inhibitors and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of medicines for the treatment of primary or secondary anemia .
在另一个方面中，本发明提供了BRAF激酶抑制剂，或者BRAF激酶抑制剂和***和/或干细胞因子，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合在扩增红系前体细胞或成红细胞中的用途。In another aspect, the invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF-β inhibitor, or a BRAF kinase inhibitor and SMAD2 /3 inhibitors, or BRAF kinase inhibitors, TGF-β inhibitors and SMAD2/3 inhibitors, and their use in combination with glucocorticoids in expanding erythroid precursor cells or erythroblasts.
在另一个方面中，本发明提供了一种扩增红系前体细胞或成红细胞的方法，其包括向受试者给药BRAF激酶抑制剂，或者BRAF激酶抑制剂和***和/或干细胞因子，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合。In another aspect, the invention provides a method of expanding erythroid precursor cells or erythroblasts comprising administering to a subject a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or Or stem cell factor, or BRAF kinase inhibitor and TGF-β inhibitor, or BRAF kinase inhibitor and SMAD2/3 inhibitor, or BRAF kinase inhibitor, TGF-β inhibitor and SMAD2/3 inhibitor, and their combination with sugar A combination of corticosteroids.
具体地，本发明提供了一种扩增红系前体细胞或成红细胞的方法，其包括向受试者给药BRAF激酶抑制剂，或者BRAF激酶抑制剂和***，或者BRAF激酶抑制剂和干细胞因子，或者BRAF激酶抑制剂、***和干细胞因子的组合。Specifically, the invention provides a method of expanding erythroid precursor cells or erythroblasts comprising administering to a subject a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin, or a BRAF kinase inhibitor agent and stem cell factor, or a combination of BRAF kinase inhibitor, erythropoietin, and stem cell factor.
在另一个方面中，本发明提供了扩增的红系前体细胞或成红细胞的方法。在另一个方面中，还提供了扩增红系前体细胞(EPs)群体的方法。在另一个实例中，本发明所述EPs具有小、圆形和非黏附的统一形态。In another aspect, the invention provides methods of expanding erythroid precursor cells or erythroblasts. In another aspect, methods of expanding a population of erythroid precursor cells (EPs) are also provided. In another example, the EPs of the present invention have a small, round and non-adhesive uniform morphology.
通过本发明的方法，能够获得足够数量的EPs、基因改造的EPs，以及可搭载药物的EPs。Through the method of the present invention, a sufficient number of EPs, genetically modified EPs, and EPs that can be loaded with drugs can be obtained.
在另一个方面中，本发明还提供了用于预防或治疗相关疾病的方法，包括输入通过本方法扩增的足够数量的EPs，以及搭载药物以治疗疾病。本发明还提供了用于治疗或预防相关疾病的药物所需的足够数量的EPs或经基因改造EPs。In another aspect, the present invention also provides a method for preventing or treating related diseases, comprising importing a sufficient number of EPs amplified by the method, and carrying drugs to treat the diseases. The present invention also provides a sufficient number of EPs or genetically modified EPs required for medicines for treating or preventing related diseases.
此外，本发明可结合发明人先前申请的人源EPs的基因修饰的方法WO2014183071，可进一步分化为带转化酶或化学药物的成熟红细胞。这些工程化红细胞有望提高治疗效果，并可用于临床，治疗遗传性疾病，如苯丙酮尿症甚至癌症。人源EPs还可以通过工程技术在红细胞表面上产生所需的酶，用于开发癌症及其他疾病的新疗法。In addition, the present invention can be combined with the method WO2014183071 of the inventor's previous application for genetic modification of human EPs, which can be further differentiated into mature red blood cells with invertase or chemical drugs. These engineered red blood cells are expected to improve therapeutic efficacy and could be used clinically to treat genetic diseases such as phenylketonuria and even cancer. Human EPs can also be engineered to produce the required enzymes on the surface of red blood cells for the development of new treatments for cancer and other diseases.
发明详述Detailed description of the invention
红细胞和巨核细胞起源于骨髓中共同的祖细胞，即巨核细胞-红细胞祖细胞(megakaryocyte-erythroid progenitor,MEP)。虽然输血已经成为非常普遍的疗法，而且改良的红细胞也被用作体内天然的药物传递载体，但它们在临床上的应用受限。因为与脐带血或骨髓相比，目前无法从外周血单核细胞(peripheral blood mononuclear cell,PBMC)等更方便的来源获得足够数量的红系前体细胞，且缺少在体外对这些细胞或其祖细胞进行基因修饰并有效扩增红细胞的手段。同时，患有骨髓衰竭症，如DBA和血液再生障碍的患者需要一种有效的治疗方法来扩增红系前体细胞。此外，还有成千上万的患者面临着慢性肾脏疾病及其并发症***(EPO)抗性的威胁。EPO抗性或是慢性炎症性贫血的原因之一。目前尚无针对EPO抗性的现有治疗方法。Erythrocytes and megakaryocytes originate from a common progenitor in the bone marrow, the megakaryocyte-erythroid progenitor (MEP). Although blood transfusions have become a very common therapy, and modified red blood cells have been used as natural drug delivery vehicles in the body, their clinical application is limited. Because compared with umbilical cord blood or bone marrow, it is currently impossible to obtain sufficient numbers of erythroid precursor cells from more convenient sources such as peripheral blood mononuclear cells (PBMC), and there is a lack of in vitro detection of these cells or their progenitors. A means of genetically modifying cells and efficiently expanding red blood cells. Meanwhile, patients suffering from bone marrow failure disorders such as DBA and aplastic aplasia require an effective therapeutic approach to expand erythroid precursor cells. In addition, thousands of patients are at risk from chronic kidney disease and its complications, erythropoietin (EPO) resistance. EPO resistance may be one of the causes of chronic inflammatory anemia. There are currently no existing treatments for EPO resistance.
本发明提供的方法解决了上述问题。本发明方法至少具有以下优势：The method provided by the present invention solves the above-mentioned problems. The inventive method has at least the following advantages:
(1)人源EPs扩增量达10倍以上，为基础研究和临床应用提供了足够的红系细胞；(1) The amplification of human EPs is more than 10 times, which provides enough erythroid cells for basic research and clinical application;
(2)人源PBMC中的EPs大量扩增，可以通过对EP进行基因改造调控红细胞发育及表达；(2) EPs in human PBMCs are amplified in large quantities, and the development and expression of red blood cells can be regulated by genetically modifying EPs;
(3)体外扩增的人源EPs可用于临床，包括但不限于各种情况下的输血。例如，体外扩增的人源EPs可用于治疗贫血性疾病或失血过多；(3) Human EPs amplified in vitro can be used clinically, including but not limited to blood transfusion in various situations. For example, human EPs expanded in vitro can be used to treat anemia or excessive blood loss;
(4)体内外给药扩增骨髓衰竭患者红系前体细胞，如DBA和AA患者的EPs，帮助缓解贫血症状；(4) In vivo and in vitro administration to expand erythroid precursor cells in patients with bone marrow failure, such as EPs in patients with DBA and AA, to help relieve symptoms of anemia;
(5)在EPO浓度相对低(或EPO耐药)或其他红系生长必要的细胞因子缺乏的情况下，可促进红系前体细胞或成红细胞分化、成熟和脱核，缓解EPO抗性或细胞因子缺乏引起的贫血症状。(5) In the case of relatively low EPO concentration (or EPO resistance) or the lack of other cytokines necessary for erythroid growth, it can promote the differentiation, maturation and denucleation of erythroid precursor cells or erythroblasts, and relieve EPO resistance or Symptoms of anemia caused by cytokine deficiency.
附图说明Description of drawings
图1A：小分子扩增红系前体细胞的高通量筛选(HTS)的示意图。Figure 1A: Schematic representation of high-throughput screening (HTS) of small molecule expansion of erythroid precursor cells.
图1B：高通量筛选的结果。Figure 1B: Results of high-throughput screening.
图2A：探究GDC-0879处理的最佳浓度。Figure 2A: Exploring the optimal concentration of GDC-0879 treatment.
图2B：多种BRAF抑制剂在宽浓度范围对红系前体细胞的扩增效果(仅给药一次)。Figure 2B: Expansion effect of various BRAF inhibitors on erythroid precursor cells over a wide concentration range (one administration only).
图2C：选定的BRAF抑制剂在宽浓度范围内对红系前体细胞的扩增效果(三次给药)。Figure 2C: Expansion effect of selected BRAF inhibitors on erythroid precursor cells over a broad concentration range (three doses).
图3A：三种小分子在最佳浓度下于红系分化体系中的生长曲线。Figure 3A: Growth curves of three small molecules at optimal concentrations in erythroid differentiation system.
图3B：小分子处理后成红细胞的生长曲线。Figure 3B: Growth curves of erythroblasts after small molecule treatment.
图4A-4E：GDC-0879在分化培养基中的FACS结果。Figures 4A-4E: FACS results of GDC-0879 in differentiation medium.
图5A-5D：集落形成结果。其中，图5A为在Methocult H4435不同给药组和对照组的红细胞集落数量(中)与单个红细胞集落面积(右)的统计，图5B显示了第14天红细胞特异性集落形成结果，图5C显示了在亮视野显微镜下使用5×物镜检测第14天红细胞特异性集落形成结果，图5D为BRAF抑制剂促进脐带血来源CD34+细胞的红系生成和各谱系发育(髓系培养基)。Figures 5A-5D: Colony formation results. Among them, Figure 5A is the statistics of the number of erythrocyte colonies (middle) and the area of a single erythrocyte colony (right) in different Methocult H4435 administration groups and the control group, and Figure 5B shows the results of erythrocyte-specific colony formation on the 14th day, and Figure 5C shows The results of erythrocyte-specific colony formation on day 14 were detected under a bright-field microscope using a 5× objective lens. Figure 5D shows that BRAF inhibitors promote erythropoiesis and lineage development of cord blood-derived CD34+ cells (myeloid medium).
图6A-6E：BRAF靶点的小分子促进红系前体细胞增殖的效果。其中，图6A显示了检测靶向BRAF的其他同靶点小分子的结果(未特别标注的小分子浓度均为1μM，处理9天)，图6B显示BRAF的其他抑制剂在仅含EPO的甲基纤维素培养基中第14天的红细胞特异性集落形成结果，图6C显示在仅含EPO的甲基纤维素培养基中，含BRAF抑制剂的其他组合在第14天的红细胞特异性集落形成结果，图6D显示了光镜视野显微镜下集落形成结果，图6E显示了图6B、6C各孔红细胞集落形成单位计数结果。Figures 6A-6E: Effects of small molecules targeting BRAF on promoting proliferation of erythroid precursor cells. Among them, Figure 6A shows the results of detection of other small molecules targeting BRAF with the same target (the concentration of small molecules not specifically marked is 1 μM, and the treatment is 9 days), and Figure 6B shows that other inhibitors of BRAF are effective in formazan containing only EPO. The results of erythrocyte-specific colony formation on day 14 in cellulose-based medium, Figure 6C shows the erythrocyte-specific colony formation on day 14 of other combinations containing BRAF inhibitors in methylcellulose medium containing EPO only As a result, Fig. 6D shows the colony formation results under the light field microscope, and Fig. 6E shows the colony forming unit counting results of each well of Fig. 6B and 6C.
图7A-7E：有效的给药周期试验。其中，图7A显示了GDC-0879从第0天到第12天不同周期给药，并在第十五天进行最后细胞数量统计的试验结果，图7B显示了0-3天处理组与对照组第13天细胞数比较，图7C显示了实验组与对照组在分化培养基中第13天FACS结果，图7D显示第13～16天对各给药3天的各组进行脱核率统计(处理组，1μM GDC-0879处理组)，图7E显示第13～16天对两个给药期(连续6天)的各组进行脱核率统计(处理组：1μM GDC-0879处理)。Figures 7A-7E: Effective dosing cycle assay. Among them, Figure 7A shows the test results of GDC-0879 administered in different cycles from the 0th day to the 12th day, and the final cell number statistics were performed on the 15th day, and Figure 7B shows the 0-3 day treatment group and the control group Comparison of the number of cells on the 13th day, Figure 7C shows the FACS results of the experimental group and the control group on the 13th day in the differentiation medium, and Figure 7D shows that the statistics of the enucleation rate of each group administered for 3 days on the 13th to 16th day ( Treatment group, 1 μM GDC-0879 treatment group), Figure 7E shows statistics on the enucleation rate of each group in the two administration periods (6 consecutive days) from day 13 to 16 (treatment group: 1 μM GDC-0879 treatment).
图8A-8K：体外扩增PBMC中的成红细胞。其中，图8A显示第9天处理组和对照组的细胞，图8B显示体外扩增健康捐献者(HD1-HD5)的PBMC中的成红细胞第9天的数量变化，图8C显示了健康捐献者(HD1-HD5)的PBMC在无血清红细胞扩增培养基中培养第9天的FACS结果，图8D显示了健康捐献者(HD1-HD5)PBMC的FACS结果在第10天细胞类群比率的统计，图8E显示了FACS第10天细胞类群比率的统计，图8F显示健康捐献者(Donor1-Donor5)的PBMC在无血清红细胞扩增培养基中培养的FACS结果，图8G显示健康捐献者(Donor1-Donor5)的PBMC在无血清红细胞扩增培养基中培养的FACS结果，图8H为一个整体的扩增能力统计，图8I为在Methocult H4435上对PBMC给药，SB和对照组的整体克隆形成图像和代表性红系单克隆集落图，图8J为在Methocult H4435不同给药组和对照组的红细胞集落在显微镜下的代表性集落。图8K为在Methocult H4435不同给药组和对照组的红细胞集落数量(左)与不同组的单个红细胞集落面积(右)的统计。Figures 8A-8K: In vitro expansion of erythroblasts in PBMCs. Wherein, Figure 8A shows the cells of the treatment group and the control group on the 9th day, Figure 8B shows the change in the number of erythroblasts in the PBMC of healthy donors (HD1-HD5) in vitro, and Figure 8C shows the changes in the number of erythroblasts in the healthy donors (HD1-HD5) on the 9th day (HD1-HD5) PBMCs were cultured in serum-free erythrocyte expansion medium on the 9th day of FACS results, and Figure 8D shows the statistics of the FACS results of healthy donors (HD1-HD5) PBMCs on the 10th day cell group ratio, Figure 8E shows the statistics of the ratio of cell groups on the 10th day of FACS, Figure 8F shows the FACS results of PBMCs from healthy donors (Donor1-Donor5) cultured in serum-free erythrocyte expansion medium, and Figure 8G shows the results of healthy donors (Donor1-Donor5) FACS results of PBMC of Donor5) cultured in serum-free erythrocyte expansion medium, Figure 8H is an overall expansion ability statistics, Figure 8I is the overall clone formation image of PBMC administered on Methocult H4435, SB and the control group And representative erythroid monoclonal colony diagram, Figure 8J is a representative colony of erythrocyte colonies in different Methocult H4435 administration groups and control groups under the microscope. Figure 8K is the statistics of the number of erythrocyte colonies (left) and the area of individual erythrocyte colonies in different groups (right) in different Methocult H4435 administration groups and control groups.
图9A-9L：低EPO条件下药物的治疗效果。其中，图9A显示GDC-0879在不同浓度***(EPO)分化培养基中的作用，图9B显示无EPO条件下培养5-10天后第14天FACS结果，图9C显示1/3浓度EPO条件下培养1-15天后第15天FACS结果，图9D显示统计13～19天各EPO浓度条件下的红细胞脱核率，图9E为用0％SCF或5％EPO的低细胞因子浓度在红系培养基中用2μM GDC-0879给药处理的UCB CD34+细胞的生长曲线，图9F为对照组(上)和GDC-0879处理组(下)中0％SCF(左)和5％EPO(右)的低浓度细胞因子在第9天分化(UCB CD34+细胞)的流式细胞术细胞表面标志物代表图像，图9G和9H为GDC处理组和对照组在第6天在不同红细胞生成条件培养基中的成红细胞的典型红细胞表面标志物表达情况(CD117、CD71和CD235)，图9I和9J为GDC处理组和对照组在第14天在不同红细胞生成条件培养基中的红细胞表面标志物(CD117、CD71和CD235)的表达情况，图9K为在不同应激条件下培养的样品的典型红细胞照片，图9L：不同压力培养条件下的代表性吉姆萨-联苯胺染色的红细胞图像。Figures 9A-9L: Therapeutic effects of drugs under low EPO conditions. Among them, Figure 9A shows the effect of GDC-0879 in different concentrations of erythropoietin (EPO) differentiation medium, Figure 9B shows the FACS results on day 14 after 5-10 days of culture without EPO, and Figure 9C shows the 1/3 concentration FACS results on the 15th day after cultured under EPO conditions for 1-15 days, Figure 9D shows the erythrocyte denucleation rate under the conditions of various EPO concentrations from 13 to 19 days, and Figure 9E shows the concentration of erythrocytes with 0% SCF or 5% EPO at low cytokine concentrations Growth curves of UCB CD34+ cells treated with 2 μM GDC-0879 in erythroid medium, and Figure 9F shows 0% SCF (left) and 5% EPO ( Right) Representative images of flow cytometry cell surface markers differentiated (UCB CD34+ cells) with low concentration of cytokines on day 9, Figure 9G and 9H are GDC treatment group and control group cultured under different erythropoietic conditions on day 6 The expression of typical erythrocyte surface markers (CD117, CD71 and CD235) of erythroblasts in the culture medium, Fig. 9I and 9J are the erythrocyte surface markers ( The expression of CD117, CD71 and CD235), Figure 9K is a typical erythrocyte photo of samples cultured under different stress conditions, Figure 9L: representative Giemsa-benzidine stained erythrocyte images under different pressure culture conditions.
图10A-10F：造血干细胞移植后纯红细胞再生缺陷导致的贫血体外治疗效果。其中，图10A显示GDC-0879在造血干细胞移植后的红细胞发育失败或纯红细胞发育不全中可能促进红细胞祖细胞的发育和自我更新，图10B为造血干细胞移植后红细胞再生缺陷(EFAHSCT)患者骨髓单个核细胞(BMMNCs)的生长曲线，图10C为EFAHSCT的BMMNC在第6天(左)和第16天(右)的红细胞发育的SB处理(下)和对照组(上)的红细胞表面标志物(CD71、CD235和Hoechst 33342)，图10D为从EFAHSCT的BMMNCs进行红系培养后获得的代表性吉姆萨-联苯胺染色红细胞，图10E为50000个EFAHSCT的BMMNCs在H4435髓系甲基纤维素培养基中的代表性图像，图10F为50000个EFAHSCT的BMMNCs在H4435髓系甲基纤维素培养基中培养出的不同谱系的集落数量。Figures 10A-10F: Effects of in vitro treatment of anemia caused by defective pure red blood cell regeneration after hematopoietic stem cell transplantation. Among them, Figure 10A shows that GDC-0879 may promote the development and self-renewal of erythroid progenitor cells in erythrocyte development failure or pure erythrocyte aplasia after hematopoietic stem cell transplantation, and Figure 10B shows the bone marrow single Growth curves of nucleated cells (BMMNCs), Figure 10C shows the erythrocyte surface markers ( CD71, CD235 and Hoechst 33342), Figure 10D is the representative Giemsa-benzidine stained erythrocytes obtained from EFAHSCT BMMNCs after erythroid culture, Figure 10E is 50000 EFAHSCT BMMNCs in H4435 myeloid methylcellulose medium Representative images in Figure 10F show the number of colonies of different lineages from 50,000 EFAHSCT-derived BMMNCs cultured in H4435 myeloid methylcellulose medium.
图11：SB-590885有效扩增DBA贫血患者外周单核细胞中的红系前体细胞。Figure 11: SB-590885 potently expands erythroid precursors in peripheral mononuclear cells from DBA anemia patients.
图12：SB-590885有效扩增DBA贫血患者外周单核细胞中的红系前体细胞的数据统计。Figure 12: Statistics of SB-590885 effectively expanding erythroid precursor cells in peripheral mononuclear cells of DBA anemia patients.
图13A-13E：BRAF抑制剂有效促进MDS患者细胞的红系分化和红系增殖。Figures 13A-13E: BRAF inhibitors effectively promote erythroid differentiation and erythroid proliferation in MDS patient cells.
图14：BRAF抑制剂对造血干祖细胞(HSPC)的扩增作用。Figure 14: Expansion of hematopoietic stem progenitor cells (HSPC) by BRAF inhibitors.
图15：BRAF抑制剂促进小鼠体内造血(正常情况)。Figure 15: BRAF inhibitors promote hematopoiesis in mice (normal conditions).
图16A-16E：BRAF抑制剂促进小鼠体内造血(急性溶血模型)。Figures 16A-16E: BRAF inhibitors promote hematopoiesis in mice (acute hemolysis model).
图17A-17B：BRAF抑制剂促进小鼠体内造血(急性溶血模型)。Figures 17A-17B: BRAF inhibitors promote hematopoiesis in mice (acute hemolysis model).
图18：BRAF抑制剂在红系分化过程中诱导MAPK通路的矛盾激活。Figure 18: BRAF inhibitors induce paradoxical activation of the MAPK pathway during erythroid differentiation.
图19：BRAF抑制剂矛盾激活MAPK/ERK通路存在浓度依赖效应。Figure 19: BRAF inhibitors paradoxically activate the MAPK/ERK pathway with a concentration-dependent effect.
图20：BRAF抑制剂矛盾激活MAPK/ERK通路同时依赖于上游细胞因子信号和下游信号转导。Figure 20: Paradoxical activation of MAPK/ERK pathway by BRAF inhibitors is dependent on both upstream cytokine signaling and downstream signaling.
图21：BRAF抑制剂矛盾激活MAPK/ERK通路主要依赖于CRAF的激活。Figure 21: BRAF inhibitors paradoxically activate the MAPK/ERK pathway primarily dependent on CRAF activation.
图22：BRAF抑制剂使红系发育在转录谱上呈现延迟但仅有限扰动转录谱。Figure 22: BRAF inhibitors delay but only limitedly perturb transcriptional profiles of erythroid development.
定义definition
如本文所用，术语“烷基”是指1至18个碳原子的饱和直链或支链烃基，其中所述烷基可任选被一个或多个以下所述取代基独立取代。所述烷基可以是一价，其实例包括C 1-C 12烷基、C 1-C 8烷基、C 1-C 6烷基，例如甲基(Me、-CH 3)、乙基(Et、-CH 2CH 3)、1-丙基(n-Pr、n-丙基、-CH 2CH 2CH 3)、2-丙基(i-Pr、i-丙基、-CH(CH 3) 2)、1-丁基(n-Bu、n-丁基、-CH 2CH 2CH 2CH 3)、2-甲基-1-丙基(i-Bu、i-丁基、-CH 2CH(CH 3) 2)、2-丁基(s-Bu、s-丁基、-CH(CH 3)CH 2CH 3)、2-甲基-2-丙基(t-Bu、t-丁基、-C(CH 3) 3)、1-戊基(n-戊基、-CH 2CH 2CH 2CH 2CH 3)、2-戊基(-CH(CH 3)CH 2CH 2CH 3)、3-戊基(-CH(CH 2CH 3) 2)、2-甲基-2-丁基(-C(CH 3) 2CH 2CH 3)、3-甲基-2-丁基(-CH(CH 3)CH(CH 3) 2)、3-甲基-1-丁基(-CH 2CH 2CH(CH 3) 2)、2-甲基-1-丁基(-CH 2CH(CH 3)CH 2CH 3)、1-己基(-CH 2CH 2CH 2CH 2CH 2CH 3)、2-己基(-CH(CH 3)CH 2CH 2CH 2CH 3)、3-己基(-CH(CH 2CH 3)(CH 2CH 2CH 3))、2-甲基-2-戊基(-C(CH 3) 2CH 2CH 2CH 3)、3-甲基-2-戊基(-CH(CH 3)CH(CH 3)CH 2CH 3)、4-甲基-2-戊基(-CH(CH 3)CH 2CH(CH 3) 2)、3-甲基-3-戊基(-C(CH 3)(CH 2CH 3) 2)、2-甲基-3-戊基(-CH(CH 2CH 3)CH(CH 3) 2)、2,3-二甲基-2-丁基(-C(CH 3) 2CH(CH 3) 2)、3,3-二甲基-2-丁基(-CH(CH 3)C(CH 3) 3)、1-庚基、1-辛基、环丙基、环丁基、环戊基、环己基、环庚基和环辛基。 As used herein, the term "alkyl" refers to a saturated linear or branched chain hydrocarbon group of 1 to 18 carbon atoms, wherein said alkyl group may be optionally substituted independently by one or more substituents described below. The alkyl group may be monovalent, and examples thereof include C 1 -C 12 alkyl, C 1 -C 8 alkyl, C 1 -C 6 alkyl, such as methyl (Me, -CH 3 ), ethyl ( Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, - CH 2 CH(CH 3 ) 2 ), 2-butyl (s-Bu, s-butyl, -CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-methyl-2-butyl (-C(CH 3 ) 2 CH 2 CH 3 ), 3-methyl- 2-butyl (-CH(CH 3 )CH(CH 3 ) 2 ), 3-methyl-1-butyl (-CH 2 CH 2 CH(CH 3 ) 2 ), 2-methyl-1-butyl group (-CH 2 CH(CH 3 )CH 2 CH 3 ), 1-hexyl group (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-hexyl group (-CH(CH 3 ) CH 2 CH 2 CH 2 CH 3 ), 3-hexyl (-CH(CH 2 CH 3 )(CH 2 CH 2 CH 3 )), 2-methyl-2-pentyl (-C(CH 3 ) 2 CH 2 CH 2 CH 3 ), 3-methyl-2-pentyl (-CH(CH 3 )CH(CH 3 )CH 2 CH 3 ), 4-methyl-2-pentyl (-CH(CH 3 )CH 2 CH(CH 3 ) 2 ), 3-methyl-3-pentyl (-C(CH 3 )(CH 2 CH 3 ) 2 ), 2-methyl-3-pentyl (-CH(CH 2 CH 3 )CH( CH 3 ) 2 ), 2,3-Dimethyl-2-butyl (-C(CH 3 ) 2 CH(CH 3 ) 2 ), 3,3-Dimethyl-2-butyl (-CH( CH 3 )C(CH 3 ) 3 ), 1-heptyl, 1-octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
所述烷基可以具有2个一价基团中心，该一价基团中心是由母烷烃的相同或两个不同碳原子上除去两个氢原子所产生的。实例包括但不限于，亚甲基(-CH 2-)、1,2-乙基(-CH 2CH 2-)、1,3-丙基(-CH 2CH 2CH 2-)、1,4-丁基(-CH 2CH 2CH 2CH 2-)等。 The alkyl group may have 2 monovalent radical centers resulting from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Examples include, but are not limited to , methylene ( -CH2- ), 1,2-ethyl (-CH2CH2-) , 1,3-propyl ( -CH2CH2CH2- ) , 1, 4-Butyl (-CH 2 CH 2 CH 2 CH 2 -) etc.
术语“烯基”指2至18个碳原子的直链或支链烃基，具有至少一个不饱和位置，即碳-碳sp 2双键，其中所述烯基可任选被一个或多个本文所述取代基独立取代，包括具有顺式和反式取向，或“E”和“Z”取向的基团。所述烯基可以是一价，其实例包括C 2-C 12烯基、C 2-C 8烯基、C 2-C 6烯基，包括但不限于，乙烯基或烯基(-CH＝CH 2)、烯丙基(-CH 2CH＝CH 2)、1-环戊-1-烯基、1-环戊-2-烯基、1-环戊-3-烯基、5-己烯基、1-环己-1-烯基、1-环己-2-烯基和1-环己-3-烯基等。 The term "alkenyl" refers to a straight or branched chain hydrocarbon radical of 2 to 18 carbon atoms, having at least one site of unsaturation, i.e. a carbon-carbon sp double bond, wherein said alkenyl radical may optionally be replaced by one or more The substituents are independently substituted and include groups with cis and trans orientations, or "E" and "Z" orientations. The alkenyl group may be monovalent, examples of which include C 2 -C 12 alkenyl, C 2 -C 8 alkenyl, C 2 -C 6 alkenyl, including but not limited to, vinyl or alkenyl (-CH= CH 2 ), allyl (-CH 2 CH=CH 2 ), 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 5-hex Alkenyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl and the like.
所述烯基可以具有2个一价基团中心，该一价基团中心是由母烯烃的相同或两个不同碳原子上除去两个氢原子所产生的。实例包括但不限于，1,2-乙烯基(-CH＝CH-)等。The alkenyl group may have two monovalent radical centers resulting from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkene. Examples include, but are not limited to, 1,2-vinyl (-CH=CH-) and the like.
所述烷基和烯基基团，单独或作为大基团如烷氧基的部分(即-OR基团，其中R为所述烷基和烯基)或作为大基团如烷硫基的部分(即-SR基团，其中R为所述烷基和烯基)。The alkyl and alkenyl groups, alone or as part of a larger group such as an alkoxy group (i.e. -OR group where R is the alkyl and alkenyl group) or as part of a larger group such as an alkylthio group moieties (ie -SR groups, where R is the alkyl and alkenyl groups described).
术语“炔基”指2至18个碳原子的直链或支链烃基，具有至少一个不饱和位置，即碳-碳sp三键，其中所述炔基可任选被一个或多个本文所述取代基独立取代。所述炔基可以是一价，其实例包括C 2-C 12炔基、C 2-C 8炔基、C 2-C 6炔基，包括但不限于，乙炔基(-C≡CH)和丙炔基(炔丙基、-CH 2C≡CH)等。所述炔基可以具有2个一价基团中心，该一价基团中心是由母炔烃的相同或两个不同碳原子上除去两个氢原子所产生的。实例包括但不限于，亚乙炔基(-C≡C-)、亚炔丙基(-CH 2C≡C-)和4-亚戊炔基(-CH 2CH 2CH 2C≡C-)等。 The term "alkynyl" refers to a straight or branched chain hydrocarbon group of 2 to 18 carbon atoms, having at least one site of unsaturation, i.e. a carbon-carbon sp triple bond, wherein said alkynyl group may optionally be replaced by one or more The above substituents are independently substituted. The alkynyl group may be monovalent, examples of which include C 2 -C 12 alkynyl, C 2 -C 8 alkynyl, C 2 -C 6 alkynyl, including but not limited to, ethynyl (-C≡CH) and propynyl (propargyl, -CH 2 C≡CH) and the like. The alkynyl group may have two monovalent radical centers resulting from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. Examples include, but are not limited to, ethynylene (-C≡C-), propargylene ( -CH2C≡C- ) , and 4-pentynylene ( -CH2CH2CH2C≡C- ) wait.
术语“芳基”是指具有6至20个碳原子的一价芳族烃基，是由母芳环***的一个碳原子上除去一个氢原子后产生的。芳基的实例包括C 6-14芳基、C 6-10芳基，示例性的结构中的一些芳基由“Ar”表示。芳基包括含有芳环的双环，所述芳环与非芳环或部分饱和环稠合。典型的芳基包括但不限于衍生自以下的基团：苯、取代的苯、萘、蒽、联苯基、茚基、茚满基、1,2-二氢萘、1,2,3,4-四氢萘基等。芳基任选被一个或多个本文所述取代基独立取代。 The term "aryl" refers to a monovalent aromatic hydrocarbon radical having 6 to 20 carbon atoms, resulting from the removal of a hydrogen atom from a carbon atom of a parent aromatic ring system. Examples of aryl groups include C 6-14 aryl, C 6-10 aryl, and some aryl groups in the exemplary structures are represented by "Ar". Aryl includes bicyclic rings containing an aromatic ring fused to a non-aromatic or partially saturated ring. Typical aryl groups include, but are not limited to, groups derived from benzene, substituted benzenes, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3, 4-tetrahydronaphthyl, etc. Aryl groups are optionally substituted independently with one or more substituents described herein.
术语“杂环”、“杂环基”、“杂芳”和“杂芳基”指具有3至25个环原子(含2至20个碳原子和1至5个选自N、O、P和S的杂原子)的饱和、部分饱和(即环内具有一个或多个双键和/或三键)或芳族碳环 基团，其中至少一个环原子是独立选自氮、氧和硫的杂原子，剩下的环原子为碳，其中一个或多个环原子任选被一个或多个本文所述取代基独立取代。杂环可为具有3至7个环原子(2至6个碳原子和1至3个选自N、O、P和S的杂原子)的单环或具有7至10个环原子(4至9个碳原子和1至3个选自N、O、P和S的杂原子)的双环，例如：双环[4,5]、[5,5]、[5,6]或[6,6]***。杂环如以下文献所述：Paquette,Leo A.；“Principles of Modern Heterocyclic Chemistry”(W.A.Benjamin,New York,1968)，特别是第1、3、4、6、7和9卷；“The Chemistry of Heterocyclic Compounds,A series of Monographs”(John Wiley&Sons,New York,1950至今)，特别是第13、14、16、19和28卷；和J.Am.Chem.Soc.(1960)82:5566。杂环基可为以碳连接的基团或以杂原子连接的基团。The terms "heterocycle", "heterocyclyl", "heteroaryl" and "heteroaryl" refer to ring atoms having 3 to 25 ring atoms (containing 2 to 20 carbon atoms and 1 to 5 ring atoms selected from N, O, P and heteroatoms of S), saturated, partially saturated (i.e., having one or more double and/or triple bonds within the ring) or aromatic carbocyclic groups in which at least one ring atom is independently selected from nitrogen, oxygen and sulfur and the remaining ring atoms are carbon, wherein one or more ring atoms are optionally independently substituted with one or more substituents described herein. The heterocycle can be a monocyclic ring having 3 to 7 ring atoms (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S) or a ring having 7 to 10 ring atoms (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example: bicyclic [4,5], [5,5], [5,6] or [6,6 ]system. Heterocycles are described in: Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968), especially volumes 1, 3, 4, 6, 7 and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950-present), especially volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. A heterocyclyl group can be a carbon-bonded group or a heteroatom-bonded group.
术语“杂环”包括杂环烷氧基。“杂环基”也包括杂环基与碳环、杂环、芳环或杂芳环稠合的基团。杂环基的实例包括但不限于，吡咯烷基、四氢呋喃基、二氢呋喃基、四氢噻吩基、四氢吡喃基、二氢吡喃基、四氢硫代吡喃基、哌啶子基、吗啉代、硫代吗啉代、噻噁烷基、哌嗪基、高哌嗪基、氮杂环丁烷基、氧杂环丁烷基、硫杂环丁烷基、高哌啶基、氧杂环庚烷基、硫杂环庚烷基(thiepanyl)、氧氮杂基、二氮杂基、硫氮杂基、2-吡咯啉基、3-吡咯啉基、吲哚啉基、2H-吡喃基、4H-吡喃基、二噁烷基、1,3-二氧戊环基、吡唑啉基、二噻烷基、二硫杂环戊烷基、二氢吡喃基、二氢噻吩基、二氢呋喃基、吡唑烷基咪唑啉基、咪唑烷基、3-氮杂双环[3.1.0]己基、3-氮杂双环[4.1.0]庚基、氮杂双环[2.2.2]己基、3H-吲哚基喹嗪基和N-吡啶基脲。螺环基团也包括在本发明范围内。其中2个环碳原子被氧代基(＝O)取代的杂环基团的实例为嘧啶酮基和1,1-二氧代-硫代吗啉基。本文的杂环基团任选被一个或多个本文所述取代基独立取代。The term "heterocycle" includes heterocycloalkoxy. "Heterocyclyl" also includes radicals in which heterocyclyl is fused to a carbocyclic, heterocyclic, aromatic or heteroaromatic ring. Examples of heterocyclic groups include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidine Base, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl , 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl Base, dihydrothienyl, dihydrofuryl, pyrazolidinyl imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, nitrogen Heterobicyclo[2.2.2]hexyl, 3H-indolylquinazinyl and N-pyridylurea. Spiro groups are also included within the scope of this invention. Examples of heterocyclic groups in which 2 ring carbon atoms are substituted by oxo (=O) are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocyclic groups herein are optionally substituted independently with one or more substituents described herein.
术语“杂芳基”包括1)含有一个或多个独立选自氮、氧和硫的杂原子的单环芳族5-、6-和7-元环；和2)8至20个原子的稠合环***，其中至少一个芳族环含有一个或多个独立选自氮、氧和硫的杂原子。杂芳基的实例为吡啶基(包括例如2-羟基吡啶基)、咪唑基、咪唑并吡啶基、嘧啶基(包括例如4-羟基嘧啶基)、吡唑基、噻唑基、吡嗪基、四唑基、呋喃基、噻吩基、异噁唑基、噻唑基、噁唑基、异噻唑基、吡咯基、喹啉基、异喹啉基、吲哚基、苯并咪唑基、苯并呋喃基、噌啉基、吲唑基、吲嗪基、酞嗪基、哒嗪基、三嗪基、异吲哚基、蝶啶基、嘌呤基、噁二唑基、***基、噻二唑基、噻二唑基、呋咱基、苯并呋咱基、苯并噻吩基、苯并噻唑基、苯并噁唑基、喹唑啉基、喹喔啉基、萘啶基和呋喃并吡啶基。杂芳基团任选被一个或多个本文所述取代基独立取代。The term "heteroaryl" includes 1) monocyclic aromatic 5-, 6- and 7-membered rings containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur; and 2) 8 to 20 atom Fused ring systems wherein at least one aromatic ring contains one or more heteroatoms independently selected from nitrogen, oxygen and sulfur. Examples of heteroaryl groups are pyridyl (including, for example, 2-hydroxypyridyl), imidazolyl, imidazopyridyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, thiazolyl, pyrazinyl, tetra Azolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolyl, indolyl, benzimidazolyl, benzofuryl , cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl , thiadiazolyl, furazanyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridyl and furopyridyl . Heteroaryl groups are optionally substituted independently with one or more substituents described herein.
杂环可以是碳连接的或氮连接的(如果可以)。例如但不限于：碳键合的杂环的结合位置在吡啶的2、3、4、5或6位；哒嗪的3、4、5或6位；嘧啶的2、4、5或6位；吡嗪的2、3、5或6位；呋喃、四氢呋喃、硫代呋喃、噻吩、吡咯或四氢吡咯的2、3、4或5位；噁唑、咪唑或噻唑的2、4或5位；异噁唑、吡唑或异噻唑的3、4或5位；氮杂环丙烷的2或3位；氮杂环丁烷的2、3或4位；喹啉的2、3、4、5、6、7或8位；或者异喹啉的1、3、4、5、6、7或8位。(2-吡啶基、3-吡啶基、4-吡啶基、5-吡啶基、6-吡啶基)。例如但不限于：氮键合的杂环的结合位置在氮杂环丙烷、氮杂环丁烷、吡咯、吡咯烷、2-吡咯啉、3-吡咯啉、咪唑、咪唑烷、2-咪唑啉、3-咪唑啉、吡唑、吡唑啉、2-吡唑啉、3-吡唑啉、哌啶、哌嗪、吲哚、吲哚啉、1H-吲唑的1位；异吲哚或异吲哚啉的2位；吗啉的4位；和咔唑或β-咔啉的9位。Heterocycles can be carbon-attached or nitrogen-attached (if applicable). For example, but not limited to: carbon-bonded heterocyclic ring binding position at 2, 3, 4, 5 or 6 positions of pyridine; 3, 4, 5 or 6 positions of pyridazine; 2, 4, 5 or 6 positions of pyrimidine ; 2, 3, 5 or 6 positions of pyrazine; 2, 3, 4 or 5 positions of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole; 2, 4 or 5 of oxazole, imidazole or thiazole position; 3, 4 or 5 positions of isoxazole, pyrazole or isothiazole; 2 or 3 positions of aziridine; 2, 3 or 4 positions of azetidine; 2, 3, 4 positions of quinoline , 5, 6, 7 or 8 positions; or 1, 3, 4, 5, 6, 7 or 8 positions of isoquinoline. (2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl). For example, but not limited to: nitrogen-bonded heterocyclic rings are bound at aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline , 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1-position of 1H-indazole; isoindole or 2-position for isoindoline; 4-position for morpholine; and 9-position for carbazole or β-carboline.
术语“碳环”、“碳环基”、“环烷基”和“环烯基”是指具有3至12个碳原子的单环的非芳族、饱和或不饱和环，或具有7至12个碳原子的双环的非芳族、饱和或不饱和环。单环碳环具有3至7个，或3至6个环原子，更通常具有5或6个环原子。双环碳环具有7至12个环原子，例如排列成双环[4,5]、 [5,5]、[5,6]或[6,6]***；或者具有9或10个环原子，排列成双环[5,6]或[6,6]***，或者成为桥环***，例如双环[2.2.1]庚烷、双环[2.2.2]辛烷和双环[3.2.2]壬烷。单环碳环的实例包括环丙基、环丁基、环戊基、1-环戊-1-烯基、1-环戊-2-烯基、1-环戊-3-烯基、环己基、1-环己-1-烯基、1-环己-2-烯基、1-环己-3-烯基、环己二烯基、环庚基、环辛基、环壬基、环癸基、环十一烷基和环十二烷基。The terms "carbocycle", "carbocyclyl", "cycloalkyl" and "cycloalkenyl" refer to a monocyclic non-aromatic, saturated or unsaturated ring having 3 to 12 carbon atoms, or a ring having 7 to A bicyclic non-aromatic, saturated or unsaturated ring of 12 carbon atoms. Monocyclic carbocycles have 3 to 7, or 3 to 6, more usually 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, for example arranged in a bicyclic [4,5], [5,5], [5,6] or [6,6] system; or have 9 or 10 ring atoms, arranged Form bicyclo[5,6] or [6,6] systems, or become bridged ring systems, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclo Hexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, Cyclodecyl, Cycloundecyl and Cyclododecyl.
本文所述的取代基包括但不限于，X、R、O-、-OR、-SR、-NR 2、-NR 3、＝NR、＝N-OR、＝O、-CX 3、-CN、-OCN、-SCN、-N＝C＝O、-NCS、-NO、-NO 2、＝N 2、-N 3、NC(＝O)R、-C(＝O)R、-C(＝O)NR 2、-SO 3 -、-SO 3H、-S(＝O) 2R、-OS(＝O) 2OR、-S(＝O) 2NR、-S(＝O)R、-OP(＝O)(OR) 2、-P(＝O)(OR) 2、-PO 3、-PO 3H 2、-C(＝O)R、-C(＝O)X、-C(＝S)R、-CO 2R、-CO 2 -、-C(＝S)OR、-C(O)SR、-C(＝S)SR、-C(＝O)NR 2、-C(＝S)NR 2和-C(＝NR)NR 2，其中各X独立为卤素(F、Cl、Br或I)，且各R独立为H、C 1-C 18烷基、C 6-C 20芳基、C 3-C 14杂环、保护基团或前药基团。 Substituents described herein include, but are not limited to, X, R, O-, -OR, -SR, -NR 2 , -NR 3 , =NR, =N-OR, =O, -CX 3 , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO 2 , =N 2 , -N 3 , NC(=O)R, -C(=O)R, -C(= O)NR 2 , -SO 3 - , -SO 3 H, -S(=O) 2 R, -OS(=O) 2 OR, -S(=O) 2 NR, -S(=O)R, -OP(=O)(OR) 2 , -P(=O)(OR) 2 , -PO 3 , -PO 3 H 2 , -C(=O)R, -C(=O)X, -C (=S)R, -CO 2 R, -CO 2 - , -C(=S)OR, -C(O)SR, -C(=S)SR, -C(=O)NR 2 , -C (=S)NR 2 and -C(=NR)NR 2 , wherein each X is independently halogen (F, Cl, Br or I), and each R is independently H, C 1 -C 18 alkyl, C 6 - C 20 aryl, C 3 -C 14 heterocycle, protecting group or prodrug group.
术语“保护基”或“Pg”指通常在化合物的其它官能团反应时，用于阻断或保护特定官能团的取代基。例如，“氨基保护基”是连接在氨基上，阻断或保护化合物中的氨基官能团的取代基。适当的氨基保护基包括乙酰基、三氟乙酰基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)和9-芴基亚甲基氧基羰基(Fmoc)。类似的，“羟基保护基”是指阻断或保护羟基官能团的羟基的取代基。适当的羟基保护基包括乙酰基、三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基和四氢吡喃基。“羧基保护基”是指阻断或保护羧基官能团的羧基取代基。通常的羧基保护基包括-CH 2CH 2SO 2Ph、氰基乙基、2-(三甲基甲硅烷基)乙基、2-(三甲基甲硅烷基)乙氧基甲基、2-(p-甲苯磺酰基)乙基、2-(p-硝基苯基亚磺酰)乙基、2-(二苯基膦基)-乙基、硝基乙基等。对于保护基团及其应用的一般描述，见T.W.Greene和P.Wuts，Protective Groups in Organic Synthesis，第3版，John Wiley&Sons，New York，1999；和P.Kocienski，Protecting Groups，第3版，Verlag，2003。 The term "protecting group" or "Pg" refers to a substituent, typically used to block or protect a particular functional group while reacting other functional groups of a compound. For example, an "amino-protecting group" is a substituent attached to an amino group that blocks or protects the amino function in a compound. Suitable amino protecting groups include acetyl, trifluoroacetyl, phthalimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc ). Similarly, "hydroxyl protecting group" refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality. Suitable hydroxy protecting groups include acetyl, trialkylsilyl, dialkylphenylsilyl, benzoyl, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl group and tetrahydropyranyl group. "Carboxy protecting group" refers to a carboxyl substituent that blocks or protects the carboxyl functionality. Common carboxyl protecting groups include -CH2CH2SO2Ph , cyanoethyl , 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2 -(p-tosyl)ethyl, 2-(p-nitrophenylsulfinyl)ethyl, 2-(diphenylphosphino)-ethyl, nitroethyl and the like. For a general description of protecting groups and their use, see TW Greene and P. Wuts, Protective Groups in Organic Synthesis, 3rd ed., John Wiley & Sons, New York, 1999; and P. Kocienski, Protecting Groups, 3rd ed., Verlag, 2003.
术语“前药”是指药用活性物质的前体或衍生形式，与母药(parent drug)相比，其对肿瘤细胞具有较小的细胞毒性，且能够被酶促激活或水解激活或被转变成更具活性的母体形式。例如，参见Wilman，“癌症化学疗法的前药”Biochemical Society Transactions，14，pp.375-382，615th Meeting Belfast(1986)和Stella等，“前药：靶向给药的化学途径”Directed Drug Delivery，Borchardt等，(ed.)，pp.247-267，Humana Press(1985)。本发明的前药包括但不限于，含磷酸酯的前药、含硫代磷酸酯的前药、含硫酸酯的前药、含肽的前药、D-氨基酸修饰的前药、糖基化的前药、含β-内酰胺的前药、含任选取代的苯氧基乙酰胺的前药或含任选取代的苯乙酰胺的前药、5-氟胞嘧啶前药和其他可被转变为更具活性的无细胞毒药物的5-氟尿苷前药。可被衍生成用于本发明的前药的细胞毒药物的例子包括但不限于以上描述的那些化疗药物。The term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and is capable of being activated enzymatically or hydrolytically or by Conversion to the more active parent form. See, eg, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp.375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery" Directed Drug Delivery , Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Prodrugs of the present invention include, but are not limited to, phosphate-containing prodrugs, phosphorothioate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine prodrugs and others that can be Conversion of 5-fluorouridine prodrugs to more active non-cytotoxic drugs. Examples of cytotoxic drugs that can be derivatized as prodrugs for use in the present invention include, but are not limited to, those chemotherapeutic drugs described above.
例如，本文所述的烷基、烯基、环烷基和环烯基可以任选地被如下取代基所取代：芳基、杂芳基、杂环基、C 1-6烷氧基、C 1-6烷硫基、芳基C 1-6烷氧基、芳基C 1-6烷硫基、氨基、单-或二-C 1-6烷基氨基、氨基磺酰基、环烷基、环烯基、羧基及其酯、酰胺、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、C 1-6酰氧基、羟基及卤素或它们的任何合并取代基。另外取代基也可为氰基。优选任选取代基包含水溶性基团；本领域的普通技术人员熟悉合适水溶性部分，包括羟基、氨基基团。更优选的任选取代基包括氨基，单或双C 1-6烷基、氨基、含氨基杂环基或羟基或它们的任何合并的取代基。 For example, the alkyl, alkenyl, cycloalkyl, and cycloalkenyl groups described herein can be optionally substituted with the following substituents: aryl, heteroaryl, heterocyclyl, C 1-6 alkoxy, C 1-6 alkylthio, aryl C 1-6 alkoxy, aryl C 1-6 alkylthio, amino, mono- or di-C 1-6 alkylamino, aminosulfonyl, cycloalkyl, Cycloalkenyl, carboxyl and its ester, amide, ureido, guanidino, C 1-6 alkyl guanidino, amidino, C 1-6 alkyl amidino, C 1-6 acyloxy, hydroxyl and halogen or Any of their combined substituents. Another substituent may also be a cyano group. Preferred optional substituents include water-soluble groups; those of ordinary skill in the art are familiar with suitable water-soluble moieties, including hydroxyl, amino groups. More preferred optional substituents include amino, mono- or di-C 1-6 alkyl, amino, amino-containing heterocyclyl or hydroxy or any combination thereof.
例如，本文所述的芳基、杂环基和杂芳基可以任选地被如下取代基所取代：卤素、羟基、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、硝基、氰基、叠氮基、氨基、单-和二-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰氧基、羧基、羧基盐、羧基酯、氨基甲酰基、单和二-N-C 1-6烷基氨基甲酰基、C 1-6烷氧基羰基、芳基氧羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、脲、氨基甲酸酯、酰基、磺酰基氨基、氨基磺酰基、C 1-6烷 硫基、C 1-6烷基亚磺酰基、C 1-6烷基磺酰基、杂环基、杂芳基、杂环基C 1-6烷基和杂芳基C 1-6烷基，及其任何合并的取代基。并且2个环碳原子可连接在一起形成二环体系。 For example, the aryl, heterocyclyl, and heteroaryl groups described herein can be optionally substituted with the following substituents: halogen, hydroxy, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1-6 alkyl , halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, nitro, cyano, azido , amino, mono- and di-NC 1-6 alkylamino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, carbamoyl, mono- and di-NC 1-6 alkyl Carbamoyl, C 1-6 alkoxycarbonyl, aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, urea, carbamic acid Esters, Acyl, Sulfonylamino, Aminosulfonyl, C 1-6 Alkylthio, C 1-6 Alkylsulfinyl, C 1-6 Alkylsulfonyl, Heterocyclyl, Heteroaryl, Heterocyclyl C 1-6 alkyl and heteroaryl C 1-6 alkyl, and any combined substituents thereof. And 2 ring carbon atoms can be linked together to form a bicyclic ring system.
如本文所用，杂C 1-6烷基-是指链末端碳原子被选自N、O或S的杂原子取代的C 1-6碳链，例如，C 1-6烷基氨基、C 1-6烷氧基、C 1-6烷硫基。 As used herein, heteroC 1-6 alkyl- refers to a C 1-6 carbon chain in which the terminal carbon atoms of the chain are replaced by heteroatoms selected from N, O or S, for example, C 1-6 alkylamino, C 1 -6 alkoxy, C 1-6 alkylthio.
如本文所用，C 1-6烷基杂C 1-6烷基是指其中一个碳原子被选自N、O或S的杂原子取代的C 3-13烷基链，例如，C 1-6烷基氨基C 1-6烷基或C 1-6烷基氨基二C 1-6烷基、C 1-6烷氧基C 1-6烷基-、C 1-6烷硫基C 1-6烷基-、或C 1-6烷硫基二C 1-6烷基。 As used herein, C 1-6 alkylheteroC 1-6 alkyl refers to a C 3-13 alkyl chain in which one carbon atom is replaced by a heteroatom selected from N, O or S, for example, C 1-6 Alkylamino C 1-6 alkyl or C 1-6 alkylaminodiC 1-6 alkyl, C 1-6 alkoxy C 1-6 alkyl-, C 1-6 alkylthio C 1- 6 alkyl-, or C 1-6 alkylthio diC 1-6 alkyl.
如本文所用，术语“立体异构体”指化学组成相同，但原子或基团的空间排列不同的化合物。术语“互变异构体”或“互变异构形式”指不同能量的结构异构体，可通过低能障而互相转化。例如，质子互变异构体(也称为质子转移互变异构体)包含通过质子转移的互变现象，例如酮-烯醇异构化和亚胺-烯胺异构化。价互变异构体包含通过一些价电子的重组的互变现象。As used herein, the term "stereoisomer" refers to compounds that have identical chemical constitution but differ in the arrangement of the atoms or groups in space. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies, which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) encompass interconversions via transfer of a proton, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers involve interconversion by recombination of some of the valence electrons.
如本文所用，术语“药学上可接受的”是指物质或组合物必须是与组成制剂的其它成分和/或接受其治疗的哺乳动物在化学上和/或毒物学上相容的。As used herein, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and/or toxicologically compatible with the other ingredients making up the formulation and/or the mammal receiving its treatment.
如本文所用，术语“可药用盐”表示本发明化合物的那些羧酸盐、氨基酸加成盐，它们在可靠的医学判断范围内适用于与患者组织接触，不会产生不恰当的毒性、刺激作用、***反应等，与合理的益处/风险比相称，就它们的预期应用而言是有效的，包括(可能的话)本发明化合物的两性离子形式。As used herein, the term "pharmaceutically acceptable salt" refers to those carboxylate, amino acid addition salts of the compounds of the present invention, which are suitable within the scope of sound medical judgment for use in contact with patient tissues without undue toxicity, irritation Effects, allergic reactions, etc., commensurate with a reasonable benefit/risk ratio, are valid for their intended application, including, where possible, zwitterionic forms of the compounds of the invention.
如本文所用，术语“同位素衍生物”是指其中一个或多个原子被原子质量或质量数与通常自然界发现的原子质量或质量数不同的原子所取代的化合物。任何具体指定的特定原子或元素的所有同位素和它们的应用都预期属于本发明化合物的范围。可被并入本发明化合物的示例性同位素包括氢、碳、氮、氧、磷、硫、氟、氯和碘的同位素，分别例如 2H、 3H、 11C、 13C、 14C、 13N、 15N、 15O、 17O、 18O、 32P、 33P、 35S、 18F、 36Cl、 123I和 125I。 As used herein, the term "isotopic derivative" refers to a compound in which one or more atoms are replaced by an atom having an atomic mass or mass number different from that normally found in nature. All isotopes of any particular atom or element specified and their use are intended to be within the scope of the compounds of the present invention. Exemplary isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 32 P, 33 P, 35 S, 18 F, 36 Cl, 123 I and 125 I.
术语“溶剂合物”是指通常由溶剂分解反应形成的与溶剂相结合的化合物或其盐的形式。这个物理缔合可包括氢键键合。常规溶剂包括包括水、甲醇、乙醇、乙酸、DMSO、THF、***等。本文所述的化合物可制备成，例如，结晶形式，且可被溶剂化。合适的溶剂合物包括药学上可接受的溶剂合物且进一步包括化学计量的溶剂合物和非化学计量的溶剂合物。在一些情况下，所述溶剂合物将能够分离，例如，当一或多个溶剂分子掺入结晶固体的晶格中时。“溶剂合物”包括溶液状态的溶剂合物和可分离的溶剂合物。代表性的溶剂合物包括水合物、乙醇合物和甲醇合物。The term "solvate" refers to a form of a compound, or a salt thereof, which is associated with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein can be prepared, for example, in crystalline forms, and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. "Solvate" includes both solution state solvates and isolatable solvates. Representative solvates include hydrates, ethanolates and methanolates.
术语“水合物”是指与水相结合的化合物。通常，包含在化合物的水合物中的水分子数与该水合物中该化合物分子数的比率确定。因此，化合物的水合物可用例如通式R×x H 2O代表，其中R是该化合物，和x是大于0的数。给定化合物可形成超过一种水合物类型，包括，例如，单水合物(x为1)、低级水合物(x是大于0且小于1的数，例如，半水合物(R×0.5H 2O))和多水合物(x为大于1的数，例如，二水合物(R×2H 2O)和六水合物(R×6H 2O))。 The term "hydrate" refers to a compound that combines with water. Generally, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Thus, a hydrate of a compound can be represented, for example, by the general formula RxxH2O , where R is the compound, and x is a number greater than zero. A given compound may form more than one hydrate type, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1, for example, hemihydrates (R× 0.5H2 O)) and polyhydrates (x is a number greater than 1, eg, dihydrate (R×2H 2 O) and hexahydrate (R×6H 2 O)).
本发明化合物可以是无定形或结晶形式(多晶型)。此外，本发明化合物可以以一种或多种结晶形式存在。因此，本发明在其范围内包括本发明化合物的所有无定形或结晶形式。术语“多晶型物”是指特定晶体堆积排列的化合物的结晶形式(或其盐、水合物或溶剂合物)。所有的多晶型物具有相同的元素组成。不同的结晶形式通常具有不同的X射线衍射图、红外光谱、熔点、密度、硬度、晶体形状、光电性质、稳定性和溶解度。重结晶溶剂、结晶速率、贮存温度和其他因素可导致一种结晶形式占优。化合物的各种多晶型物可在不同的条件下通过结晶制备。The compounds of the invention may be in amorphous or crystalline form (polymorphs). Furthermore, the compounds of the invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the invention. The term "polymorph" refers to a crystalline form of a compound (or a salt, hydrate or solvate thereof) in a particular crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms generally have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can cause one crystalline form to predominate. Various polymorphs of a compound can be prepared by crystallization under different conditions.
红系前体细胞或成红细胞及其体外培养Erythroid precursor cells or erythroblasts and their in vitro culture
通过对来自外周血单核细胞(PBMC)的体外分化和扩增的人类红系前体细胞和干细胞可用作人工 自体和同种异体血液来源。从骨髓、脐带血或iPSC/ESC获得的CD34+来源的细胞中更多扩增红细胞祖细胞和成红细胞也很有科学意义，尤其是对红系前体细胞的扩增可以让我们获得更多红系干/祖细胞，从而丰富我们对红系发育的机制的理解。Human erythroid precursors and stem cells by in vitro differentiation and expansion from peripheral blood mononuclear cells (PBMCs) can be used as artificial autologous and allogeneic blood sources. More expansion of erythroid progenitors and erythroblasts in CD34+ derived cells obtained from bone marrow, umbilical cord blood or iPSC/ESC is also of scientific interest, especially the expansion of erythroid precursors allows us to obtain more erythroid lineage stem/progenitor cells, thereby enriching our understanding of the mechanisms underlying erythroid development.
因此，在一个实例中，EPs或EBs来源于供体PBMC中的干细胞。在另一实例中，干细胞在培养之前或培养期间进行基因改造。在另一实例中，干细胞为造血干细胞。另一实例中，造血干细胞来源于骨髓或脐带血。另一实例中，实验主体为哺乳动物。另一实例中，实验对象是人、小鼠或大鼠。Thus, in one example, EPs or EBs are derived from stem cells in donor PBMCs. In another example, the stem cells are genetically modified prior to or during culture. In another example, the stem cells are hematopoietic stem cells. In another example, the hematopoietic stem cells are derived from bone marrow or umbilical cord blood. In another example, the subject of the experiment is a mammal. In another example, the subject is a human, mouse or rat.
在另一个实例中，可以在培养基中对红系前体细胞或成红细胞进行体外培养。培养基包括红系分化培养基或无血清红系分化培养基及扩增培养基。在一个具体实例中，红细胞分化培养基包括IMDM、10％的FBS、5％人血清、300μg/ml holo-transferrin、2IU/ml肝素、10μg/ml胰岛素、2mM L-谷氨酰胺；之后补充3IU/ml***、50ng/ml人类干细胞因子和10ng/ml白细胞介素(IL)-3。在另一个具体实例中，无血清培养基的红细胞分化和扩增中包括SFEM II，补充3IU/mL***、50ng/ml人类干细胞因子、10ng/ml白细胞介素(IL)-3和***(IGF)-1。In another example, erythroid precursor cells or erythroblasts can be cultured in vitro in culture medium. Media include erythroid differentiation medium or serum-free erythroid differentiation medium and expansion medium. In a specific example, the erythrocyte differentiation medium comprises IMDM, 10% FBS, 5% human serum, 300 μg/ml holo-transferrin, 2 IU/ml heparin, 10 μg/ml insulin, 2 mM L-glutamine; then supplemented with 3 IU /ml erythropoietin, 50ng/ml human stem cell factor and 10ng/ml interleukin (IL)-3. In another specific example, erythrocyte differentiation and expansion in serum-free medium includes SFEM II supplemented with 3 IU/mL erythropoietin, 50 ng/ml human stem cell factor, 10 ng/ml interleukin (IL)-3 and Insulin-like growth factor (IGF)-1.
BRAF激酶抑制剂BRAF kinase inhibitors
目前上市和临床中的BRAF激酶抑制剂对本领域技术人员是熟知的，都可以用于本发明的目的。所述BRAF激酶抑制剂例如以下专利申请中记载的那些：WO2009117080A1、WO2006024834A1、WO2007071963A2、WO2013100632A1、WO2005112932A2、WO2018107146A1、WO2009077766A1、WO2019026065A3、WO2009137391A3、WO2009137391A2、WO2011113368A1、WO2000043384A1、WO2011025927A1、WO2006084015A2、WO1999010325A1、WO2000064422A3、WO2013097224A1、WO2013134243A1、WO2014151616A1、WO2007002325A1、WO2012109075A1、WO2008153947A2、WO2007131689A2、WO2014151616A1、WO2004113274A2、WO2008043446A1、WO2005009961A2、WO2007091736A1、WO2002024680A1、WO2003022833A1、WO2000041698A1、WO2010064722A1、WO2007002325A1、WO1998022103A1；以及以下文献中记载的那些：Joshua D.Hansen et al.,Potent and selective pyrazole-based inhibitors of B-Raf kinase,Bioorganic&Medicinal Chemistry Letters 18(2008)4692–4695；Andrew K.Takle et al.,The identification of potent and selective imidazole-based inhibitors of B-Raf kinase,Bioorganic&Medicinal Chemistry Letters 16(2006)378–381。以上专利申请和文献在此并入本文作为参考。The currently marketed and clinical BRAF kinase inhibitors are well known to those skilled in the art, and all of them can be used for the purpose of the present invention.所述BRAF激酶抑制剂例如以下专利申请中记载的那些：WO2009117080A1、WO2006024834A1、WO2007071963A2、WO2013100632A1、WO2005112932A2、WO2018107146A1、WO2009077766A1、WO2019026065A3、WO2009137391A3、WO2009137391A2、WO2011113368A1、WO2000043384A1、WO2011025927A1、WO2006084015A2、WO1999010325A1、WO2000064422A3、WO2013097224A1、WO2013134243A1 、WO2014151616A1、WO2007002325A1、WO2012109075A1、WO2008153947A2、WO2007131689A2、WO2014151616A1、WO2004113274A2、WO2008043446A1、WO2005009961A2、WO2007091736A1、WO2002024680A1、WO2003022833A1、WO2000041698A1、WO2010064722A1、WO2007002325A1、WO1998022103A1；以及以下文献中记载的那些：Joshua D.Hansen et al.,Potent and selective pyrazole-based inhibitors of B-Raf kinase, Bioorganic&Medicinal Chemistry Letters 18(2008)4692–4695; Andrew K.Takle et al., The identification of potent and selective imidazole-based inhibitors of B-Rafkinaseed, 16 (2006) 378–381. The above patent applications and documents are hereby incorporated by reference.
具体地，优选的BRAF激酶抑制剂为下式的化合物：Specifically, preferred BRAF kinase inhibitors are compounds of the formula:
Figure PCTCN2022122562-appb-000001
Figure PCTCN2022122562-appb-000001
其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、- NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8 炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
或者，BRAF激酶抑制剂为下式的化合物：Alternatively, the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000002
Figure PCTCN2022122562-appb-000002
IIII
其中，in,
X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基， 其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
Ar为式a)或b)的基团：Ar is a group of formula a) or b):
Figure PCTCN2022122562-appb-000003
Figure PCTCN2022122562-appb-000003
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
R 15为O或N-OH； R 15 is O or N-OH;
X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
或者，BRAF激酶抑制剂为下式的化合物：Alternatively, the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000004
Figure PCTCN2022122562-appb-000004
IIIIII
其中，in,
X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
Ar是下式a)或b)：Ar is the following formula a) or b):
Figure PCTCN2022122562-appb-000005
Figure PCTCN2022122562-appb-000005
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
更优选地，所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：More preferably, the BRAF kinase inhibitor induces paradoxical activation of MAPK; Preferably, the BRAF kinase inhibitor promotes RAF protein dimerization; Preferably, the BRAF kinase inhibitor induces αC-helix and DFG domain in BRAF kinase IN conformation of at least one of them, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof:
Figure PCTCN2022122562-appb-000006
Figure PCTCN2022122562-appb-000006
Figure PCTCN2022122562-appb-000007
Figure PCTCN2022122562-appb-000007
Figure PCTCN2022122562-appb-000008
Figure PCTCN2022122562-appb-000008
Figure PCTCN2022122562-appb-000009
Figure PCTCN2022122562-appb-000009
优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、L-779450、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
TGF-β抑制剂TGF-β inhibitors
目前上市和临床中的TGF-β抑制剂对本领域技术人员是熟知的，都可以用于本发明的目的。所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：TGF-β inhibitors currently on the market and in clinical practice are well known to those skilled in the art, and all of them can be used for the purpose of the present invention. The TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof:
Figure PCTCN2022122562-appb-000010
Figure PCTCN2022122562-appb-000010
Figure PCTCN2022122562-appb-000011
Figure PCTCN2022122562-appb-000011
优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
SMAD2/3抑制剂SMAD2/3 inhibitors
目前上市和临床中的SMAD2/3抑制剂对本领域技术人员是熟知的，都可以用于本发明的目的。所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Currently marketed and clinical SMAD2/3 inhibitors are well known to those skilled in the art, and all of them can be used for the purpose of the present invention. The SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof:
Figure PCTCN2022122562-appb-000012
Figure PCTCN2022122562-appb-000012
糖皮质激素Glucocorticoids
糖皮质激素(Glucocorticoid)是一种肾上腺皮质激素，是由肾上腺皮质中层的束状带分泌的类固醇激素，也可体外由化学方法人工合成。它对机体的发育、生长、代谢以及免疫功能等起着重要调节作用，是机体应激反应最重要的调节激素，同时也是临床上使用最为广泛而有效的抗炎和免疫抑制剂。在紧急或危重情况下，糖皮质激素往往为首选药物。Glucocorticoid (Glucocorticoid) is a kind of adrenocortical hormone, which is a steroid hormone secreted by the zona fascicularis in the middle layer of the adrenal cortex, and can also be synthesized artificially in vitro by chemical methods. It plays an important role in regulating the body's development, growth, metabolism, and immune function. It is the most important regulatory hormone for the body's stress response, and it is also the most widely used and effective anti-inflammatory and immunosuppressant clinically. In urgent or critical situations, glucocorticoids are often the drug of choice.
糖皮质激素类药物主要作用于细胞内糖皮质受体下游通路，增加红细胞和血红蛋白的生成，使血小板和纤维蛋白原增加，使单核细胞和嗜中性白细胞进入血液循环的量增加，淋巴细胞和嗜酸性和嗜碱性白细胞减少。糖皮质激素类药物是目前用于治疗贫血尤其是难治性贫血，骨髓衰竭类贫血(如DBA贫血)的临床药物，其可以体外扩增红系前体细胞(BFU-E和CFU-E)，也可以用于体内治疗贫血并促进体内红细胞的生成。Glucocorticoid drugs mainly act on the downstream pathway of intracellular glucocorticoid receptors, increase the production of red blood cells and hemoglobin, increase platelets and fibrinogen, increase the amount of monocytes and neutrophils entering the blood circulation, and lymphocytes and decreased eosinophils and basophils. Glucocorticoids are clinical drugs currently used to treat anemia, especially refractory anemia, bone marrow failure anemia (such as DBA anemia), which can expand erythroid precursor cells (BFU-E and CFU-E) in vitro , can also be used in vivo to treat anemia and promote the production of red blood cells in the body.
经过RNA-seq和BRAF抑制剂连用等实验证明，BRAF抑制剂和糖皮质激素类药物的作用机制不同，二者协同连用可以实现增加红细胞生成的效果。Experiments such as the combined use of RNA-seq and BRAF inhibitors have proved that BRAF inhibitors and glucocorticoids have different mechanisms of action, and the synergistic combination of the two can achieve the effect of increasing erythropoiesis.
贫血性疾病anemic disease
贫血性疾病主要分为由红细胞生成减少或缺陷引起的贫血、由于红细胞破坏引起的贫血、失血过多引起的贫血。Anemia diseases are mainly classified into anemia caused by decreased or defective red blood cell production, anemia caused by destruction of red blood cells, and anemia caused by excessive blood loss.
所述由红细胞生成减少或缺陷引起的贫血可为原发性的，包括原发性再生障碍性贫血、Diamond-Blackfan贫血(DBA)、Shwachman-Diamond综合征、先天性角化不良、Fanconi贫血、先天性红细胞生成障碍性贫血(CDA)、地中海型贫血、镰刀状细胞性贫血、骨髓增生异常综合征导致的贫血。The anemia caused by reduced or defective erythropoiesis may be primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome, dyskeratosis congenita, Fanconi anemia, Anemia due to congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, myelodysplastic syndrome.
所述由红细胞生成减少或缺陷引起的贫血也可为继发性的，包括继发性骨髓增生异常综合征导致的贫血、继发性再生障碍贫血、维生素缺乏型贫血、缺铁性贫血、慢性炎症性贫血、内分泌疾病性贫血、肾功能衰竭导致EPO分泌不足的继发性贫血、造血干细胞移植后血液谱系重建不良；优选地，其中所述继发性再生障碍贫血由下列原因导致：自身免疫类疾病(如***性红斑狼疮、类风湿性关节炎、炎症性肠病等)、其他免疫机制导致的贫血(如ABO血型不合、干细胞移植、坏疽性脓皮病)、淋巴增生性疾病(如慢性淋巴细胞白血病、LGL白血病、霍奇金病、非霍奇淋巴瘤等)、其他血液***恶性肿瘤(如慢性粒细胞白血病、慢性粒单核细胞白血病)、实体瘤(如胸腺瘤、胃癌、乳腺癌、甲状腺癌、肾癌等)、病毒感染(如B19细小病毒、HIV、T细胞白血病淋巴瘤病毒等)、细菌感染(如结核菌、细菌性败血病)、药物和毒素导致的免疫反应(如外源人EPO诱导的抗体相关的纯红再障、铅/苯中毒)、对内/外源EPO抵抗所造成的贫血及各种原因导致的对***抵抗的贫血。The anemia caused by the reduction or defect of red blood cell production can also be secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency anemia, iron deficiency anemia, chronic Inflammatory anemia, anemia of endocrine disease, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary aplastic anemia is caused by: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO incompatibility, stem cell transplantation, pyoderma gangrenosum), lymphoproliferative diseases (such as Chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin lymphoma, etc.), other hematological malignancies (such as chronic myelogenous leukemia, chronic myelomonocytic leukemia), solid tumors (such as thymoma, gastric cancer, Breast cancer, thyroid cancer, kidney cancer, etc.), viral infection (such as B19 parvovirus, HIV, T-cell leukemia lymphoma virus, etc.), bacterial infection (such as tuberculosis, bacterial septicemia), immunity caused by drugs and toxins Reactions (such as antibody-related pure red aplastic anemia induced by exogenous human EPO, lead/benzene poisoning), anemia caused by resistance to endogenous/exogenous EPO, and anemia caused by resistance to erythropoietin due to various reasons.
所述由于红细胞破坏引起的贫血可为原发性的，包括遗传性球形红细胞增多症、遗传性椭圆细胞增多症、无β脂蛋白血症、酶缺乏症导致的贫血(如丙酮酸激酶和己糖激酶缺陷导致糖酵解缺陷型贫血、葡萄糖6-磷酸脱氢酶缺乏症和谷胱甘肽合成酶缺乏症、氧化应激增加导致的贫血)。The anemia caused by the destruction of red blood cells may be primary, including hereditary spherocytosis, hereditary ellipsocytosis, abeta lipoproteinemia, anemia caused by enzyme deficiency (such as pyruvate kinase and hexose Kinase deficiency leads to glycolysis deficient anemia, glucose 6-phosphate dehydrogenase deficiency and glutathione synthetase deficiency, anemia due to increased oxidative stress).
所述由于红细胞破坏引起的贫血也可为继发性的，包括抗体介导的温性自身免疫性溶血性贫血、冷凝集素溶血性贫血、溶血性的疾病(如新生儿溶血症)、红细胞机械损伤(如微血管病性溶血性贫血，包括血栓性血小板减少性紫癜和弥散性血管内凝血)、感染导致的溶血性贫血(如疟疾感染)。The anemia caused by the destruction of red blood cells can also be secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic diseases (such as hemolytic disease of newborns), red blood cell Mechanical injury (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), infection-induced hemolytic anemia (eg, malaria infection).
所述失血过多引起的贫血包括早产儿贫血(如实验室检测的频繁采血，并且任选地合并红细胞生成不足)、外/内伤(各种创伤或手术导致的急性失血)、胃肠道病变导致的急性出血(如静脉曲张病变、消化性溃疡)或慢性失血(如血管发育不良)、妇科疾病导致的慢性失血、癌症(包括结肠直肠癌和膀胱癌导致的急性或慢性失血，尤其是在晚期)、以血液为食的肠道线虫感染(如钩虫和鞭虫导致失血性贫血)、医源性贫血、反复抽血和医疗程序造成的失血。The anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood sampling in laboratory tests, and optionally combined with insufficient erythropoiesis), external/internal injuries (acute blood loss caused by various traumas or operations), gastrointestinal lesions Acute bleeding (eg, varicose lesions, peptic ulcers) or chronic bleeding (eg, angiodysplasia), chronic bleeding due to gynecological disorders, acute or chronic bleeding due to cancer (including colorectal and bladder cancers, especially in late stage), blood-feeding intestinal nematode infections (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws and medical procedures.
而且，所述贫血性疾病可对***或糖皮质激素或其他治疗贫血的药物具有耐药性。Furthermore, the anemic disease may be resistant to erythropoietin or glucocorticoids or other drugs for the treatment of anemia.
具体实施方案specific implementation plan
在一个具体实施方案中，本发明提供一种用于治疗原发性或继发性贫血的药物，其包含BRAF激酶抑制剂和一种或多种药学上可接受的赋形剂。In a specific embodiment, the present invention provides a medicament for treating primary or secondary anemia, which comprises a BRAF kinase inhibitor and one or more pharmaceutically acceptable excipients.
在该具体实施方案的优选方案中，其中所述药物还包含TGF-β抑制剂和/或SMAD2/3抑制剂和/或糖皮质激素。In a preferred version of this specific embodiment, the drug further comprises a TGF-β inhibitor and/or a SMAD2/3 inhibitor and/or a glucocorticoid.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：In a preferred version of this specific embodiment, wherein said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the αC-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives:
Figure PCTCN2022122562-appb-000013
Figure PCTCN2022122562-appb-000013
Figure PCTCN2022122562-appb-000014
Figure PCTCN2022122562-appb-000014
Figure PCTCN2022122562-appb-000015
Figure PCTCN2022122562-appb-000015
Figure PCTCN2022122562-appb-000016
Figure PCTCN2022122562-appb-000016
优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、L-779450、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000017
Figure PCTCN2022122562-appb-000017
其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、 -NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保 护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000018
Figure PCTCN2022122562-appb-000018
IIII
其中，in,
X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
Ar为式a)或b)的基团：Ar is a group of formula a) or b):
Figure PCTCN2022122562-appb-000019
Figure PCTCN2022122562-appb-000019
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
R 15为O或N-OH； R 15 is O or N-OH;
X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000020
Figure PCTCN2022122562-appb-000020
IIIIII
其中，in,
X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
Ar是下式a)或b)：Ar is the following formula a) or b):
Figure PCTCN2022122562-appb-000021
Figure PCTCN2022122562-appb-000021
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：
Figure PCTCN2022122562-appb-000022
Figure PCTCN2022122562-appb-000023
Figure PCTCN2022122562-appb-000024
In a preferred version of this specific embodiment, wherein said TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof:
Figure PCTCN2022122562-appb-000022
Figure PCTCN2022122562-appb-000023
Figure PCTCN2022122562-appb-000024
优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
在该具体实施方案的优选方案中，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：
Figure PCTCN2022122562-appb-000025
Figure PCTCN2022122562-appb-000026
In a preferred version of this specific embodiment, wherein said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof:
Figure PCTCN2022122562-appb-000025
Figure PCTCN2022122562-appb-000026
在该具体实施方案的优选方案中，其中所述贫血为由红细胞生成减少或缺陷引起的贫血。In a preferred version of this particular embodiment, wherein said anemia is anemia caused by decreased or defective erythropoiesis.
在该具体实施方案的优选方案中，其中所述由红细胞生成减少或缺陷引起的贫血为原发性的，包括原发性再生障碍性贫血、Diamond-Blackfan贫血(DBA)、Shwachman-Diamond综合征、先天性角化不良、Fanconi贫血、先天性红细胞生成障碍性贫血(CDA)、地中海型贫血、镰刀状细胞性贫血、骨髓增生异常综合征导致的贫血。In a preferred version of this specific embodiment, wherein said anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome , dyskeratosis congenita, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, anemia caused by myelodysplastic syndrome.
在该具体实施方案的优选方案中，其中所述由红细胞生成减少或缺陷引起的贫血为继发性的，包括继发性骨髓增生异常综合征导致的贫血、继发性再生障碍贫血、维生素缺乏型贫血、缺铁性贫血、慢性炎症性贫血、内分泌疾病性贫血、肾功能衰竭导致EPO分泌不足的继发性贫血、造血干细胞移植后血液谱系重建不良；优选地，其中所述继发性再生障碍贫血由下列原因导致：自身免疫类疾病(如***性红斑狼疮、类风湿性关节炎、炎症性肠病等)、其他免疫机制导致的贫血(如ABO血型不合、干细胞移植、坏疽性脓皮病)、淋巴增生性疾病(如慢性淋巴细胞白血病、LGL白血病、霍奇金病、非霍奇淋巴瘤等)、其他血液***恶性肿瘤(如慢性粒细胞白血病、慢性粒单核细胞白血病)、实体瘤(如胸腺瘤、胃癌、乳腺癌、甲状腺癌、肾癌等)、病毒感染(如B19细小病毒、HIV、T细胞白血病淋巴瘤病毒等)、细菌感染(如结核菌、细菌性败血病)、药物和毒素导致的免疫反应(如外源人EPO诱导的抗体相关的纯红再障、铅/苯中毒)、对内/外源EPO抵抗所造成的贫血及各种原因导致的对***抵抗的贫血。In a preferred version of this specific embodiment, wherein said anemia caused by reduced or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency type anemia, iron deficiency anemia, chronic inflammatory anemia, endocrine disease anemia, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary regeneration Obstacle anemia is caused by the following reasons: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, gangrenous pyoderma, etc.) disease), lymphoproliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, etc.), other hematological malignancies (such as chronic myeloid leukemia, chronic myelomonocytic leukemia), Solid tumors (such as thymoma, gastric cancer, breast cancer, thyroid cancer, kidney cancer, etc.), viral infections (such as B19 parvovirus, HIV, T-cell leukemia lymphoma virus, etc.), bacterial infections (such as Mycobacterium tuberculosis, bacterial sepsis diseases), immune reactions caused by drugs and toxins (such as antibody-related pure red aplastic anemia induced by exogenous human EPO, lead/benzene poisoning), anemia caused by resistance to endogenous/exogenous EPO, and various causes of Erythropoietin-resistant anemia.
在该具体实施方案的优选方案中，其中所述贫血为由于红细胞破坏引起的贫血。In a preferred version of this particular embodiment, wherein said anemia is anemia due to destruction of red blood cells.
在该具体实施方案的优选方案中，其中所述由于红细胞破坏引起的贫血为原发性的，包括遗传性 球形红细胞增多症、遗传性椭圆细胞增多症、无β脂蛋白血症、酶缺乏症导致的贫血(如丙酮酸激酶和己糖激酶缺陷导致糖酵解缺陷型贫血、葡萄糖6-磷酸脱氢酶缺乏症和谷胱甘肽合成酶缺乏症、氧化应激增加导致的贫血)。In a preferred version of this specific embodiment, wherein said anemia due to destruction of red blood cells is primary, including hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, enzyme deficiency Anemias resulting from (eg, glycolysis deficient anemia due to pyruvate kinase and hexokinase deficiencies, glucose 6-phosphate dehydrogenase and glutathione synthase deficiencies, anemia due to increased oxidative stress).
在该具体实施方案的优选方案中，其中所述由于红细胞破坏引起的贫血为继发性的，包括抗体介导的温性自身免疫性溶血性贫血、冷凝集素溶血性贫血、溶血性的疾病(如新生儿溶血症)、红细胞机械损伤(如微血管病性溶血性贫血，包括血栓性血小板减少性紫癜和弥散性血管内凝血)、感染导致的溶血性贫血(如疟疾感染)。In a preferred version of this specific embodiment, wherein the anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
在该具体实施方案的优选方案中，其中所述贫血为失血过多引起的贫血。In a preferred version of this specific embodiment, wherein the anemia is anemia caused by excessive blood loss.
在该具体实施方案的优选方案中，其中所述失血过多引起的贫血包括早产儿贫血(如实验室检测的频繁采血，并且任选地合并红细胞生成不足)、外/内伤(各种创伤或手术导致的急性失血)、胃肠道病变导致的急性出血(如静脉曲张病变、消化性溃疡)或慢性失血(如血管发育不良)、妇科疾病导致的慢性失血、癌症(包括结肠直肠癌和膀胱癌导致的急性或慢性失血，尤其是在晚期)、以血液为食的肠道线虫感染(如钩虫和鞭虫导致失血性贫血)、医源性贫血、反复抽血和医疗程序造成的失血。In a preferred version of this specific embodiment, wherein the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood collection as detected by the laboratory, and optionally combined with insufficient erythropoiesis), external/internal injuries (various trauma or Acute blood loss due to surgery), acute bleeding due to gastrointestinal lesions (eg, variceal lesions, peptic ulcer) or chronic blood loss (eg, angiodysplasia), chronic blood loss due to gynecological diseases, cancer (including colorectal cancer and bladder cancer) Acute or chronic blood loss from cancer, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
在该具体实施方案的优选方案中，其中所述疾病对***或糖皮质激素或其他现有治疗贫血的药物具有耐药性。In a preferred version of this particular embodiment, wherein the disease is resistant to erythropoietin or glucocorticoids or other currently available drugs for the treatment of anemia.
在另一个具体实施方案中，本发明提供一种试剂盒，其包括：In another specific embodiment, the invention provides a kit comprising:
a)BRAF激酶抑制剂，和任选地，a) a BRAF kinase inhibitor, and optionally,
b)***。b) Erythropoietin.
在该具体实施方案的优选方案中，其还包括：In a preferred version of this specific embodiment, it also includes:
c)TGF-β抑制剂和/或SMAD2/3抑制剂和/或糖皮质激素和/或***和/或干细胞因子。c) TGF-β inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or erythropoietin and/or stem cell factor.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、BRAF抑制剂1(化合物13)、RAF709、L-779450、LY3009120、Belvarafenib(HM95573)、RO5126766(CH5126766)、TAK-632、PLX-4720、Agerafenib(RXDX-105)、Regorafenib(BAY 73-4506)、Sorafenib(BAY 43-9006)、Donafenib(Sorafenib D3)、Lifirafenib(BGB-283)、BRAF IN1、Vemurafenib(PLX4032)、RAF265(chir265)、AZ 628、AZ304、CCT196969、Doramapimod(BIRB796)、Encorafenib(LGX818)、Naporafenib(LXH254)、BMS-908662、BGB659、GW5074、MLN2480(TAK580)、ARQ-736或ZM336372；In a preferred version of this specific embodiment, wherein said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the αC-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BAY 73-4506), Sorafenib(BAY 43-9006), Donafenib(Sorafenib D3), Lifirafenib(BGB-283), BRAF IN1, Vemurafenib(PLX4032), RAF265( chir265), AZ 628, AZ304, CCT196969, Doramapimod (BIRB796), Encorafenib (LGX818), Naporafenib (LXH254), BMS-908662, BGB659, GW5074, MLN2480 (TAK580), ARQ-736, or ZM336372;
优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、L-779450、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000027
Figure PCTCN2022122562-appb-000027
其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、- P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000028
Figure PCTCN2022122562-appb-000028
IIII
其中，in,
X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
Ar为式a)或b)的基团：Ar is a group of formula a) or b):
Figure PCTCN2022122562-appb-000029
Figure PCTCN2022122562-appb-000029
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
R 15为O或N-OH； R 15 is O or N-OH;
X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000030
Figure PCTCN2022122562-appb-000030
IIIIII
其中，in,
X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
Ar是下式a)或b)：Ar is the following formula a) or b):
Figure PCTCN2022122562-appb-000031
Figure PCTCN2022122562-appb-000031
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761、Galunisertib(LY2157299)、LY364947、SB431542、LDN-193189、SB525334、SB505124、GW788388、RepSox(E-616452)、K02288、BIBF-0775、TP0427736、A-83-01、LDN-214117、SD-208、Vactosertib(TEW-7197)、LDN-212854、Dorsomorphin(Compound C)或LY3200882；In a preferred version of this specific embodiment, wherein said TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
在该具体实施方案的优选方案中，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Luspatercept、Sotatercept、SIS3HCl、Alantolactone、Halofuginone和AUDA。In the preferred version of this specific embodiment, wherein said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
在另一个具体实施方案中，本发明提供BRAF激酶抑制剂，或者BRAF激酶抑制剂和***和/或干细胞因子，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合在制备用于扩增红系前体细胞或成红细胞的药物中的用途。In another specific embodiment, the present invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF-β inhibitor, or a BRAF kinase inhibitor and Use of SMAD2/3 inhibitors, or BRAF kinase inhibitors, TGF-β inhibitors and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of a medicament for expanding erythroid precursor cells or erythroblasts use.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂延缓红细胞的终末期分化进程。In a preferred aspect of this specific embodiment, wherein said BRAF kinase inhibitor delays the progression of terminal differentiation of erythrocytes.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂促使成红细胞具有更多的自我更新。In a preferred version of this embodiment, wherein said BRAF kinase inhibitor promotes greater self-renewal of erythroblasts.
在该具体实施方案的优选方案中，其中所述红系前体细胞扩增后产生成熟脱核的红细胞。In a preferred version of this specific embodiment, wherein said erythroid precursor cells are expanded to produce mature denucleated erythrocytes.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、BRAF抑制剂1(化合物13)、RAF709、L-779450、LY3009120、Belvarafenib(HM95573)、RO5126766(CH5126766)、TAK-632、PLX-4720、Agerafenib(RXDX-105)、Regorafenib(BAY 73-4506)、Sorafenib(BAY 43-9006)、Donafenib(Sorafenib D3)、Lifirafenib(BGB-283)、BRAF IN1、Vemurafenib(PLX4032)、RAF265(chir265)、AZ 628、AZ304、CCT196969、Doramapimod(BIRB796)、Encorafenib(LGX818)、Naporafenib(LXH254)、BMS-908662、BGB659、GW5074、MLN2480(TAK580)、ARQ-736或ZM336372；In a preferred version of this specific embodiment, wherein said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the αC-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BAY 73-4506), Sorafenib(BAY 43-9006), Donafenib(Sorafenib D3), Lifirafenib(BGB-283), BRAF IN1, Vemurafenib(PLX4032), RAF265( chir265), AZ 628, AZ304, CCT196969, Doramapimod (BIRB796), Encorafenib (LGX818), Naporafenib (LXH254), BMS-908662, BGB659, GW5074, MLN2480 (TAK580), ARQ-736, or ZM336372;
优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、L-779450、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000032
Figure PCTCN2022122562-appb-000032
其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、 -SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000033
Figure PCTCN2022122562-appb-000033
IIII
其中，in,
X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
Ar为式a)或b)的基团：Ar is a group of formula a) or b):
Figure PCTCN2022122562-appb-000034
Figure PCTCN2022122562-appb-000034
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
R 15为O或N-OH； R 15 is O or N-OH;
X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000035
Figure PCTCN2022122562-appb-000035
IIIIII
其中，in,
X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
Ar是下式a)或b)：Ar is the following formula a) or b):
Figure PCTCN2022122562-appb-000036
Figure PCTCN2022122562-appb-000036
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761、Galunisertib(LY2157299)、LY364947、SB431542、LDN-193189、SB525334、SB505124、GW788388、RepSox(E-616452)、K02288、BIBF-0775、TP0427736、A-83-01、LDN-214117、SD-208、Vactosertib(TEW-7197)、LDN-212854、Dorsomorphin(Compound C)或LY3200882；In a preferred version of this specific embodiment, wherein said TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
在该具体实施方案的优选方案中，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Luspatercept、Sotatercept、SIS3 HCl、Alantolactone、Halofuginone和AUDA。In the preferred version of this specific embodiment, wherein said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives: Luspatercept, Sotatercept, SIS3 HCl , Alantolactone, Halofuginone, and AUDA.
在另一个具体实施方案中，本发明提供BRAF激酶抑制剂，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合在制备用于治疗治疗原发性或继发性贫血的药物中的用途。In another specific embodiment, the present invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and a TGF-beta inhibitor, or a BRAF kinase inhibitor and a SMAD2/3 inhibitor, or a BRAF kinase inhibitor, a TGF-beta inhibitor Agents and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of medicines for the treatment of primary or secondary anemia.
在该具体实施方案的优选方案中，其中所述贫血为由红细胞生成减少或缺陷引起的贫血。In a preferred version of this particular embodiment, wherein said anemia is anemia caused by decreased or defective erythropoiesis.
在该具体实施方案的优选方案中，其中所述由红细胞生成减少或缺陷引起的贫血为原发性的，包括原发性再生障碍性贫血、Diamond-Blackfan贫血(DBA)、Shwachman-Diamond综合征、先天性角化不良、Fanconi贫血、先天性红细胞生成障碍性贫血(CDA)、地中海型贫血、镰刀状细胞性贫血、骨髓增生异常综合征导致的贫血。In a preferred version of this specific embodiment, wherein said anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome , dyskeratosis congenita, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, anemia caused by myelodysplastic syndrome.
在该具体实施方案的优选方案中，其中所述由红细胞生成减少或缺陷引起的贫血为继发性的，包括继发性骨髓增生异常综合征导致的贫血、继发性再生障碍贫血、维生素缺乏型贫血、缺铁性贫血、慢性炎症性贫血、内分泌疾病性贫血、肾功能衰竭导致EPO分泌不足的继发性贫血、造血干细胞移植后血液谱系重建不良；优选地，其中所述继发性再生障碍贫血由下列原因导致：自身免疫类疾病(如***性红斑狼疮、类风湿性关节炎、炎症性肠病等)、其他免疫机制导致的贫血(如ABO血型不合、干细胞移植、坏疽性脓皮病)、淋巴增生性疾病(如慢性淋巴细胞白血病、LGL白血病、霍奇金病、非霍奇淋巴瘤等)、其他血液***恶性肿瘤(如慢性粒细胞白血病、慢性粒单核细胞白血病)、实体瘤(如胸腺瘤、胃癌、乳腺癌、甲状腺癌、肾癌等)、病毒感染(如B19细小病毒、HIV、T细胞白血病淋巴瘤病毒等)、细菌感染(如结核菌、细菌性败血病)、药物和毒素导致的免疫反应(如外源人EPO诱导的抗体相关的纯红再障、铅/苯中毒)、对内/外源EPO抵抗所造成的贫血及各种原因导致的对***抵抗的贫血。In a preferred version of this specific embodiment, wherein said anemia caused by reduced or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency type anemia, iron deficiency anemia, chronic inflammatory anemia, endocrine disease anemia, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary regeneration Obstacle anemia is caused by the following reasons: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, gangrenous pyoderma, etc.) disease), lymphoproliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, etc.), other hematological malignancies (such as chronic myeloid leukemia, chronic myelomonocytic leukemia), Solid tumors (such as thymoma, gastric cancer, breast cancer, thyroid cancer, kidney cancer, etc.), viral infections (such as B19 parvovirus, HIV, T-cell leukemia lymphoma virus, etc.), bacterial infections (such as Mycobacterium tuberculosis, bacterial sepsis diseases), immune reactions caused by drugs and toxins (such as antibody-related pure red aplastic anemia induced by exogenous human EPO, lead/benzene poisoning), anemia caused by resistance to endogenous/exogenous EPO, and various causes of Erythropoietin-resistant anemia.
在该具体实施方案的优选方案中，其中所述贫血为由于红细胞破坏引起的贫血。In a preferred version of this particular embodiment, wherein said anemia is anemia due to destruction of red blood cells.
在该具体实施方案的优选方案中，其中所述由于红细胞破坏引起的贫血为原发性的，包括遗传性球形红细胞增多症、遗传性椭圆细胞增多症、无β脂蛋白血症、酶缺乏症导致的贫血(如丙酮酸激酶和己糖激酶缺陷导致糖酵解缺陷型贫血、葡萄糖6-磷酸脱氢酶缺乏症和谷胱甘肽合成酶缺乏症、氧化应激增加导致的贫血)。In a preferred version of this specific embodiment, wherein said anemia due to destruction of red blood cells is primary, including hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, enzyme deficiency Anemias resulting from (eg, glycolysis deficient anemia due to pyruvate kinase and hexokinase deficiencies, glucose 6-phosphate dehydrogenase and glutathione synthase deficiencies, anemia due to increased oxidative stress).
在该具体实施方案的优选方案中，其中所述由于红细胞破坏引起的贫血为继发性的，包括抗体介导的温性自身免疫性溶血性贫血、冷凝集素溶血性贫血、溶血性的疾病(如新生儿溶血症)、红细胞机械损伤(如微血管病性溶血性贫血，包括血栓性血小板减少性紫癜和弥散性血管内凝血)、感染导致的溶血性贫血(如疟疾感染)。In a preferred version of this specific embodiment, wherein the anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
在该具体实施方案的优选方案中，其中所述贫血为失血过多引起的贫血。In a preferred version of this specific embodiment, wherein the anemia is anemia caused by excessive blood loss.
在该具体实施方案的优选方案中，其中所述失血过多引起的贫血包括早产儿贫血(如实验室检测的频繁采血，并且任选地合并红细胞生成不足)、外/内伤(各种创伤或手术导致的急性失血)、胃肠道病变导致的急性出血(如静脉曲张病变、消化性溃疡)或慢性失血(如血管发育不良)、妇科疾病导致的慢性失血、癌症(包括结肠直肠癌和膀胱癌导致的急性或慢性失血，尤其是在晚期)、以血液为食的肠道线虫感染(如钩虫和鞭虫导致失血性贫血)、医源性贫血、反复抽血和医疗程序造成的失血。In a preferred version of this specific embodiment, wherein the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood collection as detected by the laboratory, and optionally combined with insufficient erythropoiesis), external/internal injuries (various trauma or Acute blood loss due to surgery), acute bleeding due to gastrointestinal lesions (eg, variceal lesions, peptic ulcer) or chronic blood loss (eg, angiodysplasia), chronic blood loss due to gynecological diseases, cancer (including colorectal cancer and bladder cancer) Acute or chronic blood loss from cancer, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
在该具体实施方案的优选方案中，其中所述疾病对***或糖皮质激素或其他治疗贫血的药物具有耐药性。In a preferred version of this particular embodiment, wherein the disease is resistant to erythropoietin or glucocorticoids or other drugs for the treatment of anemia.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地， 所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、BRAF抑制剂1(化合物13)、RAF709、L-779450、LY3009120、Belvarafenib(HM95573)、RO5126766(CH5126766)、TAK-632、PLX-4720、Agerafenib(RXDX-105)、Regorafenib(BAY 73-4506)、Sorafenib(BAY 43-9006)、Donafenib(Sorafenib D3)、Lifirafenib(BGB-283)、BRAF IN1、Vemurafenib(PLX4032)、RAF265(chir265)、AZ 628、AZ304、CCT196969、Doramapimod(BIRB796)、Encorafenib(LGX818)、Naporafenib(LXH254)、BMS-908662、BGB659、GW5074、MLN2480(TAK580)、ARQ-736或ZM336372；In a preferred version of this specific embodiment, wherein said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the αC-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BAY 73-4506), Sorafenib(BAY 43-9006), Donafenib(Sorafenib D3), Lifirafenib(BGB-283), BRAF IN1, Vemurafenib(PLX4032), RAF265( chir265), AZ 628, AZ304, CCT196969, Doramapimod (BIRB796), Encorafenib (LGX818), Naporafenib (LXH254), BMS-908662, BGB659, GW5074, MLN2480 (TAK580), ARQ-736, or ZM336372;
优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, Regorafenib, or ZM336372;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000037
Figure PCTCN2022122562-appb-000037
其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、 -P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2- C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000038
Figure PCTCN2022122562-appb-000038
IIII
其中，in,
X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
Ar为式a)或b)的基团：Ar is a group of formula a) or b):
Figure PCTCN2022122562-appb-000039
Figure PCTCN2022122562-appb-000039
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
R 15为O或N-OH； R 15 is O or N-OH;
X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000040
Figure PCTCN2022122562-appb-000040
IIIIII
其中，in,
X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
Ar是下式a)或b)：Ar is the following formula a) or b):
Figure PCTCN2022122562-appb-000041
Figure PCTCN2022122562-appb-000041
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761、Galunisertib(LY2157299)、LY364947、SB431542、LDN-193189、SB525334、SB505124、GW788388、RepSox(E-616452)、K02288、BIBF-0775、TP0427736、A-83-01、LDN-214117、SD-208、Vactosertib(TEW-7197)、LDN-212854、Dorsomorphin(Compound C)或LY3200882；In a preferred version of this specific embodiment, wherein said TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
在该具体实施方案的优选方案中，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Luspatercept、Sotatercept、SIS3HCl、Alantolactone、Halofuginone和AUDA。In the preferred version of this specific embodiment, wherein said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
在另一个具体实施方案中，本发明提供BRAF激酶抑制剂，或者BRAF激酶抑制剂和***和/或干细胞因子，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合在扩增红系前体细胞或成红细胞中的用途。In another specific embodiment, the present invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF-β inhibitor, or a BRAF kinase inhibitor and SMAD2/3 inhibitors, or BRAF kinase inhibitors, TGF-beta inhibitors and SMAD2/3 inhibitors, and their use in combination with glucocorticoids for expanding erythroid precursor cells or erythroblasts.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂延缓了红细胞的终末期分化进程。In a preferred aspect of this specific embodiment, wherein said BRAF kinase inhibitor delays the progression of terminal phase differentiation of erythrocytes.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂促进了成红细胞具有更多的自我更新。In a preferred version of this embodiment, wherein said BRAF kinase inhibitor promotes greater self-renewal of erythroblasts.
在该具体实施方案的优选方案中，其中所述红系前体细胞扩增后产生成熟脱核的红细胞。In a preferred version of this specific embodiment, wherein said erythroid precursor cells are expanded to produce mature denucleated erythrocytes.
在该具体实施方案的优选方案中，其中所述红系前体细胞选自红细胞集落形成单位(CFU-E)、爆发形成单位红细胞祖细胞(BFU-E)和原成红细胞。In a preferred version of this particular embodiment, wherein said erythroid precursor cells are selected from the group consisting of erythroid colony forming units (CFU-E), burst forming unit erythroid progenitors (BFU-E) and proerythroblasts.
在该具体实施方案的优选方案中，其中所述红系前体细胞为基因改造的红系前体细胞或可搭载药物的红系前体细胞。In a preferred version of this specific embodiment, the erythroid precursor cells are genetically modified erythroid precursor cells or drug-loadable erythroid precursor cells.
在该具体实施方案的优选方案中，其中所述基因改造的红系前体细胞能够治疗遗传性疾病，如苯丙酮尿症和癌症。In a preferred version of this specific embodiment, wherein said genetically modified erythroid precursor cells are capable of treating genetic diseases, such as phenylketonuria and cancer.
在该具体实施方案的优选方案中，其中所述红系前体细胞或成红细胞来自血液来源，包括哺乳动物例如小鼠、大鼠、人类等的血液和血液制品。In a preferred version of this specific embodiment, wherein said erythroid precursor cells or erythroblasts are derived from blood sources, including blood and blood products of mammals such as mice, rats, humans, and the like.
在该具体实施方案的优选方案中，其中所述红系前体细胞或成红细胞为从骨髓、脐带血或胚胎/诱导多能性干细胞(iPSC/ESC)获得的CD34+来源的细胞。In a preferred embodiment of this specific embodiment, wherein said erythroid precursor cells or erythroblasts are CD34+ derived cells obtained from bone marrow, umbilical cord blood or embryonic/induced pluripotent stem cells (iPSC/ESC).
在该具体实施方案的优选方案中，其中所述红系前体细胞或成红细胞来自外周血单核细胞(PBMC)的体外分化和扩增的人红系前体细胞和干细胞，或者来自PBMC中的干细胞。In a preferred version of this specific embodiment, wherein said erythroid precursor cells or erythroblasts are derived from in vitro differentiated and expanded human erythroid precursor cells and stem cells from peripheral blood mononuclear cells (PBMC), or from PBMC of stem cells.
在该具体实施方案的优选方案中，其中所述干细胞为造血干细胞。In a preferred version of this specific embodiment, wherein said stem cells are hematopoietic stem cells.
在该具体实施方案的优选方案中，其中所述造血干细胞来源于骨髓或脐带血。In a preferred version of this specific embodiment, wherein said hematopoietic stem cells are derived from bone marrow or umbilical cord blood.
在该具体实施方案的优选方案中，其中所述干细胞在培养之前或培养期间进行基因改造。In a preferred version of this specific embodiment, wherein said stem cells are genetically modified before or during culturing.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、BRAF抑制剂1(化合物13)、RAF709、L-779450、LY3009120、Belvarafenib(HM95573)、RO5126766(CH5126766)、TAK-632、PLX-4720、Agerafenib(RXDX-105)、Regorafenib(BAY 73-4506)、Sorafenib(BAY 43-9006)、Donafenib(Sorafenib D3)、Lifirafenib(BGB-283)、BRAF IN1、Vemurafenib(PLX4032)、RAF265(chir265)、AZ 628、AZ304、CCT196969、Doramapimod(BIRB796)、Encorafenib(LGX818)、Naporafenib(LXH254)、BMS-908662、BGB659、GW5074、MLN2480(TAK580)、ARQ-736或ZM336372；In a preferred version of this specific embodiment, wherein said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the αC-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BAY 73-4506), Sorafenib(BAY 43-9006), Donafenib(Sorafenib D3), Lifirafenib(BGB-283), BRAF IN1, Vemurafenib(PLX4032), RAF265( chir265), AZ 628, AZ304, CCT196969, Doramapimod (BIRB796), Encorafenib (LGX818), Naporafenib (LXH254), BMS-908662, BGB659, GW5074, MLN2480 (TAK580), ARQ-736, or ZM336372;
优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, Regorafenib, or ZM336372;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000042
Figure PCTCN2022122562-appb-000042
其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、- OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、 -S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000043
Figure PCTCN2022122562-appb-000043
IIII
其中，in,
X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以 任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
Ar为式a)或b)的基团：Ar is a group of formula a) or b):
Figure PCTCN2022122562-appb-000044
Figure PCTCN2022122562-appb-000044
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
R 15为O或N-OH； R 15 is O or N-OH;
X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000045
Figure PCTCN2022122562-appb-000045
IIIIII
其中，in,
X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
Ar是下式a)或b)：Ar is the following formula a) or b):
Figure PCTCN2022122562-appb-000046
Figure PCTCN2022122562-appb-000046
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761、Galunisertib(LY2157299)、LY364947、SB431542、LDN-193189、SB525334、SB505124、GW788388、RepSox(E-616452)、K02288、BIBF-0775、TP0427736、A-83-01、LDN-214117、SD-208、Vactosertib(TEW-7197)、LDN-212854、Dorsomorphin(Compound C)或LY3200882；In a preferred version of this specific embodiment, wherein said TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
在该具体实施方案的优选方案中，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Luspatercept、Sotatercept、SIS3HCl、Alantolactone、Halofuginone和AUDA。In the preferred version of this specific embodiment, wherein said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂以1nM至100μM，优选100nM至10μM的浓度、更优选1μM至10μM的浓度使用。In a preferred version of this particular embodiment, wherein said BRAF kinase inhibitor is used at a concentration of 1 nM to 100 μM, preferably 100 nM to 10 μM, more preferably 1 μM to 10 μM.
在该具体实施方案的优选方案中，其中还同时使用***。In a preferred version of this particular embodiment, erythropoietin is also used concomitantly.
在另一个具体实施方案中，本发明提供一种扩增红系前体细胞或成红细胞的方法，其包括向受试者给药BRAF激酶抑制剂，或者BRAF激酶抑制剂和***和/或干细胞因子，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合。In another specific embodiment, the invention provides a method of expanding erythroid precursor cells or erythroblasts comprising administering to a subject a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and /or stem cell factor, or BRAF kinase inhibitor and TGF-β inhibitor, or BRAF kinase inhibitor and SMAD2/3 inhibitor, or BRAF kinase inhibitor, TGF-β inhibitor and SMAD2/3 inhibitor, and their combination Combinations of glucocorticoids.
在该具体实施方案的优选方案中，其中所述方法延缓了红细胞的终末期分化进程。In a preferred version of this embodiment, wherein said method delays the progression of terminal differentiation of erythrocytes.
在该具体实施方案的优选方案中，其中所述方法促进了成红细胞具有更多的自我更新。In a preferred version of this embodiment, wherein said method promotes greater self-renewal of erythroblasts.
在该具体实施方案的优选方案中，其中所述方法产生成熟脱核的红细胞。In a preferred version of this specific embodiment, wherein said method produces mature denucleated red blood cells.
在该具体实施方案的优选方案中，其中所述红系前体细胞选自红细胞集落形成单位(CFU-E)、爆发形成单位红细胞祖细胞(BFU-E)和原成红细胞。In a preferred version of this particular embodiment, wherein said erythroid precursor cells are selected from the group consisting of erythroid colony forming units (CFU-E), burst forming unit erythroid progenitors (BFU-E) and proerythroblasts.
在该具体实施方案的优选方案中，其中所述受试者为哺乳动物，例如小鼠、大鼠、人类等。In a preferred version of this specific embodiment, wherein the subject is a mammal, such as a mouse, a rat, a human, or the like.
在该具体实施方案的优选方案中，其中所述方法用于治疗原发性或继发性贫血。In a preferred version of this specific embodiment, wherein said method is for the treatment of primary or secondary anemia.
在该具体实施方案的优选方案中，其中所述贫血为由红细胞生成减少或缺陷引起的贫血。In a preferred version of this particular embodiment, wherein said anemia is anemia caused by decreased or defective erythropoiesis.
在该具体实施方案的优选方案中，其中所述由红细胞生成减少或缺陷引起的贫血为原发性的，包括原发性再生障碍性贫血、Diamond-Blackfan贫血(DBA)、Shwachman-Diamond综合征、先天性角化不良、Fanconi贫血、先天性红细胞生成障碍性贫血(CDA)、地中海型贫血、镰刀状细胞性贫血、骨髓增生异常综合征导致的贫血。In a preferred version of this specific embodiment, wherein said anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome , dyskeratosis congenita, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, anemia caused by myelodysplastic syndrome.
在该具体实施方案的优选方案中，其中所述由红细胞生成减少或缺陷引起的贫血为继发性的，包括继发性骨髓增生异常综合征导致的贫血、继发性再生障碍贫血、维生素缺乏型贫血、缺铁性贫血、慢性炎症性贫血、内分泌疾病性贫血、肾功能衰竭导致EPO分泌不足的继发性贫血、造血干细胞移植后血液谱系重建不良；优选地，其中所述继发性再生障碍贫血由下列原因导致：自身免疫类疾病(如***性红斑狼疮、类风湿性关节炎、炎症性肠病等)、其他免疫机制导致的贫血(如ABO血型不合、干细胞移植、坏疽性脓皮病)、淋巴增生性疾病(如慢性淋巴细胞白血病、LGL白血病、霍奇金病、非霍奇淋巴瘤等)、其他血液***恶性肿瘤(如慢性粒细胞白血病、慢性粒单核细胞白血病)、实体瘤(如胸腺瘤、胃癌、乳腺癌、甲状腺癌、肾癌等)、病毒感染(如B19细小病毒、HIV、T细胞白血病淋巴瘤病毒等)、细菌感染(如结核菌、细菌性败血病)、药物和毒素导致的免疫反应(如外源人EPO诱导的抗体相关的纯红再障、铅/苯中毒)、对内/外源EPO抵抗所造成的贫血及各种原因导致的对***抵抗的贫血。In a preferred version of this specific embodiment, wherein said anemia caused by reduced or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency type anemia, iron deficiency anemia, chronic inflammatory anemia, endocrine disease anemia, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary regeneration Obstacle anemia is caused by the following reasons: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, gangrenous pyoderma, etc.) disease), lymphoproliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, etc.), other hematological malignancies (such as chronic myeloid leukemia, chronic myelomonocytic leukemia), Solid tumors (such as thymoma, gastric cancer, breast cancer, thyroid cancer, kidney cancer, etc.), viral infections (such as B19 parvovirus, HIV, T-cell leukemia lymphoma virus, etc.), bacterial infections (such as Mycobacterium tuberculosis, bacterial sepsis diseases), immune reactions caused by drugs and toxins (such as antibody-related pure red aplastic anemia induced by exogenous human EPO, lead/benzene poisoning), anemia caused by resistance to endogenous/exogenous EPO, and various causes of Erythropoietin-resistant anemia.
在该具体实施方案的优选方案中，其中所述贫血为由于红细胞破坏引起的贫血。In a preferred version of this particular embodiment, wherein said anemia is anemia due to destruction of red blood cells.
在该具体实施方案的优选方案中，其中所述由于红细胞破坏引起的贫血为原发性的，包括遗传性球形红细胞增多症、遗传性椭圆细胞增多症、无β脂蛋白血症、酶缺乏症导致的贫血(如丙酮酸激酶和己糖激酶缺陷导致糖酵解缺陷型贫血、葡萄糖6-磷酸脱氢酶缺乏症和谷胱甘肽合成酶缺乏症、氧化应激增加导致的贫血)。In a preferred version of this specific embodiment, wherein said anemia due to destruction of red blood cells is primary, including hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, enzyme deficiency Anemias resulting from (eg, glycolysis deficient anemia due to pyruvate kinase and hexokinase deficiencies, glucose 6-phosphate dehydrogenase and glutathione synthase deficiencies, anemia due to increased oxidative stress).
在该具体实施方案的优选方案中，其中所述由于红细胞破坏引起的贫血为继发性的，包括抗体介导的温性自身免疫性溶血性贫血、冷凝集素溶血性贫血、溶血性的疾病(如新生儿溶血症)、红细胞机械损伤(如微血管病性溶血性贫血，包括血栓性血小板减少性紫癜和弥散性血管内凝血)、感染导致的溶血性贫血(如疟疾感染)。In a preferred version of this specific embodiment, wherein the anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
在该具体实施方案的优选方案中，其中所述贫血为失血过多引起的贫血。In a preferred version of this specific embodiment, wherein the anemia is anemia caused by excessive blood loss.
在该具体实施方案的优选方案中，其中所述失血过多引起的贫血包括早产儿贫血(如实验室检测的频繁采血，并且任选地合并红细胞生成不足)、外/内伤(各种创伤或手术导致的急性失血)、胃肠道病变导致的急性出血(如静脉曲张病变、消化性溃疡)或慢性失血(如血管发育不良)、妇科疾病导致的慢性失血、癌症(包括结肠直肠癌和膀胱癌导致的急性或慢性失血，尤其是在晚期)、以血液为食的肠道线虫感染(如钩虫和鞭虫导致失血性贫血)、医源性贫血、反复抽血和医疗程序造成的失血。In a preferred version of this specific embodiment, wherein the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood collection as detected by the laboratory, and optionally combined with insufficient erythropoiesis), external/internal injuries (various trauma or Acute blood loss due to surgery), acute bleeding due to gastrointestinal lesions (eg, variceal lesions, peptic ulcer) or chronic blood loss (eg, angiodysplasia), chronic blood loss due to gynecological diseases, cancer (including colorectal cancer and bladder cancer) Acute or chronic blood loss from cancer, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
在该具体实施方案的优选方案中，其中所述疾病对***或糖皮质激素或其他治疗贫血的药物具有耐药性。In a preferred version of this particular embodiment, wherein the disease is resistant to erythropoietin or glucocorticoids or other drugs for the treatment of anemia.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选 自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、BRAF抑制剂1(化合物13)、RAF709、L-779450、LY3009120、Belvarafenib(HM95573)、RO5126766(CH5126766)、TAK-632、PLX-4720、Agerafenib(RXDX-105)、Regorafenib(BAY 73-4506)、Sorafenib(BAY 43-9006)、Donafenib(Sorafenib D3)、Lifirafenib(BGB-283)、BRAF IN1、Vemurafenib(PLX4032)、RAF265(chir265)、AZ 628、AZ304、CCT196969、Doramapimod(BIRB796)、Encorafenib(LGX818)、Naporafenib(LXH254)、BMS-908662、BGB659、GW5074、MLN2480(TAK580)、ARQ-736或ZM336372；In a preferred version of this specific embodiment, wherein said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the αC-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BAY 73-4506), Sorafenib(BAY 43-9006), Donafenib(Sorafenib D3), Lifirafenib(BGB-283), BRAF IN1, Vemurafenib(PLX4032), RAF265( chir265), AZ 628, AZ304, CCT196969, Doramapimod (BIRB796), Encorafenib (LGX818), Naporafenib (LXH254), BMS-908662, BGB659, GW5074, MLN2480 (TAK580), ARQ-736, or ZM336372;
优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、L-779450、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000047
Figure PCTCN2022122562-appb-000047
其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6- C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 - C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、 -SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000048
Figure PCTCN2022122562-appb-000048
IIII
其中，in,
X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
Ar为式a)或b)的基团：Ar is a group of formula a) or b):
Figure PCTCN2022122562-appb-000049
Figure PCTCN2022122562-appb-000049
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
R 15为O或N-OH； R 15 is O or N-OH;
X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂为下式的化合物：In a preferred version of this specific embodiment, wherein the BRAF kinase inhibitor is a compound of the formula:
Figure PCTCN2022122562-appb-000050
Figure PCTCN2022122562-appb-000050
IIIIII
其中，in,
X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
Ar是下式a)或b)：Ar is the following formula a) or b):
Figure PCTCN2022122562-appb-000051
Figure PCTCN2022122562-appb-000051
其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、 C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
在该具体实施方案的优选方案中，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761、Galunisertib(LY2157299)、LY364947、SB431542、LDN-193189、SB525334、SB505124、GW788388、RepSox(E-616452)、K02288、BIBF-0775、TP0427736、A-83-01、LDN-214117、SD-208、Vactosertib(TEW-7197)、LDN-212854、Dorsomorphin(Compound C)或LY3200882；In a preferred version of this specific embodiment, wherein said TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
在该具体实施方案的优选方案中，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Luspatercept、Sotatercept、SIS3HCl、Alantolactone、Halofuginone和AUDA。In the preferred version of this specific embodiment, wherein said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
在该具体实施方案的优选方案中，其中所述BRAF激酶抑制剂以1nM至100μM，优选100nM至10μM的浓度、更优选1μM至10μM的浓度使用。In a preferred version of this particular embodiment, wherein said BRAF kinase inhibitor is used at a concentration of 1 nM to 100 μM, preferably 100 nM to 10 μM, more preferably 1 μM to 10 μM.
在该具体实施方案的优选方案中，其中还同时使用***和/或干细胞因子。In a preferred version of this particular embodiment, erythropoietin and/or stem cell factor are also used concomitantly.
实施例Example
培养基体系：Culture medium system:
1.分化培养基1. Differentiation Medium
分化培养基包括：IMDM、10％的FBS、5％人血清、300μg/ml holo-transferrin(Sigma-Aldrich,St.Louis,MO,USA,Cat T0665)、2IU/ml肝素(Sigma-Aldrich,St.Louis,MO,USA,Cat H3149)、10μg/ml胰岛素(Sigma-Aldrich,St.Louis,MO,USA,Cat I9278)、2mM L-谷氨酰胺；之后补充3IU/ml***(Amgen,Thousand Oaks,CA,Cat 55513-144-10)、50ng/ml人类干细胞因子(StemCell,Vancouver,BC,CA,Cat 78062)和10ng/ml白细胞介素(IL)-3(StemCell,Vancouver,BC,CA,Cat 78042)。The differentiation medium includes: IMDM, 10% FBS, 5% human serum, 300 μg/ml holo-transferrin (Sigma-Aldrich, St.Louis, MO, USA, Cat T0665), 2IU/ml heparin (Sigma-Aldrich, St. .Louis, MO, USA, Cat H3149), 10μg/ml insulin (Sigma-Aldrich, St.Louis, MO, USA, Cat I9278), 2mM L-glutamine; then add 3IU/ml erythropoietin (Amgen , Thousand Oaks, CA, Cat 55513-144-10), 50ng/ml human stem cell factor (StemCell, Vancouver, BC, CA, Cat 78062) and 10ng/ml interleukin (IL)-3 (StemCell, Vancouver, BC , CA, Cat 78042).
2.无血清分化培养基2. Serum-free Differentiation Medium
无血清分化培养基包括：SFEM II(StemCell Technologies,Vancouver,BC,Canada,Cat 09655)，补充3IU/mL***(Amgen,Thousand Oaks,CA,Cat 55513-144-10)、50ng/ml人类干细胞因子(StemCell,Vancouver,BC,CA,Cat 78062)、10ng/ml白细胞介素(IL)-3(StemCell,Vancouver,BC,CA,Cat 78042)和***(IGF)-1(40ng/ml,PeproTech,Rocky Hill,NJ,USA,Cat AF-100-11)。Serum-free differentiation medium includes: SFEM II (StemCell Technologies, Vancouver, BC, Canada, Cat 09655), supplemented with 3IU/mL erythropoietin (Amgen, Thousand Oaks, CA, Cat 55513-144-10), 50ng/ml Human stem cell factor (StemCell, Vancouver, BC, CA, Cat 78062), 10ng/ml interleukin (IL)-3 (StemCell, Vancouver, BC, CA, Cat 78042) and insulin-like growth factor (IGF)-1 ( 40ng/ml, PeproTech, Rocky Hill, NJ, USA, Cat AF-100-11).
3.人造血干细胞/祖细胞无血清扩增培养基3. Human hematopoietic stem/progenitor cell serum-free expansion medium
造血干祖细胞无血清培养基包括：SFEM II(StemCell Technologies,Vancouver,BC,Canada,Cat 09655)，补充有100ng/ml人类干细胞因子(StemCell,Vancouver,BC,CA,Cat 78062)，100ng/ml FLT3-L(StemCell,Vancouver,BC,CA,Cat 78137)，50ng/ml血小板生成素(TPO)(StemCell,Vancouver,BC,CA,Cat 78210)，10μg/ml人低密度脂蛋白(Low density lipoprotein)(StemCell,Vancouver,BC,CA,Cat 02698)。Hematopoietic stem and progenitor cell serum-free medium includes: SFEM II (StemCell Technologies, Vancouver, BC, Canada, Cat 09655), supplemented with 100 ng/ml human stem cell factor (StemCell, Vancouver, BC, CA, Cat 78062), 100 ng/ml FLT3-L (StemCell, Vancouver, BC, CA, Cat 78137), 50ng/ml thrombopoietin (TPO) (StemCell, Vancouver, BC, CA, Cat 78210), 10μg/ml human low density lipoprotein (Low density lipoprotein) ) (StemCell, Vancouver, BC, CA, Cat 02698).
4.CD34 +细胞的分离和红系分化培养 4. Isolation of CD34 + cells and culture of erythroid differentiation
新鲜脐带血与磷酸缓冲液(DPBS)1：1混合，混匀后小心用移液器注入到下层注满淋巴细胞分离剂(Lymphoprep)的50mL SepMate分离管中，在预冷至4℃的离心机1200g离心10min。随后将上层 血清和外周单核细胞转移至新的50mL管中，2000rpm离心10min。吸去上清，每管向下层细胞沉淀中加入10mL红细胞裂解液，在室温裂解十分钟，随后在预冷至4℃的离心机2000rpm离心5min。按照每300000000外周单核细胞加入1mL MACS缓冲液(2％FBS,2mM EDTA in PBS)重悬。随后每毫升细胞悬液加入300μL的Human IgG用于阻断(block)，在室温孵育十分钟后，再按照每毫升细胞悬液加入300μL的CD34 +细胞株(Microbeads)，在4℃孵育30min，孵育期间应间隔一段时间保持悬液混匀。随后每毫升混合液加入14mL的MACS缓冲液，4℃的离心机2000rpm离心5min后，按200000000/mL MACS缓冲液重悬细胞。随后先用3mL MACS缓冲液润洗已经放置于磁力架上的LS磁力柱，后将细胞悬液加入到LS磁力柱内，随后分3次每次加入4mL的MACS缓冲液洗涤磁力柱。完成洗涤后，将LS磁力柱从磁力架上取下放在15mL离心管上，用6mL MACS缓冲液冲洗掉磁力柱上磁附的CD34 +细胞。使用CD34-PE和Lin-APC检测洗脱获得的CD34 +细胞的纯度，确认纯度高于95％后，离心沉淀细胞，使用无血清冻存液冻存细胞。将复苏的CD34 +接种于基于分化培养基中，隔3日更换一次新培养基，12天后可以收集用于分析终末分化的细胞。 Fresh umbilical cord blood was mixed with phosphate buffered solution (DPBS) at a ratio of 1:1, and then carefully poured into a 50mL SepMate separation tube filled with lymphocyte separation agent (Lymphoprep) in the lower layer with a pipette, and centrifuged in a precooled to 4°C Centrifuge at 1200g for 10min. Then the supernatant serum and peripheral mononuclear cells were transferred to a new 50mL tube and centrifuged at 2000rpm for 10min. Aspirate the supernatant, add 10 mL of erythrocyte lysate to each tube to the cell pellet in the lower layer, lyse at room temperature for 10 minutes, and then centrifuge at 2000 rpm for 5 minutes in a centrifuge precooled to 4°C. Add 1mL MACS buffer (2% FBS, 2mM EDTA in PBS) for every 300 million peripheral mononuclear cells to resuspend. Then add 300 μL of Human IgG per ml of cell suspension for blocking (block), incubate at room temperature for ten minutes, then add 300 μL of CD34 + cell line (Microbeads) per ml of cell suspension, and incubate at 4 °C for 30 min, Keep the suspension well mixed at intervals during the incubation period. Then 14 mL of MACS buffer was added to each mL of the mixture, centrifuged in a centrifuge at 2000 rpm for 5 min at 4°C, and the cells were resuspended at 200000000/mL MACS buffer. Then rinse the LS magnetic column that has been placed on the magnetic stand with 3mL MACS buffer, then add the cell suspension to the LS magnetic column, and then add 4mL of MACS buffer three times to wash the magnetic column. After washing, remove the LS magnetic column from the magnetic stand and place it on a 15mL centrifuge tube, and wash away the magnetically attached CD34 + cells on the magnetic column with 6mL MACS buffer. CD34-PE and Lin-APC were used to detect the purity of the eluted CD34 + cells. After confirming that the purity was higher than 95%, the cells were centrifuged and frozen in a serum-free freezing solution. The revived CD34 + were inoculated in the differentiation-based medium, and the new medium was replaced every 3 days. After 12 days, the terminally differentiated cells could be collected for analysis.
5.BRAF抑制剂的体内给药实验5. In vivo administration experiments of BRAF inhibitors
配制BRAF抑制剂溶液：SB-590885用2％Cremophor EL,2％N,N-二甲基乙酰胺,pH 5.0溶解，溶解至30mg/ml。GDC-0879用50％PGE 300和50％PBS溶解，溶解至50mg/ml。Encorafenib用50％PGE 300和50％PBS溶解，溶解至30mg/ml。Prepare BRAF inhibitor solution: Dissolve SB-590885 in 2% Cremophor EL, 2% N,N-dimethylacetamide, pH 5.0, to 30mg/ml. GDC-0879 was dissolved in 50 % PGE 300 and 50% PBS to 50mg/ml. Encorafenib was dissolved in 50 % PGE 300 and 50% PBS to 30mg/ml.
体内给药：SB-590885通过腹腔注射给药，除特别提到外，每日给药一次。GDC-0879和Encorafenib通过管饲法灌胃给药。In vivo administration: SB-590885 is administered by intraperitoneal injection, once a day unless otherwise mentioned. GDC-0879 and Encorafenib were administered by gavage.
诱导急性溶血性贫血：用PBS溶解苯肼，60mg/kg对小鼠进行腹腔注射。To induce acute hemolytic anemia: Dissolve phenylhydrazine in PBS and inject 60 mg/kg intraperitoneally into mice.
小鼠血液组分检测：通过眼眶，每次取血30μL至K2EDTA包被的取血管中。通过迈瑞全自动血液分析仪对血液成分进行检测。Detection of blood components in mice: through the orbit, 30 μL of blood was drawn each time into a K2EDTA-coated blood vessel. Blood components were detected by Mindray automatic blood analyzer.
实施例1：小分子扩增红系前体细胞的高通量筛选Example 1: High-throughput screening of small molecule expansion of erythroid precursor cells
实验前，所有CD34+细胞在添加CC100(StemCell Technologies,Vancouver,BC,CA,Cat 02690)的SFEM II(StemCell Technologies,Vancouver,BC,CA,Cat 09655)中预培养3天，然后进行化合物文库筛选。Before the experiment, all CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 02690) for 3 days, and then screened the compound library.
在第三天时计数，并将包含300个CD34+细胞的200μL分化培养基加入96孔板中，随后向每个孔中加入小分子化合物。此筛选中每种小分子准备3个生物学重复，此后不更换培养基持续培养7天。在第七天时往每个孔中加入40μL Cell 
Figure PCTCN2022122562-appb-000052
Cell Viability试剂，在37℃避光孵育2小时后于BioTek多功能微孔板检测仪检测于560nM及590nM的吸光值，计算每孔560/590的比值，减去背景比值后计算每个实验组相对对照组平均值的比值。 On the third day, counts were made and 200 μL of differentiation medium containing 300 CD34+ cells were added to the 96-well plate, followed by the addition of small molecule compounds to each well. Three biological replicates were prepared for each small molecule in this screening, and culture was continued for 7 days without changing the medium thereafter. Add 40 μL Cell to each well on the seventh day
Figure PCTCN2022122562-appb-000052
Cell Viability reagent, after incubating at 37°C in the dark for 2 hours, detect the absorbance at 560nM and 590nM on the BioTek multi-functional microplate detector, calculate the ratio of 560/590 in each well, subtract the background ratio and calculate for each experimental group Ratio relative to the mean of the control group.
通过高通量筛选测试了122种潜在化学分子，最终得到GDC-0879，其仅在最开始时给药便会在七天时产生超过2倍的细胞扩增。GDC-0879是一种有效的BRAF抑制剂，IC 50为0.13nM。 122 potential chemical molecules were tested through a high-throughput screen, culminating in GDC-0879, which produced more than 2-fold cell expansion at seven days with just the initial administration. GDC-0879 is a potent BRAF inhibitor with IC 50 of 0.13nM.
如图1A所示，为高通量筛选(HTS)的示意图。在Day0时将200个hUCB-CD34+造血干祖细胞种在96孔板的每孔中，每孔补充200μL红系培养基和对应的小分子。每种小分子设置三个生物学重复。在96孔板中培养7天之后通过CellTiter-Glo荧光法进行细胞数量和活力检测。As shown in Fig. 1A, it is a schematic diagram of high-throughput screening (HTS). On Day 0, 200 hUCB-CD34+ hematopoietic stem and progenitor cells were seeded in each well of a 96-well plate, and each well was supplemented with 200 μL of erythroid medium and corresponding small molecules. Three biological replicates were set up for each small molecule. Cell number and viability were detected by CellTiter-Glo fluorescence method after 7 days of culture in 96-well plates.
如图1B所示，在高通量筛选(HTS)的122种潜在化学分子中，仅仅鉴定了BRAF激酶抑制剂GDC-0879为潜在小分子，可以使CD34+细胞在红系分化培养基中7天扩增2倍，而其他分子几乎没有作用。As shown in Figure 1B, among the 122 potential chemical molecules in the high-throughput screening (HTS), only the BRAF kinase inhibitor GDC-0879 was identified as a potential small molecule that can induce CD34+ cells in erythroid differentiation medium for 7 days Amplified 2-fold, while other molecules had little effect.
实施例2-1：探究GDC-0879处理的最佳浓度Example 2-1: Explore the optimal concentration of GDC-0879 treatment
实验前，所有CD34+细胞在添加CC100(StemCell Technologies,Vancouver,BC,CA,Cat 02690)的SFEM II(StemCell Technologies,Vancouver,BC,CA,Cat 09655)中预培养3天。Before the experiment, all CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药，每次换液时保持细胞密度在100000/ml以下。在第九天时往每个孔中加入20％体积的Cell 
Figure PCTCN2022122562-appb-000053
Cell Viability试剂，在37℃避光孵育2小时后于BioTek多功能微孔板检测仪检测于560nM及590nM的吸光值，计算每孔560/590的比值，减去背景比值后计算每个实验组相对对照组平均值的比值。每组数据以平均值±SD表示，N＝3个生物重复。 The cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cell density was kept below 100,000/ml every time the medium was changed. Add 20% volume of Cell to each well on the ninth day
Figure PCTCN2022122562-appb-000053
Cell Viability reagent, after incubating at 37°C in the dark for 2 hours, detect the absorbance at 560nM and 590nM on the BioTek multi-functional microplate detector, calculate the ratio of 560/590 in each well, subtract the background ratio and calculate for each experimental group Ratio relative to the mean of the control group. Each group of data is expressed as mean ± SD, N = 3 biological repeats.
实验使用了浓度为0.1μM、0.5μM、1μM、5μM、10μM和20μM GDC-0879和浓度为0.1μM、1μM的Galunisertib(LY2157299)以及0.1μM的GDC-0879和1μM的Galunisertib的组合。The experiments used concentrations of 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM and 20 μM GDC-0879 and concentrations of 0.1 μM, 1 μM Galunisertib (LY2157299) and combinations of 0.1 μM GDC-0879 and 1 μM Galunisertib.
结果如图2A所示，GDC-0879具有较广的有效浓度范围。在分化培养基中，100nM至10μM的GDC-0879对红系前体细胞的扩增最为有效。GDC-0879的最佳有效浓度在1～10μM范围内，对脐血来源CD34+细胞的红系扩增可达5倍以上。Results As shown in Figure 2A, GDC-0879 has a wide range of effective concentrations. In differentiation medium, GDC-0879 at 100nM to 10μM is most effective for the expansion of erythroid precursor cells. The optimal effective concentration of GDC-0879 is in the range of 1-10μM, and the erythroid expansion of cord blood-derived CD34+ cells can reach more than 5 times.
TGF-β抑制剂之前被报道为潜在的成红细胞刺激剂。Galunisertib(LY2157299)是一种口服的选择性TGF-βI型受体(TGF-βRI)激酶抑制剂，IC50为56nM。GDC-0879联合Galunisertib处理第9天出现约6倍的扩增量。培养基和小分子每3天更换一次。图中，GDC：GDC-0879；Gal：Galunisertib；Dex：***。数据以平均值±SD表示，N＝3个生物重复。TGF-β inhibitors were previously reported as potential erythroblast stimulators. Galunisertib (LY2157299) is an oral, selective TGF-β receptor type I (TGF-βRI) kinase inhibitor with IC50 of 56nM. GDC-0879 combined with Galunisertib showed about 6-fold amplification on the 9th day. Medium and small molecules were changed every 3 days. In the figure, GDC: GDC-0879; Gal: Galunisertib; Dex: dexamethasone. Data are presented as mean ± SD, N = 3 biological replicates.
实施例2-2：探究BRAF抑制剂处理的最佳浓度Example 2-2: Explore the optimal concentration of BRAF inhibitor treatment
实验前，所有CD34+细胞在添加CC100(StemCell Technologies,Vancouver,BC,CA,Cat 02690)的SFEM II(StemCell Technologies,Vancouver,BC,CA,Cat 09655)中预培养3天。Before the experiment, all CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
图2B为BRAF抑制剂仅在起始时单次给药对红系前体细胞扩增的效果。图2B显示，在扩增培养3天后，将包含300个CD34+细胞的200μL分化培养基加入96孔板中，随后向每个孔中加入小分子化合物。此筛选中每种小分子准备3个生物学重复，此后不更换培养基持续培养7天。在第七天时往每个孔中加入40μL
Figure PCTCN2022122562-appb-000054
Cell Viability试剂，在37℃避光孵育2小时后于BioTek多功能微孔板检测仪检测于560nM及590nM的吸光值，计算每孔560/590的比值，减去背景比值后计算每个实验组相对对照组平均值的比值。每组数据以平均值±SD表示，N＝3个生物重复。 Figure 2B shows the effect of a single initial dose of BRAF inhibitor on the expansion of erythroid precursors only. Figure 2B shows that after 3 days of expansion culture, 200 μL of differentiation medium containing 300 CD34+ cells was added to a 96-well plate, and then small molecule compounds were added to each well. Three biological replicates were prepared for each small molecule in this screening, and culture was continued for 7 days without changing the medium thereafter. Add 40 μL to each well on the seventh day
Figure PCTCN2022122562-appb-000054
Cell Viability reagent, after incubating at 37°C in the dark for 2 hours, detect the absorbance at 560nM and 590nM on the BioTek multi-functional microplate detector, calculate the ratio of 560/590 in each well, subtract the background ratio and calculate for each experimental group Ratio relative to the mean of the control group. Each group of data is expressed as mean ± SD, N = 3 biological repeats.
图2B为多种BRAF抑制剂在宽浓度范围对红系前体细胞的扩增效果(仅给药一次)。Figure 2B shows the expansion effect of various BRAF inhibitors on erythroid precursor cells in a wide range of concentrations (administered only once).
具体地，在红系培养Day 0时于96孔板种下300颗hUCB-CD34+造血干祖细胞后，在Day 7时各给药组细胞数量的扩增倍数差异。图2B显示了96孔板检测获得的不同浓度的BRAF抑制剂在Day7时促红系祖细胞增殖的结果。Vemu：Vemurafenib；Enco：Encorafenib；Dabra：Dabrafenib；SB：SB-590885；PLX：PLX-8394；GDC：GDC-0879。统计数据柱状图为平均值±SD，**：P<0.01，***：P<0.001。Specifically, after planting 300 hUCB-CD34+ hematopoietic stem and progenitor cells in a 96-well plate on Day 0 of erythroid culture, the differences in the expansion folds of the number of cells in each administration group on Day 7. Fig. 2B shows the results obtained by the 96-well plate assay that different concentrations of BRAF inhibitors stimulate the proliferation of erythroid progenitor cells on Day 7. Vemu: Vemurafenib; Enco: Encorafenib; Dabra: Dabrafenib; SB: SB-590885; PLX: PLX-8394; GDC: GDC-0879. Statistical data histograms are mean±SD, **: P<0.01, ***: P<0.001.
实验使用了浓度为30nM，100nM，300nM，1000nM，3000nM，10000nM，30000nM的各式BRAF抑制剂。该实验结果说明SB和GDC是较好促进早期红系干祖细胞增殖的BRAF抑制剂。Various BRAF inhibitors with concentrations of 30nM, 100nM, 300nM, 1000nM, 3000nM, 10000nM and 30000nM were used in the experiment. The experimental results indicated that SB and GDC are BRAF inhibitors that can better promote the proliferation of early erythroid stem progenitor cells.
图2C为选定的BRAF抑制剂在宽浓度范围内对红系前体细胞的扩增效果(三次给药)。Figure 2C shows the expansion effect of selected BRAF inhibitors on erythroid precursor cells over a wide range of concentrations (three doses).
在正常的培养体系下，对SB-590885、GDC-0879和Encorafenib三种BRAF抑制剂在30nM-30μM的浓度范围给药，并在第十天进行细胞数量统计。第十天时，在1μM的浓度条件下，三种药物均具备显著的扩增效果。其中SB-590885和GDC-0879可以相对于对照组扩增细胞数量十倍以上，而Encorafenib可以扩增超四倍的细胞数量。Under the normal culture system, three BRAF inhibitors, SB-590885, GDC-0879 and Encorafenib, were administered in the concentration range of 30nM-30μM, and the number of cells was counted on the tenth day. On the tenth day, under the concentration condition of 1 μM, all three drugs had significant amplification effects. Among them, SB-590885 and GDC-0879 can expand the number of cells by more than ten times compared with the control group, while Encorafenib can expand the number of cells by more than four times.
结果如图2C所示，GDC-0879具有较广的有效浓度范围。在分化培养基中，100nM至10μM的 GDC-0879对红系前体细胞的扩增最为有效。GDC-0879的最佳有效浓度在300nM～10μM范围内，对脐血来源CD34+细胞的红系扩增可达5-10倍。The results are shown in Figure 2C, GDC-0879 has a wide effective concentration range. GDC-0879 at 100 nM to 10 μM in differentiation medium is most effective for the expansion of erythroid precursor cells. The optimal effective concentration of GDC-0879 is in the range of 300nM-10μM, and the erythroid expansion of cord blood-derived CD34+ cells can reach 5-10 times.
培养基和小分子每3天更换一次。图中，数据以平均值±SD表示，N＝3个生物重复。Medium and small molecules were changed every 3 days. In the figure, data are presented as mean ± SD, N = 3 biological replicates.
实施例3：成红细胞的生长曲线Example 3: Growth curve of erythroblasts
实验前，所有CD34+细胞在添加CC100(StemCell Technologies,Vancouver,BC,CA,Cat 02690)的SFEM II(StemCell Technologies,Vancouver,BC,CA,Cat 09655)中预培养3天。Before the experiment, all CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药，换液时保持细胞密度在200000/ml以下。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药。在第3、6、9、11、14天时取出每孔的一半细胞进行计数，另一半细胞继续培养。每组数据以平均值±SD表示，N＝3个生物重复。The cells were counted at the beginning of Day 0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day 0, and the cell density was kept below 200,000/ml when changing the medium. The medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. On day 3, 6, 9, 11, and 14, half of the cells in each well were taken out for counting, and the other half of the cells continued to be cultured. Each group of data is expressed as mean ± SD, N = 3 biological repeats.
图3A为BRAF抑制剂对成红细胞的扩增效果。Figure 3A shows the effect of BRAF inhibitors on the expansion of erythroblasts.
在最佳浓度条件下(1μM)，十四天的红系发育生长曲线证明BRAF抑制剂给药均可延长细胞的对数生长时间，使得红系发育过程推迟到更晚的时间点出现细胞数量下降。在对照组发育至红系分化终点(Day 14)时，GDC(21.39倍)和SB(16.99倍)均可以实现超一个数量级的细胞数量扩增，而Enco可以实现约5.21倍的细胞数量扩增。Under the condition of optimal concentration (1μM), the growth curve of erythroid development for fourteen days proves that the administration of BRAF inhibitors can prolong the logarithmic growth time of cells, so that the erythroid development process is delayed until the number of cells appears at a later time point decline. When the control group developed to the end of erythroid differentiation (Day 14), both GDC (21.39 times) and SB (16.99 times) could achieve a cell number expansion of more than one order of magnitude, while Enco could achieve a cell number expansion of about 5.21 times .
图3A为三种小分子在最佳浓度下于红系分化体系中的生长曲线。每个条件设置三个生物学重复，统计数据为平均值±SD，***：P<0.001，****：P<0.0001。Figure 3A is the growth curves of three small molecules in the erythroid differentiation system at the optimal concentration. Three biological replicates were set for each condition, and statistical data are mean±SD, ***: P<0.001, ****: P<0.0001.
图3B为BRAF抑制剂和TGF-β抑制剂协同起效对成红细胞的扩增效果。Fig. 3B shows the synergistic effect of BRAF inhibitors and TGF-β inhibitors on the expansion of erythroblasts.
分化培养基中小分子处理后的成红细胞生长曲线如图3B所示。5μM GDC-0879处理12天使红细胞扩增约10倍。GDC-0879联合Galunisertib给药在第12天使红细胞扩增约12倍。图中，GDC：GDC-0879；Gal：Galunisertib；LY：LY2109761。数据以平均值±SD表示。The growth curve of erythroblasts after small molecule treatment in differentiation medium is shown in Fig. 3B. After 12 days of treatment with 5μM GDC-0879, red blood cells expanded about 10 times. Administration of GDC-0879 in combination with Galunisertib resulted in approximately 12-fold expansion of red blood cells at day 12. In the figure, GDC: GDC-0879; Gal: Galunisertib; LY: LY2109761. Data are presented as mean ± SD.
实施例4：GDC-0879在分化培养基中对分化(基于FACS)的影响Example 4: Effect of GDC-0879 on Differentiation (FACS Based) in Differentiation Medium
实验前，所有CD34+细胞在添加CC100(StemCell Technologies,Vancouver,BC,CA,Cat 02690)的SFEM II(StemCell Technologies,Vancouver,BC,CA,Cat 09655)中预培养3天。Before the experiment, all CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药。总共给药共9天，在第9天后不再给药处理。The cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. The medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. A total of 9 days were administered, and no treatment was administered after the 9th day.
在每次换液时取出一半细胞用于流式细胞术检测，在第六天和第十二天时，重悬细胞并过滤，对细胞进行计数和细胞密度分析，以约1000000/mL细胞重悬于PBS中，按照1：100加入Mouse IgG于室温封闭细胞10min，随后再按照1：200的比例加入CD235a-APC，CD71-FITC，10μg/mL Hoechst 33342在37℃染色孵育30min。完成后，使用BD Fortessa进行流式分析，随后用FlowJo对FCS文件进行数据处理。Take out half of the cells at each medium change for flow cytometry detection. On the sixth and twelfth days, resuspend the cells and filter, count the cells and analyze the cell density. Suspend in PBS, add Mouse IgG at a ratio of 1:100 to block the cells at room temperature for 10 min, then add CD235a-APC, CD71-FITC, and 10 μg/mL Hoechst 33342 at a ratio of 1:200 and incubate at 37°C for 30 min. Once complete, flow analysis was performed using BD Fortessa, followed by data processing of the FCS files with FlowJo.
图4A的FACS结果显示第6天不同浓度的GDC-0879对分化培养基中红细胞发育及红系表面标记物CD235a(Glycophorin A)表达的影响(小分子处理6天)。The FACS results in Figure 4A show the effects of different concentrations of GDC-0879 on the development of erythrocytes and the expression of the erythroid surface marker CD235a (Glycophorin A) in the differentiation medium on day 6 (small molecule treatment for 6 days).
图4B的FACS结果显示，第6天不同浓度GDC-0879对分化培养基中红细胞发育及红系表面标记物CD235a(Glycophorin A)表达的影响(小分子处理6天)。其中1μM GDC-0879处理组中主要细胞类群仍为CD235a阴性，而对照组主要细胞类群为CD235a阳性，即GDC-0879延缓了红细胞的终末期分化进程，使成红细胞有了更多的自我更新。The FACS results in Figure 4B show the effects of different concentrations of GDC-0879 on the development of erythrocytes and the expression of the erythroid surface marker CD235a (Glycophorin A) in the differentiation medium on day 6 (small molecule treatment for 6 days). Among them, the main cell group in the 1 μM GDC-0879 treatment group was still CD235a-negative, while the main cell group in the control group was CD235a-positive, that is, GDC-0879 delayed the terminal differentiation process of erythrocytes, allowing more self-renewal of erythroblasts.
图4C显示了第12天分化培养基中不同浓度的GDC-0879对红细胞发育和红系表面标记物 CD235a(Glycophorin A)表达的影响(小分子处理6天)。对照组在第12天CD71表达下降直至红细胞成熟。此时处理组仍有CD71高表达细胞类群，说明GDC-0879处理组红细胞分化延迟。Figure 4C shows the effects of different concentrations of GDC-0879 in the differentiation medium on day 12 on the development of erythrocytes and the expression of the erythroid surface marker CD235a (Glycophorin A) (small molecule treatment for 6 days). In the control group, CD71 expression decreased on day 12 until erythrocytes matured. At this time, the treatment group still had high CD71 expression cell population, indicating that the red blood cell differentiation in the GDC-0879 treatment group was delayed.
图4D可以看出，GDC-0879处理组在第12天红细胞脱核率较对照组略有下降(小分子处理9天)。但如下图9D所示，脱核期延长，脱核细胞数不变，故GDC-0879处理对脱核率没有直接的负面影响。It can be seen from Figure 4D that the denucleation rate of erythrocytes in the GDC-0879 treatment group was slightly lower than that in the control group on day 12 (small molecule treatment for 9 days). However, as shown in Figure 9D below, the enucleation period was prolonged and the number of enucleated cells remained unchanged, so GDC-0879 treatment had no direct negative effect on the enucleation rate.
图4E可以看出，GDC-0879处理组会略微降低脐带血来源的CD34+细胞的脱核比例，但这个降低可能是由于分化的延迟导致的脱核比例下降。It can be seen from Figure 4E that the GDC-0879 treatment group will slightly reduce the denucleation ratio of CD34+ cells derived from umbilical cord blood, but this decrease may be due to the decrease in the denucleation ratio caused by the delay in differentiation.
基于以上的结果可证明，BRAF抑制剂给药在红系分化培养过程中延迟了细胞的分化，维持了更高比例的具备更强增殖潜能的早期红系干祖细胞。同时，在终末分化上仅呈现延迟分化，并未阻滞红系分化进行。在终末阶段呈现出一定比例的脱核比例下降。但一方面，这一脱核比例下降和分化延迟有关；另一方面，脱核比例下降有限，BRAF抑制剂对脱核的影响远低于其对细胞数量的扩增效果。Based on the above results, it can be proved that administration of BRAF inhibitors delayed cell differentiation during erythroid differentiation culture and maintained a higher proportion of early erythroid stem progenitor cells with stronger proliferation potential. At the same time, terminal differentiation only showed delayed differentiation, and did not block erythroid differentiation. In the terminal stage, a certain proportion of denucleation ratio decreased. But on the one hand, this decrease in the proportion of denucleation is related to the delay in differentiation; on the other hand, the decrease in the proportion of denucleation is limited, and the effect of BRAF inhibitors on denucleation is much lower than its effect on the expansion of cell number.
实施例5：集落形成结果Example 5: Colony Formation Results
图5A为BRAF抑制剂促进PBMC来源的集落形成。Figure 5A shows that BRAF inhibitors promote PBMC-derived colony formation.
在实验前一天复苏健康供者的PBMC，并在分化培养基中过夜，在第一天时按照每孔(35mm)100000个PBMC细胞接种于含髓系细胞发育必须的细胞因子的甲基纤维素培养基Methocult H4435(StemCell,Vancouver,BC,CA,Cat H4435)中，对给药组药物预混均匀，于培养箱中培养14天。在14天时使用Leica倒置相差显微镜对集落进行拍照，计数和面积统计。Resuscitate PBMC from a healthy donor one day before the experiment, and overnight in the differentiation medium. On the first day, 100,000 PBMC cells per well (35 mm) are seeded in methylcellulose containing cytokines necessary for the development of myeloid cells In the medium Methocult H4435 (StemCell, Vancouver, BC, CA, Cat H4435), the medicines of the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed, counted and area counted using a Leica inverted phase-contrast microscope at 14 days.
图5A为在Methocult H4435不同给药组和对照组的红细胞集落数量(中)与单个红细胞集落面积(右)的统计。最左图相片中，左，比例尺＝10mm；图右，比例尺＝500μm。GDC：GDC-0879；Dex：***。每个条件设置3个生物学重复，统计数据为平均值±SD；ns：无显著性差异，P>0.05；****：P<0.0001。Figure 5A is the statistics of the number of erythrocyte colonies (middle) and the area of a single erythrocyte colony (right) in different administration groups of Methocult H4435 and the control group. In the photo of the leftmost picture, left, scale bar=10 mm; right picture, scale bar=500 μm. GDC: GDC-0879; Dex: Dexamethasone. Three biological repetitions were set for each condition, and statistical data are mean ± SD; ns: no significant difference, P>0.05; ****: P<0.0001.
图5B为BRAF抑制剂促进脐带血来源CD34+细胞的红系生成(EPO only培养基)。Figure 5B shows that BRAF inhibitors promote erythropoiesis of CD34+ cells derived from umbilical cord blood (EPO only medium).
在实验前一天复苏CD34+细胞，并在分化培养基中过夜，在第一天时按照每孔(35mm)600个CD34+细胞接种于仅含EPO的甲基纤维素培养基Methocult H4330(StemCell,Vancouver,BC,CA,Cat H4330)中，对给药组药物预混均匀，于培养箱中培养14天。在14天时使用Leica倒置相差显微镜对集落进行拍照。The CD34+ cells were revived one day before the experiment, and overnight in the differentiation medium. On the first day, 600 CD34+ cells per well (35 mm) were inoculated in the methylcellulose medium Methocult H4330 containing only EPO (StemCell, Vancouver, BC, CA, Cat H4330), the drugs in the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed at 14 days using a Leica inverted phase contrast microscope.
图5B显示了第14天红细胞特异性集落形成结果。其中将600个CD34+细胞在仅含有EPO的红系分化培养基中预培养过夜，第二天将其培养至35mm孔中仅含EPO的甲基纤维素培养基中，并在培养的第14天拍摄培养基照片。对照组肉眼和图片上均看不到大的BFU-E集落，而GDC-0879处理组肉眼可见大的BFU-E集落。Figure 5B shows the results of erythrocyte-specific colony formation at day 14. Among them, 600 CD34+ cells were pre-cultured overnight in erythroid differentiation medium containing only EPO, and cultured in 35 mm wells in methylcellulose medium containing only EPO the next day, and on the 14th day of culture Take photos of the medium. In the control group, no large BFU-E colonies could be seen with naked eyes and pictures, but large BFU-E colonies could be seen with the naked eye in the GDC-0879 treatment group.
图5C显示了在亮视野显微镜下使用5×物镜检测第14天红细胞特异性集落形成结果，处理组与对照组在集落面积和每个集落的细胞数上均有明显差异。比例尺，500μm。Figure 5C shows the results of red blood cell-specific colony formation on day 14 detected under a bright-field microscope using a 5× objective lens. There were significant differences in the colony area and the number of cells per colony between the treatment group and the control group. Scale bar, 500 μm.
图5D为BRAF抑制剂促进脐带血来源CD34+细胞的红系生成和各谱系发育(髓系培养基)。Figure 5D shows that BRAF inhibitors promote erythropoiesis and lineage development of cord blood-derived CD34+ cells (myeloid medium).
在实验前一天复苏CD34+细胞，并在造血干细胞维持培养基中过夜，在第一天时按照每孔(35mm)200个CD34+细胞接种于含髓系发育各谱系所需细胞因子的甲基纤维素培养基Methocult H4435(StemCell,Vancouver,BC,CA,Cat H4435)中，对给药组药物预混均匀，于培养箱中培养14天。在14天时使用Leica倒置相差显微镜对集落进行拍照。值得注意的是，BRAF抑制剂并未导致显著的造血谱系偏移(图D)。The CD34+ cells were revived one day before the experiment, and kept overnight in the hematopoietic stem cell maintenance medium. On the first day, 200 CD34+ cells per well (35mm) were inoculated in methylcellulose containing cytokines required for each lineage of myeloid development In the medium Methocult H4435 (StemCell, Vancouver, BC, CA, Cat H4435), the medicines of the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed at 14 days using a Leica inverted phase contrast microscope. Notably, BRAF inhibitors did not result in a significant hematopoietic lineage shift (Panel D).
A：起始(Day 0)在处理组和对照组培养基上均接种100个CD34+细胞，然后在第十四天用PBS洗脱下并对每孔进行计数的结果。B：每个孔中红细胞(Non CD11b+)的数量。C：每个孔中粒细胞(CD11b+)的占比。D：在Methocult H4435上对UCB-hCD34+给药，GDC和对照组的长出的集落数 量统计。E：在Methocult H4435上每组三个重复孔的示意图。GDC，GDC-0879。比例尺＝10mm。每个条件设置3个生物学重复，统计数据为平均值±SD，ns：无显著性差异，P>0.05；*：P<0.05；**：P<0.01。A: At the beginning (Day 0), 100 CD34+ cells were inoculated on the culture medium of the treatment group and the control group, and then eluted with PBS on the fourteenth day and counted for each well. B: Number of red blood cells (Non CD11b+) in each well. C: The proportion of granulocytes (CD11b+) in each well. D: On Methocult H4435, UCB-hCD34+ was administered, and the number of colonies grown from GDC and the control group was counted. E: Schematic representation of each set of triplicate wells on Methocult H4435. GDC, GDC-0879. Scale bar = 10mm. Three biological replicates were set for each condition, and statistical data were mean ± SD, ns: no significant difference, P>0.05; *: P<0.05; **: P<0.01.
以上的集落形成实验结果证明，仅一次的BRAF抑制剂给药足以极大促进红系的增殖(单个克隆面积和总细胞数量)。且在仅含EPO的培养基中更明显的促进了红系集落的形成。此外，本实施例证明BRAF抑制剂对红系集落的扩增效果明显强于粒细胞集落，显示出BRAF抑制剂对不同谱系的扩增存在差异。The results of the above colony formation experiments prove that only one administration of BRAF inhibitor is enough to greatly promote the proliferation of erythroid (individual colony area and total cell number). And the formation of erythroid colonies was more obviously promoted in the medium containing only EPO. In addition, this example proves that the effect of BRAF inhibitors on the expansion of erythroid colonies is significantly stronger than that of granulocyte colonies, showing that there are differences in the expansion of BRAF inhibitors on different lineages.
实施例6：BRAF靶点的小分子促进红系前体细胞增殖的效果Example 6: The effect of small molecules targeting BRAF on promoting the proliferation of erythroid precursor cells
实施例6-1：相同靶点的小分子试验结果Example 6-1: Small molecule test results for the same target
实验前，所有CD34+细胞在添加CC100(StemCell Technologies,Vancouver,BC,CA,Cat 02690)的SFEM II(StemCell Technologies,Vancouver,BC,CA,Cat 09655)中预培养3天。Before the experiment, all CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药。总共给药共9天，在第9天后不再给药处理。The cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. The medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. A total of 9 days were administered, and no treatment was administered after the 9th day.
在第10天时往每个孔中加入20％体积的Cell
Figure PCTCN2022122562-appb-000055
Cell Viability试剂，在37℃避光孵育2小时后于BioTek多功能微孔板检测仪检测于560nM及590nM的吸光值，计算每孔560/590的比值，减去背景比值后计算每个实验组相对对照组平均值的比值。每组数据以平均值±SD表示，N＝3个生物重复。 On day 10, add 20% volume of Cell to each well
Figure PCTCN2022122562-appb-000055
Cell Viability reagent, after incubating at 37°C in the dark for 2 hours, detect the absorbance at 560nM and 590nM on the BioTek multi-functional microplate detector, calculate the ratio of 560/590 in each well, subtract the background ratio and calculate for each experimental group Ratio relative to the mean of the control group. Each group of data is expressed as mean ± SD, N = 3 biological repeats.
图6A显示了检测靶向BRAF的其他同靶点小分子的结果(未特别标注的小分子浓度均为1μM，处理9天)。其中1μM的Encorafenib和Dabrafenib联合处理组在第10天红细胞扩增数超过3倍。另一种高选择性BRAF抑制剂SB-590885的扩增能力(第8天为8倍)略微高于GDC-0879(第10天超过6倍)，虽然SB-590885对BRAF的IC 50为0.16nM，而GDC-0879的IC 50为0.13nM，但总体而言，SB和GDC同属Type 1型抑制剂，同样诱导BRAF的生理激活构象，其有相似的促增殖结果也可以理解。该结果说明BRAF抑制剂对细胞的扩增效果以及BRAF抑制剂的矛盾激活是普遍的。以上数据以平均值±SD表示，N＝3个生物重复。 Figure 6A shows the results of detection of other small molecules targeting BRAF with the same target (the concentration of small molecules not specifically marked is 1 μM, treated for 9 days). Among them, the combined treatment group of 1 μM Encorafenib and Dabrafenib had more than 3 times the number of red blood cell expansion on the 10th day. Another highly selective BRAF inhibitor, SB-590885, had a slightly higher amplification capacity (8-fold at day 8) than GDC-0879 (more than 6-fold at day 10), although the IC50 of SB-590885 for BRAF was 0.16 nM, while the IC 50 of GDC-0879 is 0.13nM, but in general, SB and GDC are both Type 1 inhibitors, and they also induce the physiologically activated conformation of BRAF, and it is understandable that they have similar pro-proliferation results. This result illustrates that the expansion effect of BRAF inhibitors on cells and the paradoxical activation of BRAF inhibitors are general. The above data are expressed as mean ± SD, N = 3 biological replicates.
实施例6-2：BRAF靶点的小分子促进红系前体细胞增殖的效果Example 6-2: The effect of small molecules targeting BRAF on promoting the proliferation of erythroid precursor cells
在实验前一天复苏CD34+细胞，并在分化培养基中过夜，在第一天时按照每孔(22mm)300个CD34+细胞接种于仅含EPO的甲基纤维素培养基Methocult H4330(StemCell Technologies,Vancouver,BC,CA,Cat H4330)中，对给药组药物预混均匀，于培养箱中培养14天。在14天时使用Leica倒置相差显微镜对集落进行拍照和数量统计。The CD34+ cells were revived one day before the experiment, and overnight in the differentiation medium. On the first day, 300 CD34+ cells per well (22mm) were inoculated in EPO-only methylcellulose medium Methocult H4330 (StemCell Technologies, Vancouver) , BC, CA, Cat H4330), the drugs in the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed and counted using a Leica inverted phase-contrast microscope at 14 days.
图6B显示BRAF的其他抑制剂在仅含EPO的甲基纤维素培养基中第14天的红细胞特异性集落形成结果。其中300个CD34+细胞在添加了CC100的SFEM II(StemCell Technologies,Vancouver,BC,CA,Cat 09655)中预培养过夜，然后在第二天将其置于35mm孔中，并在第14天拍摄培养基照片。每个处理组2个重复。图中Gal：2μM Galunisertib处理；Enco：5μM Encorafenib处理；Dabr：5μM Dabrafenib处理；GDC：5μM GDC-0879处理；SB：5μM SB-590885处理；Ctrl：对照(DMSO)。Figure 6B shows the results of erythrocyte-specific colony formation on day 14 in EPO-only methylcellulose medium with other inhibitors of BRAF. Among them, 300 CD34+ cells were pre-cultured overnight in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100, and then placed in 35mm wells the next day, and cultured on the 14th day base photo. 2 replicates per treatment group. In the figure, Gal: treated with 2 μM Galunisertib; Enco: treated with 5 μM Encorafenib; Dabr: treated with 5 μM Dabrafenib; GDC: treated with 5 μM GDC-0879; SB: treated with 5 μM SB-590885; Ctrl: control (DMSO).
图6C显示在仅含EPO的甲基纤维素培养基中，含BRAF抑制剂的其他组合在第14天的红细胞特异性集落形成结果。其中300个CD34+细胞在添加了CC100的SFEM II(StemCell Technologies,Vancouver,BC,CA,Cat 09655)中预培养过夜，然后在第二天将其置于35mm孔中，并在第14天拍摄培养基照片。每个处理组2/3个重复。图中GDC Gal：2μM galunisertib和5μM GDC-0879处理；SB  Gal：2μM galunisertib和5μM SB-590885处理；Ctrl：对照。Figure 6C shows the results of erythrocyte-specific colony formation at day 14 for other combinations containing BRAF inhibitors in EPO-only methylcellulose media. Among them, 300 CD34+ cells were pre-cultured overnight in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100, and then placed in 35mm wells the next day, and cultured on the 14th day base photo. 2/3 replicates per treatment group. In the figure, GDC Gal: treated with 2 μM galunisertib and 5 μM GDC-0879; SB Gal: treated with 2 μM galunisertib and 5 μM SB-590885; Ctrl: control.
图6D显示了光镜视野显微镜下集落形成结果，用5×物镜下检测第14天红细胞特异性集落形成结果。在显微镜下，图6B和6C中红细胞最大集落见图D。处理组与对照组的集落面积和每个集落的细胞数有明显差异。图中Gal：2μM Galunisertib处理；Enco：5μM Encorafenib处理；Dabr：5μM Dabrafenib处理；GDC：5μM GDC-0879处理；SB：5μM SB-590885处理；Gal+GDC：2μM Galunisertib和5μM GDC-0879处理；Gal+SB：2μM Galunisertib和5μM SB-590885处理；Ctrl：对照。比例尺，500μm。Figure 6D shows the colony formation results under the light field microscope, and the erythrocyte-specific colony formation results on day 14 were detected under the 5× objective lens. Under the microscope, the largest colony of erythrocytes in Figures 6B and 6C is shown in Figure D. The colony area and the number of cells per colony were significantly different between the treatment group and the control group. In the figure, Gal: treated with 2μM Galunisertib; Enco: treated with 5μM Encorafenib; Dabr: treated with 5μM Dabrafenib; GDC: treated with 5μM GDC-0879; SB: treated with 5μM SB-590885; +SB: 2 μM Galunisertib and 5 μM SB-590885 treatment; Ctrl: control. Scale bar, 500 μm.
图6E显示了图6B、6C各孔红细胞集落形成单位计数结果，数据以平均值±SD表示，处理组N＝2个生物重复(对照组4个)。Gal：2μM Galunisertib处理；Enco：5μM Encorafenib处理；Dabr：5μM Dabrafenib处理；GDC：5μM GDC-0879处理；SB：5μM SB-590885处理；Gal+GDC：2μM Galunisertib和5μM GDC-0879处理；Gal+SB：2μM Galunisertib和5μM SB-590885处理；Ctrl：对照(DMSO)。Figure 6E shows the counting results of erythrocyte colony forming units in each well of Figures 6B and 6C, and the data are expressed as mean ± SD, and N=2 biological replicates in the treatment group (4 in the control group). Gal: treated with 2 μM Galunisertib; Enco: treated with 5 μM Encorafenib; Dabr: treated with 5 μM Dabrafenib; GDC: treated with 5 μM GDC-0879; SB: treated with 5 μM SB-590885; Gal+GDC: treated with 2 μM Galunisertib and 5 μM GDC-0879; : 2 μM Galunisertib and 5 μM SB-590885 treatment; Ctrl: control (DMSO).
以上的实施例证明，BRAF抑制剂普遍具有矛盾激活效果，且在集落形成能力中具备和其他小分子协同促进红系增殖的潜能。The above examples prove that BRAF inhibitors generally have paradoxical activation effects, and have the potential to promote erythroid proliferation synergistically with other small molecules in the colony formation ability.
实施例7：有效的给药周期试验Embodiment 7: effective dosing cycle test
实施例7-1：有效的给药周期试验结果1Embodiment 7-1: Effective administration cycle test result 1
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药。不同给药时间组分别给药0-3天、3-6天、6-9天和9-12天。The cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. The medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. Different administration time groups were administered for 0-3 days, 3-6 days, 6-9 days and 9-12 days respectively.
在第12和第13天时往每个孔中加入20％体积的Cell
Figure PCTCN2022122562-appb-000056
Cell Viability试剂，在37℃避光孵育2小时后于BioTek多功能微孔板检测仪检测于560nM及590nM的吸光值，计算每孔560/590的比值，减去背景比值后计算每个实验组相对对照组平均值的比值。每组数据以平均值±SD表示，N＝3/6个生物重复。 On the 12th and 13th day, add 20% volume of Cell to each well
Figure PCTCN2022122562-appb-000056
Cell Viability reagent, after incubating at 37°C in the dark for 2 hours, detect the absorbance at 560nM and 590nM on the BioTek multi-functional microplate detector, calculate the ratio of 560/590 in each well, subtract the background ratio and calculate for each experimental group Ratio relative to the mean of the control group. Each group of data is expressed as mean ± SD, N = 3/6 biological repeats.
图7A显示了GDC-0879从第0天到第12天不同周期给药，并在第十五天进行最后细胞数量统计的试验结果，以3天为给药周期，探讨持续给药的药物依赖效应。图7B显示了0-3天处理组与对照组第13天细胞数比较。在分化培养基中处理红细胞不同周期，6-9天的处理组扩增能力最强。该图证明了GDC-0879扩增的持续效应。单剂量处理组细胞数量也明显增加，说明GDC-0879扩增了所有阶段的红系前体细胞。但这一结果提示，该药物在红系发育的后期(增殖下降期)有更明显的作用。图7B右图显示，仅0-3天1μM GDC-0879处理组在第13天细胞数量增加近2倍。数据以平均值±SD表示，N＝3/6个生物重复。Figure 7A shows the test results of GDC-0879 administered in different cycles from the 0th day to the 12th day, and the final cell count was counted on the 15th day, with 3 days as the administration period, to explore the drug dependence of continuous administration effect. Figure 7B shows the comparison of the number of cells in the 0-3 day treatment group and the control group on day 13. In the different cycles of red blood cells treated in the differentiation medium, the expansion ability of the 6-9 day treatment group was the strongest. This figure demonstrates the persistent effect of GDC-0879 expansion. The number of cells in the single-dose treatment group also increased significantly, indicating that GDC-0879 expanded erythroid precursor cells at all stages. However, this result suggests that the drug has a more pronounced effect in the later stages of erythroid development (declining proliferation). The right panel of Figure 7B shows that only 0-3 days of 1 μM GDC-0879 treatment group increased the number of cells by nearly 2 times on day 13. Data are presented as mean ± SD, N = 3/6 biological replicates.
以上实施例的结果说明，BRAF抑制剂的促增殖作用是时间依赖的，且在相对靠后期(即正常发育过程中增殖能力下降的时期)中其起效更加明显(尤其day 6到day 9中)。但即便如此，在早期的给药(图7B)依然可以使得细胞获得更强的增殖。The results of the above examples show that the proliferation-promoting effect of BRAF inhibitors is time-dependent, and its onset is more obvious (especially in day 6 to day 9) in the relatively late stage (that is, the period when the proliferative ability decreases in the normal development process). ). But even so, administration in the early stage (Fig. 7B) can still make cells proliferate stronger.
实施例7-2：有效的给药周期试验结果2Embodiment 7-2: Effective administration cycle test result 2
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药。给药3天，在第3天后不在给药处理。The cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. The medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. Dosing for 3 days, after the 3rd day, no more dosing treatment.
在每次换液时取出一半细胞用于流式细胞术检测，在第13天时，重悬细胞并过滤，对细胞进行计数和细胞密度分析，以约1000000/mL细胞重悬于PBS中，按照1：100加入小鼠IgG于室温封闭 细胞10min，随后再按照1：200的比例加入CD235a-APC，CD71-FITC，10μg/mL Hoechst 33342在37℃染色孵育30min。完成后，使用BD Fortessa进行流式分析，随后用FlowJo对FCS文件进行数据处理。Take out half of the cells at each medium change for flow cytometry detection. On the 13th day, resuspend the cells and filter, count the cells and analyze the cell density. Resuspend the cells in PBS at about 1,000,000/mL. Mouse IgG was added at a ratio of 1:100 to block the cells at room temperature for 10 min, and then CD235a-APC, CD71-FITC, and 10 μg/mL Hoechst 33342 were added at a ratio of 1:200 and incubated at 37°C for 30 min. Once complete, flow analysis was performed using BD Fortessa, followed by data processing of the FCS files with FlowJo.
图7C显示了实验组与对照组在分化培养基中第13天FACS结果。处理组从第0～3天给药。由FACS图可知，0～3天1μM GDC-0879处理组在第13天的分化率几乎与对照组相同，甚至有更高的脱核率。处理组，1μM GDC-0879仅在第1天至第3天给药。这说明早期的给药依旧促进增殖，但完全不影响分化，甚至促进终末成熟和脱核。Fig. 7C shows the FACS results of the experimental group and the control group on day 13 in the differentiation medium. The treatment group was administered from the 0th to the 3rd day. It can be seen from the FACS graph that the differentiation rate of the 1μM GDC-0879 treatment group for 0-3 days was almost the same as that of the control group on the 13th day, and even had a higher enucleation rate. In the treatment group, 1 μM GDC-0879 was administered only from day 1 to day 3. This indicated that early drug administration still promoted proliferation, but did not affect differentiation at all, and even promoted terminal maturation and denucleation.
实施例7-3：有效的给药周期试验结果3Embodiment 7-3: Effective administration cycle test result 3
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药，换液时保持细胞密度在100000/ml以下。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药。不同给药时间组分别给药0-3天、3-6天、0-6天、3-9天、6-9天。The cells were counted at the beginning of Day 0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day 0, and the cell density was kept below 100,000/ml when changing the medium. The medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. Different administration time groups were administered for 0-3 days, 3-6 days, 0-6 days, 3-9 days and 6-9 days respectively.
在每次换液时取出一半细胞用于流式细胞术检测，在第13-16天时，重悬细胞并过滤，对细胞进行计数和细胞密度分析，以约1000000/mL细胞重悬于PBS中，按照1：100加入小鼠IgG于室温封闭细胞10min，随后再按照1：200的比例加入CD235a-APC，CD71-FITC，10μg/mL Hoechst 33342在37℃染色孵育30min。完成后，使用BD Fortessa进行流式分析，随后用FlowJo对FCS文件进行数据处理，计算CD235a-APC+和Hoechst 33342-的细胞群的比例即为脱核细胞比例。Take out half of the cells at each medium change for flow cytometry detection. On day 13-16, resuspend the cells and filter, count the cells and analyze the cell density, resuspend in PBS at about 1,000,000/mL cells , add mouse IgG at a ratio of 1:100 to block the cells at room temperature for 10 min, then add CD235a-APC, CD71-FITC, and 10 μg/mL Hoechst 33342 at a ratio of 1:200 and incubate for 30 min at 37°C for staining. After completion, use BD Fortessa for flow analysis, and then use FlowJo for data processing of the FCS file, and calculate the ratio of CD235a-APC+ and Hoechst 33342- cell populations as the ratio of denucleated cells.
图7D显示第13～16天对各给药3天的各组进行脱核率统计(处理组，1μM GDC-0879处理组)。从图中可以看出，GDC-0879并不直接影响脱核过程和红细胞的终末成熟。纵坐标为细胞百分比。Figure 7D shows statistics on the enucleation rate of each group administered for 3 days from day 13 to day 16 (treatment group, 1 μM GDC-0879 treatment group). It can be seen from the figure that GDC-0879 does not directly affect the process of denucleation and terminal maturation of erythrocytes. The vertical axis is the percentage of cells.
图7E显示第13～16天对两个给药期(连续6天)的各组进行脱核率统计(处理组：1μM GDC-0879处理)。从图中可以看出，GDC-0879并不直接影响脱核过程和红细胞的终末成熟。纵坐标为细胞百分比。Figure 7E shows statistics on the enucleation rate of each group in the two administration periods (6 consecutive days) from day 13 to day 16 (treatment group: 1 μM GDC-0879 treatment). It can be seen from the figure that GDC-0879 does not directly affect the process of denucleation and terminal maturation of erythrocytes. The vertical axis is the percentage of cells.
实施例8：体外扩增PBMC中的成红细胞Example 8: Expansion of erythroblasts in PBMCs in vitro
实施例8-1：在无血清分化培养基中体外扩增来自健康供体PBMC的成红细胞Example 8-1: In vitro expansion of erythroblasts from healthy donor PBMCs in serum-free differentiation medium
在起始Day0时对细胞进行计数，在每孔的无血清分化培养基中接种500000个来自健康捐献者(Healthy Donor 1-Healthy Donor 5,HD1-HD5)的PBMC细胞，并在Day0时按照相应的浓度进行给药。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药，给药第九天后对不同给药组进行离心并对细胞沉淀进行拍照The cells were counted at the start of Day 0, and 500,000 PBMC cells from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) were inoculated in each well of serum-free differentiation medium, and at Day 0, according to the corresponding concentration for administration. The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced. After the ninth day of administration, the different administration groups were centrifuged and the cell pellets were photographed.
图8A显示第9天处理组和对照组的细胞。健康捐献者(Healthy Donor 1-Healthy Donor 5,HD1-HD5)的PBMC在无血清红细胞扩增培养基(SFEM II(StemCell Technologies,Vancouver,BC,Canada,Cat 09655)中培养9天，添加3IU/ml***(Amgen,Thousand Oaks,CA,Cat 55513-144-10)、50ng/ml人干细胞因子(StemCell,Vancouver,BC,CA,Cat 78062)、10ng/ml白细胞介素(IL)-3(StemCell，Vancouver BC CA Cat 78042)和***(IGF)-1(40ng/mL,PeproTech,Rocky Hill,NJ,USA,Cat AF-100-11))。经1μM SB-590885处理后，处理组细胞明显多于对照组。Figure 8A shows the cells of the treated and control groups at day 9. PBMCs from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) were cultured in serum-free erythrocyte expansion medium (SFEM II (StemCell Technologies, Vancouver, BC, Canada, Cat 09655)) for 9 days, and 3 IU/ ml erythropoietin (Amgen, Thousand Oaks, CA, Cat 55513-144-10), 50ng/ml human stem cell factor (StemCell, Vancouver, BC, CA, Cat 78062), 10ng/ml interleukin (IL)- 3 (StemCell, Vancouver BC CA Cat 78042) and insulin-like growth factor (IGF)-1 (40ng/mL, PeproTech, Rocky Hill, NJ, USA, Cat AF-100-11)). After treatment with 1 μM SB-590885, the cells in the treatment group were significantly more than those in the control group.
实施例8-2：体外扩增PBMC中的成红细胞结果1Example 8-2: In vitro expansion of erythroblasts in PBMC Result 1
在起始Day0时对细胞进行计数，在每孔的无血清分化培养基中接种500000个来自健康捐献者(Healthy Donor 1-Healthy Donor 5,HD1-HD5)的PBMC细胞，并在Day0时按照相应的浓度进行给药。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药，在给药第九天时对细胞数量进行 统计。The cells were counted at the start of Day 0, and 500,000 PBMC cells from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) were inoculated in each well of serum-free differentiation medium, and at Day 0, according to the corresponding concentration for administration. The culture medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the number of cells was counted on the ninth day of administration.
在第9天时往每个孔中加入20％体积的Cell 
Figure PCTCN2022122562-appb-000057
Cell Viability试剂，在37℃避光孵育2小时后于BioTek多功能微孔板检测仪检测于560nM及590nM的吸光值，计算每孔560/590的比值，减去背景比值后计算每个实验组相对对照组平均值的比值。每组数据以平均值±SD表示，N＝3/6个生物重复。 Add 20% volume of Cell to each well on day 9
Figure PCTCN2022122562-appb-000057
Cell Viability reagent, after incubating at 37°C in the dark for 2 hours, detect the absorbance at 560nM and 590nM on the BioTek multi-functional microplate detector, calculate the ratio of 560/590 in each well, subtract the background ratio and calculate for each experimental group Ratio relative to the mean of the control group. Each group of data is expressed as mean ± SD, N = 3/6 biological repeats.
图8B显示体外扩增健康捐献者(Healthy Donor 1-Healthy Donor 5,HD1-HD5)的PBMC中的成红细胞第9天的数量变化。GDC：GDC-0879；SB：SB-590885。数据以平均值±SD表示。Figure 8B shows the change in the number of erythroblasts in the PBMCs of healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) expanded in vitro on day 9. GDC: GDC-0879; SB: SB-590885. Data are presented as mean ± SD.
以上实施例证明，在PBMC中，BRAF抑制剂可以高效扩增其中的红系前体细胞，并无需任何针对红系前体细胞的分离手段即可获得大量的红细胞。The above examples prove that in PBMCs, BRAF inhibitors can efficiently expand the erythroid precursor cells therein, and a large number of erythrocytes can be obtained without any separation means for erythroid precursor cells.
实施例8-3：体外扩增PBMC中的成红细胞结果2Example 8-3: In vitro expansion of erythroblasts in PBMC results 2
在起始Day0时对细胞进行计数，在每孔的无血清分化培养基中接种500000个来自健康捐献者的PBMC细胞，并在Day0时按照相应的浓度进行给药，换液时保持细胞密度在1000000/ml以下。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药，在给药第九天时对细胞进行流式细胞术检测。The cells were counted at the beginning of Day 0, and 500,000 PBMC cells from healthy donors were inoculated in each well of serum-free differentiation medium, and administered at the corresponding concentration on Day 0, and the cell density was kept at Below 1000000/ml. The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cells were detected by flow cytometry on the ninth day of administration.
在第9天时，重悬细胞并过滤，对细胞进行计数和细胞密度分析，以约1000000/mL细胞重悬于PBS中，按照1：100加入小鼠IgG于室温封闭细胞10min，随后再按照1：200的比例加入CD235a-APC，CD71-PE，10μg/mL Hoechst 33342在37℃染色孵育30min。完成后，使用BD Fortessa进行流式分析，随后用FlowJo对FCS文件进行数据处理，并统计在第九天时各捐赠者处理组双阴性(CD235a/CD71双阴性)和双阳性(CD235a/CD71双阳性)占统计活细胞的比例进行统计并进行未配对学生t检验(unpaired student-t test)，并计算统计学意义是否显著。数据以平均值±SD表示，N＝5个生物重复；p-值<0.05被认为具有显著性。On the 9th day, resuspend the cells and filter, count the cells and analyze the cell density, resuspend the cells in PBS at about 1,000,000/mL, add mouse IgG at a ratio of 1:100, block the cells at room temperature for 10 minutes, and then follow the 1 Add CD235a-APC, CD71-PE, 10μg/mL Hoechst 33342 at a ratio of 200 and incubate at 37°C for 30min. After completion, use BD Fortessa for flow cytometry analysis, and then use FlowJo to perform data processing on the FCS file, and count the double negative (CD235a/CD71 double negative) and double positive (CD235a/CD71 double positive) of each donor treatment group on the ninth day Sex) accounted for the proportion of statistical living cells for statistics and unpaired student t test (unpaired student-t test), and calculate whether the statistical significance is significant. Data are presented as mean ± SD, N = 5 biological replicates; p-values < 0.05 were considered significant.
图8C显示了健康捐献者(Healthy Donor 1-Healthy Donor 5,HD1-HD5)的PBMC在无血清红细胞扩增培养基中培养第9天的FACS结果。处理组，1μM SB-590885。Figure 8C shows the FACS results of PBMCs from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) cultured in serum-free erythrocyte expansion medium on day 9. Treatment group, 1 μM SB-590885.
图8D显示了健康捐献者(Healthy Donor 1-Healthy Donor 5,HD1-HD5)PBMC的FACS结果在第10天细胞类群比率的统计。纵坐标为细胞百分比。双阴性：CD71和CD235a双阴性；双阳性：CD71和CD235a双阳性；SB：SB-590885；GDC：GDC-0879。Figure 8D shows the statistics of the FACS results of the PBMC of healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) on day 10 of the cell population ratio. The vertical axis is the percentage of cells. Double negative: CD71 and CD235a double negative; Double positive: CD71 and CD235a double positive; SB: SB-590885; GDC: GDC-0879.
图8E显示了FACS第10天细胞类群比率的统计。双阴性，CD71和CD235a双阴性；双阳性，CD71和CD235a双阳性；SB，SB-590885；GDC，GDC-0879。未配对双尾学生t检验具有统计学意义。数据以平均值±SD表示，N＝5个生物重复；p-值<0.05被认为具有显著性。Figure 8E shows FACS day 10 cell population ratio statistics. Double negative, CD71 and CD235a double negative; Double positive, CD71 and CD235a double positive; SB, SB-590885; GDC, GDC-0879. Statistical significance was determined by unpaired two-tailed Student's t-test. Data are presented as mean ± SD, N = 5 biological replicates; p-values < 0.05 were considered significant.
实施例8-4：体外扩增PBMC中的成红细胞结果3Example 8-4: In vitro expansion of erythroblasts in PBMC results 3
在起始Day0时对细胞进行计数，在每孔的无血清分化培养基中接种500000个来自健康捐献者(Donor1-Donor5)的PBMC细胞，并在Day0时按照相应的浓度进行给药，换液时保持细胞密度在1000000/ml以下。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药，在给药第九天时对细胞进行流式细胞术检测。Count the cells at the start of Day0, inoculate 500,000 PBMC cells from healthy donors (Donor1-Donor5) in serum-free differentiation medium per well, and administer the corresponding concentration on Day0, and change the medium Keep the cell density below 1000000/ml. The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cells were detected by flow cytometry on the ninth day of administration.
在第9天时，重悬细胞并过滤，对细胞进行计数和细胞密度分析，以约1000000/mL细胞重悬于PBS中，按照1：100加入小鼠IgG于室温封闭细胞10min，随后再按照1：200的比例加入CD235a-APC，CD71-PE，10μg/mL Hoechst 33342在37℃染色孵育30min。完成后，使用BD Fortessa进行流式分析，随后用FlowJo对FCS文件进行数据处理。On the 9th day, resuspend the cells and filter, count the cells and analyze the cell density, resuspend the cells in PBS at about 1,000,000/mL, add mouse IgG at a ratio of 1:100, block the cells at room temperature for 10 minutes, and then follow the 1 Add CD235a-APC, CD71-PE, 10μg/mL Hoechst 33342 at a ratio of 200 and incubate at 37°C for 30min. Once complete, flow analysis was performed using BD Fortessa, followed by data processing of the FCS files with FlowJo.
图8F显示健康捐献者(Donor1-Donor5)的PBMC在无血清红细胞扩增培养基中培养的FACS结 果。处理组：1μM SB-590885。Figure 8F shows the FACS results of PBMC from healthy donors (Donor1-Donor5) cultured in serum-free erythrocyte expansion medium. Treatment group: 1 μM SB-590885.
图8G显示健康捐献者(Donor1-Donor5)的PBMC在无血清红细胞扩增培养基中培养的FACS结果。处理组：1μM SB-590885。Figure 8G shows the FACS results of PBMC from healthy donors (Donor1-Donor5) cultured in serum-free erythrocyte expansion medium. Treatment group: 1 μM SB-590885.
以上的结果证明，BRAF抑制剂可以极大扩增PBMC中的红系前体细胞占比，更好的促进在PBMC中的红系前体细胞增殖。The above results prove that BRAF inhibitors can greatly expand the proportion of erythroid precursor cells in PBMC, and better promote the proliferation of erythroid precursor cells in PBMC.
实施例8-5：体外扩增PBMC中的成红细胞结果4Example 8-5: In vitro expansion of erythroblasts in PBMC Result 4
将100000个来源于健康人PBMC和BRAF抑制剂以及目前唯一一种被广泛用于多种贫血治疗的药物，即糖皮质激素***(Dexamethasone，Dex)在培养起始时与PBMC同时接种/给药于髓系细胞半固体完全培养基中(Methocult H4435)，以比较药物对红系前体细胞扩增能力。给药后，培养皿上长出的肉眼可见的红细胞集落明显变多，单个集落明显变大。对长出的红系单克隆的面积进行统计，发现GDC和Dex给药均使得单个红系克隆面积提高一倍左右，而GDC给药组和Dex给药组相比，红系单克隆面积没有明显变化。而GDC和Dex的联合给药下，红系单克隆面积大小约是对照组四倍，比单独给药获得的红系克隆面积增大一倍(图8H)。图8H为一个整体的扩增能力统计。100,000 PBMCs from healthy people and BRAF inhibitors and the only drug widely used in the treatment of various anemias, namely the glucocorticoid dexamethasone (Dex), were inoculated with PBMCs at the beginning of the culture /Administered in myeloid cell semi-solid complete medium (Methocult H4435) to compare the drug's ability to expand erythroid precursor cells. After administration, the number of visible erythrocyte colonies grown on the culture dish increased significantly, and the individual colonies became significantly larger. The area of the grown erythroid monoclonal was counted, and it was found that the administration of GDC and Dex increased the area of a single erythroid clone by about one time, while the area of the erythroid monoclonal in the GDC administration group and the Dex administration group had no difference. obvious change. Under the combined administration of GDC and Dex, the size of the erythroid monoclonal area was about four times that of the control group, and doubled that obtained by single administration ( FIG. 8H ). Figure 8H is an overall amplification capacity statistics.
A：在Methocult H4435上对PBMC给药，GDC和对照组的整体克隆形成图像和代表性红系单克隆集落图。A: Whole-body clonogenic images and representative erythroid monoclonal colonies of GDC and control group administered to PBMC on Methocult H4435.
B：在Methocult H4435不同给药组和对照组的红细胞集落数量(左)与单个红细胞集落面积(右)的统计。B: The statistics of the number of erythrocyte colonies (left) and the area of a single erythrocyte colony (right) in different administration groups of Methocult H4435 and the control group.
C左，比例尺＝10mm；C右，比例尺＝500μm。GDC：GDC-0879；Dex：***。每个条件设置3个生物学重复，统计数据为平均值±SD；ns：无显著性差异，P>0.05；****：P<0.0001。C left, scale bar = 10 mm; C right, scale bar = 500 μm. GDC: GDC-0879; Dex: Dexamethasone. Three biological repetitions were set for each condition, and statistical data are mean ± SD; ns: no significant difference, P>0.05; ****: P<0.0001.
实施例8-6：体外扩增PBMC中的成红细胞结果5(SB-590885)Example 8-6: In vitro expansion of erythroblasts in PBMC Result 5 (SB-590885)
将200000个来源于健康人PBMC和BRAF抑制剂在培养起始时与PBMC同时接种/给药于髓系细胞半固体完全培养基中(Methocult H4435)，以验证BRAF抑制剂SB-590885对PBMC中红系前体细胞扩增能力。200,000 PBMCs from healthy people and BRAF inhibitors were inoculated/administered at the same time as PBMCs in the semi-solid complete medium of myeloid cells (Methocult H4435) at the beginning of culture to verify the effect of BRAF inhibitor SB-590885 on PBMCs Erythroid precursor cell expansion capacity.
图8I：在Methocult H4435上对PBMC给药，SB和对照组的整体克隆形成图像和代表性红系单克隆集落图。Figure 8I: Whole-body clonogenic images and representative erythroid monoclonal colony images of SB and controls administered to PBMCs on Methocult H4435.
图8J：在Methocult H4435不同给药组和对照组的红细胞集落在显微镜下的代表性集落。比例尺＝500μm。Figure 8J: Representative colonies of erythrocyte colonies in different Methocult H4435 administration groups and control groups under the microscope. Scale bar = 500 μm.
图8K：在Methocult H4435不同给药组和对照组的红细胞集落数量(左)与不同组的单个红细胞集落面积(右)的统计。Figure 8K: Statistics of the number of erythrocyte colonies (left) and the area of individual erythrocyte colonies in different groups (right) in different Methocult H4435 administration groups and control groups.
实施例9：低EPO条件下药物的治疗效果Embodiment 9: Therapeutic effect of medicine under low EPO condition
实施例9-1：低EPO条件下药物的治疗效果1Embodiment 9-1: Therapeutic effect of medicine under low EPO condition 1
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药。在分化培养基中先预先培养4天，在第五天时将各组分别换至仅改变EPO浓度的分化培养基(Full EPO为3IU，1/3EPO为1IU，NO EPO不添加EPO)中再培养六天。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药，在给药第6天时对细胞进行细胞数量进行统计计数。数据以平均值±SD表示，N＝3个生物重复。The cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. Pre-culture in the differentiation medium for 4 days, and on the fifth day, change each group to the differentiation medium that only changes the EPO concentration (Full EPO is 3IU, 1/3EPO is 1IU, NO EPO does not add EPO) and then culture six days. The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the number of cells was statistically counted on the 6th day of administration. Data are presented as mean ± SD, N = 3 biological replicates.
图9A显示GDC-0879在不同浓度***(EPO)分化培养基中的作用。前4天，各组在正常分化培养基(3IU/ml EPO)中培养。后6天，正常EPO组添加3IU/ml***(Amgen, Thousand Oaks,CA,Cat 55513-144-10)；1/3EPO浓度组补充1.5IU/ml EPO；不添加EPO组不补充EPO。第10天进行细胞计数。数据以平均值±SD表示，统计结果由三个独立的生物学重复计算。Figure 9A shows the effect of GDC-0879 in different concentrations of erythropoietin (EPO) differentiation medium. For the first 4 days, each group was cultured in normal differentiation medium (3IU/ml EPO). After 6 days, the normal EPO group was supplemented with 3 IU/ml erythropoietin (Amgen, Thousand Oaks, CA, Cat 55513-144-10); the 1/3 EPO concentration group was supplemented with 1.5 IU/ml EPO; the no EPO group was supplemented with no EPO . Cell counts were performed on day 10. Data are expressed as mean ± SD, and statistical results are calculated from three independent biological replicates.
实施例9-2：低EPO条件下药物的治疗效果2Embodiment 9-2: Therapeutic effect of medicine under low EPO condition 2
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药。在分化培养基中先预先培养4天，在第五天时将各组分别换至不添加EPO的培养基中再培养十天。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药，共给药六天，在六天后不再给药4天。在培养的第14天进行流式细胞术检测。The cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. Pre-cultured in the differentiation medium for 4 days, and on the fifth day, each group was changed to the medium without EPO and cultured for another ten days. The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, for a total of six days, and no further administration for 4 days after six days. Flow cytometry detection was performed on the 14th day of culture.
在培养的第14天时，重悬细胞并过滤，对细胞进行计数和细胞密度分析，以约1000000/mL细胞重悬于PBS中，按照1：100加入小鼠IgG于室温封闭细胞10min，随后再按照1：200的比例加入CD235a-APC，CD71-FITC，10μg/mL Hoechst 33342在37℃染色孵育30min。完成后，使用BD Fortessa进行流式分析，随后用FlowJo对FCS文件进行数据处理。On the 14th day of culture, resuspend the cells and filter, count the cells and analyze the cell density, resuspend the cells in PBS at about 1,000,000/mL, add mouse IgG at a ratio of 1:100, block the cells at room temperature for 10 minutes, and then Add CD235a-APC, CD71-FITC, 10 μg/mL Hoechst 33342 at a ratio of 1:200 and incubate at 37°C for 30 minutes. Once complete, flow analysis was performed using BD Fortessa, followed by data processing of the FCS files with FlowJo.
图9B显示无EPO条件下培养5-10天后第14天FACS结果。左：第5天至第10天1μM GDC-0879处理；右：对照。处理组前4天采用完全***分化培养基(3IU/ml***,EPO)，5～10天采用无***(EPO)分化培养基。处理组第5～10天在培养液中加入1μM GDC-0879。Figure 9B shows the FACS results on day 14 after 5-10 days of culture in the absence of EPO. Left: 1 μM GDC-0879 treatment from day 5 to day 10; right: control. The treatment group used complete erythropoietin differentiation medium (3IU/ml erythropoietin, EPO) for the first 4 days, and used erythropoietin (EPO)-free differentiation medium for 5 to 10 days. In the treatment group, 1 μM GDC-0879 was added to the culture medium on the 5th to 10th day.
实施例9-3：低EPO条件下药物的治疗效果3Example 9-3: Therapeutic Effect of Drugs under Low EPO Conditions 3
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药。在仅含1/3EPO浓度(即1IU EPO)中培养。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药，给药组仅给药处理9天。总共在此1/3EPO浓度的分化培养基中培养15天。The cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. Culture in only 1/3 EPO concentration (i.e. 1IU EPO). The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the treatment group was only administered for 9 days. A total of 15 days were cultured in this 1/3 EPO concentration differentiation medium.
在培养的第15天时，重悬细胞并过滤，对细胞进行计数和细胞密度分析，以约1000000/mL细胞重悬于PBS中，按照1：100加入小鼠IgG于室温封闭细胞10min，随后再按照1：200的比例加入CD235a-APC，CD71-FITC，10μg/mL Hoechst 33342在37℃染色孵育30min。完成后，使用BD Fortessa进行流式分析，随后用FlowJo对FCS文件进行数据处理。On the 15th day of culture, resuspend the cells and filter, count the cells and analyze the cell density, resuspend the cells in PBS at about 1,000,000/mL, add mouse IgG at a ratio of 1:100, block the cells at room temperature for 10 minutes, and then Add CD235a-APC, CD71-FITC, 10 μg/mL Hoechst 33342 at a ratio of 1:200 and incubate at 37°C for 30 minutes. Once complete, flow analysis was performed using BD Fortessa, followed by data processing of the FCS files with FlowJo.
图9C显示1/3浓度EPO条件下培养1-15天后第15天FACS结果。从第1天到第14天均使用1/3EPO分化培养基(1IU/ml EPO)。处理组从第1天至第9天在培养液中添加1μM GDC-0879。Figure 9C shows the FACS results on the 15th day after culturing for 1-15 days under the condition of 1/3 concentration of EPO. From day 1 to day 14, 1/3 EPO differentiation medium (1IU/ml EPO) was used. In the treatment group, 1 μM GDC-0879 was added to the culture medium from day 1 to day 9.
实施例9-4：低EPO条件下药物的治疗效果4Embodiment 9-4: Therapeutic effect of medicine under low EPO condition 4
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药。在仅含1/3EPO浓度(即1IU EPO)和正常分化培养基中培养。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药，给药组给药处理12天。总共在此两种EPO浓度的分化培养基中培养19天。The cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. Cultured in normal differentiation medium containing only 1/3 EPO concentration (i.e. 1IU EPO). The culture medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the administration group was administered for 12 days. A total of 19 days were cultured in the differentiation medium of these two EPO concentrations.
在培养的第13-19天时，重悬细胞并过滤，对细胞进行计数和细胞密度分析，以约1000000/mL细胞重悬于PBS中，按照1：100加入小鼠IgG于室温封闭细胞10min，随后再按照1：200的比例加入CD235a-APC，CD71-FITC，10μg/mL Hoechst 33342在37℃染色孵育30min。完成后，使用BD Fortessa进行流式分析，随后用FlowJo对FCS文件进行数据处理。计算CD235a-APC+和Hoechst 33342-的细胞群的比例即为脱核比例。红细胞在终末分化时期不再进行自我更新，在进行数次流式细胞术检测后细胞数量不足，故处理组在15天后不再进行流式细胞术检测。On the 13th-19th day of culture, resuspend the cells and filter, count the cells and analyze the cell density, resuspend the cells in PBS at about 1,000,000/mL, add mouse IgG at a ratio of 1:100, and block the cells at room temperature for 10 minutes. Then add CD235a-APC, CD71-FITC, 10 μg/mL Hoechst 33342 at a ratio of 1:200 and incubate at 37°C for 30 minutes. Once complete, flow analysis was performed using BD Fortessa, followed by data processing of the FCS files with FlowJo. The ratio of CD235a-APC+ and Hoechst 33342- cell populations was calculated as the ratio of denucleation. Red blood cells no longer undergo self-renewal during the terminal differentiation stage, and the number of cells was insufficient after several flow cytometry tests, so the treatment group did not perform flow cytometry tests after 15 days.
图9D显示统计13～19天各EPO浓度条件下的红细胞脱核率。对照组大部分细胞在第15天凋 亡，因此第15天后没有进一步的数据。处理组，从第1天至第12天以1μM GDC-0879处理。FEPO，完全EPO组在分化培养基中添加3IU/ml***；1/3EPO组在分化培养基中添加1.5IU/ml EPO。Fig. 9D shows the statistics of the denucleation rate of red blood cells under the conditions of various EPO concentrations from day 13 to day 19. Most of the cells in the control group were apoptotic by day 15, therefore no further data were available after day 15. The treatment group was treated with 1 μM GDC-0879 from day 1 to day 12. In the FEPO and complete EPO groups, 3 IU/ml erythropoietin was added to the differentiation medium; in the 1/3 EPO group, 1.5 IU/ml EPO was added to the differentiation medium.
以上实施例证明，在低EPO的培养状况下，BRAF抑制剂促进了正常红系发育，促进了红系增殖，维持了红系的脱核比例。The above examples prove that under low EPO culture conditions, BRAF inhibitors can promote normal erythroid development, promote erythroid proliferation, and maintain erythroid denucleation ratio.
实施例9-5：在低细胞因子浓度的条件下BRAF抑制剂治疗和促进红系分化的效果Example 9-5: Effect of BRAF Inhibitor Treatment and Promotion of Erythroid Differentiation under Low Cytokine Concentration Conditions
实施例9-5a：低EPO和SCF条件下药物的治疗效果1Example 9-5a: Therapeutic effect of drugs under low EPO and SCF conditions 1
在起始Day0时对细胞进行计数，在每个孔的分化培养基中接种10000个CD34+细胞，并在Day0时按照相应的浓度进行给药。将各组分别改变EPO浓度和SCF浓度的分化培养基(Full Ctrl为3IU，5％EPO为0.15IU，NO SCF不添加SCF)。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药。数据以平均值±SD表示，N＝3个生物重复。The cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. Differentiation medium with EPO concentration and SCF concentration were changed in each group (3 IU for Full Ctrl, 0.15 IU for 5% EPO, no SCF for NO SCF). The medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. Data are presented as mean ± SD, N = 3 biological replicates.
图9E为用0％SCF或5％EPO的低细胞因子浓度在红系培养基中用2μM GDC-0879给药处理的UCB CD34+细胞的生长曲线。所有条件的给药组均比对照组细胞数量高一个数量级。Figure 9E is a growth curve of UCB CD34+ cells treated with 2 μM GDC-0879 administration in erythroid medium with low cytokine concentrations of 0% SCF or 5% EPO. In all conditions, the number of cells in the administration group was an order of magnitude higher than that in the control group.
图9F为对照组(上)和GDC-0879处理组(下)中0％SCF(左)和5％EPO(右)的低浓度细胞因子在第9天分化(UCB CD34+细胞)的流式细胞术细胞表面标志物代表图像。给药组相比对照组，红系发育过程正常，未发育的组别比例较低。Figure 9F is the flow cytometry of 0% SCF (left) and 5% EPO (right) low concentrations of cytokines differentiated (UCB CD34+ cells) on day 9 in the control group (top) and GDC-0879 treatment group (bottom) Representative images of cell surface markers. Compared with the control group, the erythroid development process of the administration group was normal, and the proportion of the undeveloped group was lower.
以上实施例证明，在极端的低细胞因子浓度条件下，BRAF抑制剂给药依然可以维持一个数量级的扩增，且给药组可以维持正常的红系发育。The above examples prove that under extremely low cytokine concentration conditions, BRAF inhibitor administration can still maintain an order of magnitude expansion, and the administration group can maintain normal erythroid development.
实施例9-7：在低细胞因子浓度的条件下BRAF抑制剂治疗和促进红系分化的效果-2Example 9-7: Effect of BRAF Inhibitor Treatment and Promotion of Erythroid Differentiation under Low Cytokine Concentration-2
图9G和9H：GDC处理组和对照组在第6天在不同红细胞生成条件培养基中的成红细胞的典型红细胞表面标志物表达情况(CD117、CD71和CD235)。Figures 9G and 9H: Expression of typical erythrocyte surface markers (CD117, CD71 and CD235) of erythroblasts in different erythropoietic conditioned media of GDC treatment group and control group on day 6.
图9I和9J：GDC处理组和对照组在第14天在不同红细胞生成条件培养基中的红细胞表面标志物(CD117、CD71和CD235)的表达情况。图9J显示了GDC和对照组在不同压力条件下的CD235表达量差异。Figures 9I and 9J: Expression of erythrocyte surface markers (CD117, CD71 and CD235) in different erythropoietic conditioned media in the GDC treatment group and control group on day 14. Figure 9J shows the difference in the expression of CD235 between GDC and the control group under different stress conditions.
图9K：5％E.：5％EPO条件培养基离心14天后在不同应激条件下培养的样品的典型红细胞照片(每个EP管中的细胞数相等)；S.Free：无SCF条件培养基。除了提到的细胞因子差异外，条件培养基中的其他成分与红细胞生成培养基中的相同。Figure 9K: 5% E.: Typical photographs of red blood cells of samples cultured under different stress conditions after centrifugation of 5% EPO-conditioned medium for 14 days (the number of cells in each EP tube is equal); S.Free: cultured without SCF base. Apart from the cytokine differences mentioned, the other components in the conditioned medium were the same as in the erythropoietic medium.
图9L：不同压力培养条件下的代表性吉姆萨-联苯胺染色的红细胞图像。比例尺＝50μm。Figure 9L: Representative Giemsa-benzidine stained RBC images under different stress culture conditions. Scale bar = 50 μm.
以上实施例从更多角度证明，在极端的低细胞因子浓度条件下，BRAF抑制剂给药组可以维持正常的红系发育，相比于对照组的发育的更加成熟。The above examples prove from more perspectives that under extremely low cytokine concentration conditions, the BRAF inhibitor administration group can maintain normal erythroid development, which is more mature than that of the control group.
实施例10：造血干细胞移植后纯红细胞再生缺陷导致的贫血体外治疗效果Example 10: In vitro therapeutic effect of anemia caused by pure red blood cell regeneration defects after hematopoietic stem cell transplantation
在起始Day0时对细胞进行计数，在每孔的无血清分化培养基中接种500000个来自患有造血干细胞移植后红系发育障碍的患者的PBMC细胞，并在Day0时按照相应的浓度进行给药，换液时保持细胞密度在1000000/ml以下。培养基和小分子每3天更换一次，处理组在更换培养基的同时给药，在给药第10天时对细胞进行流式细胞术和细胞数量检测。The cells were counted at the start of Day0, and 500,000 PBMC cells from patients with erythroid dysplasia after hematopoietic stem cell transplantation were inoculated in serum-free differentiation medium per well, and administered at the corresponding concentration on Day0. When changing the medium, keep the cell density below 1,000,000/ml. The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cells were detected by flow cytometry and cell number on the 10th day of administration.
A.在第10天时往每个孔中加入20％体积的Cell
Figure PCTCN2022122562-appb-000058
Cell Viability试剂，在37℃避光孵育2小时后于BioTek多功能微孔板检测仪检测于560nM及590nM的吸光值，计算每孔560/590的比值，减去背景比值后计算每个实验组相对对照组平均值的比值。每组数据以平均值±SD表示，N＝3 个生物重复。 A. Add 20% volume of Cell to each well on day 10
Figure PCTCN2022122562-appb-000058
Cell Viability reagent, after incubating at 37°C in the dark for 2 hours, detect the absorbance at 560nM and 590nM on the BioTek multi-functional microplate detector, calculate the ratio of 560/590 in each well, subtract the background ratio and calculate for each experimental group Ratio relative to the mean of the control group. Each group of data is expressed as mean ± SD, N = 3 biological repeats.
B.在第10天时，重悬细胞并过滤，对细胞进行计数和细胞密度分析，以约1000000/mL细胞重悬于PBS中，按照1：100加入小鼠IgG于室温封闭细胞10min，随后再按照1：200的比例加入CD235a-APC，CD71-PE，10μg/mL Hoechst 33342在37℃染色孵育30min。完成后，使用BD Fortessa进行流式分析，随后用FlowJo对FCS文件进行数据处理。B. On the 10th day, resuspend the cells and filter, count the cells and analyze the cell density, resuspend the cells in PBS at about 1000000/mL, add mouse IgG at a ratio of 1:100, block the cells at room temperature for 10 minutes, and then Add CD235a-APC, CD71-PE, 10 μg/mL Hoechst 33342 at a ratio of 1:200 and incubate at 37°C for 30 minutes. Once complete, flow analysis was performed using BD Fortessa, followed by data processing of the FCS files with FlowJo.
图10A显示GDC-0879在造血干细胞移植后的红细胞发育失败或纯红细胞发育不全中可能促进红细胞祖细胞的发育和自我更新。该患者曾是白血病患者，在接受造血干细胞移植手术后，其红细胞计数仍然偏低。在分化培养基中使用GDC-0879处理该患者的PBMC 10天后，不仅细胞数量扩大到约6倍(图10A)，而且FACS显示低比例的CD71/CD235a阴性细胞群和良好的红细胞发育结果。(图10B)培养基和小分子每3天更换一次。数据以平均值±SD表示，统计结果由三个独立的生物学重复计算。Figure 10A shows that GDC-0879 may promote the development and self-renewal of erythroid progenitors in erythroid failure or pure erythroid aplasia after hematopoietic stem cell transplantation. The patient had a history of leukemia and had a low red blood cell count after a hematopoietic stem cell transplant. After 10 days of treatment of this patient's PBMC with GDC-0879 in differentiation medium, not only the cell number expanded to about 6-fold (Fig. 10A), but also FACS showed a low proportion of CD71/CD235a negative cell population and good erythroid development outcome. (FIG. 10B) Media and small molecules were changed every 3 days. Data are expressed as mean ± SD, and statistical results are calculated from three independent biological replicates.
图10B：造血干细胞移植后红细胞再生缺陷(EFAHSCT)患者骨髓单个核细胞(BMMNCs)的生长曲线。*代表不同治疗组和对照组之间的成对比较。GDC：GDC-0879；SB：SB-590885；***：P<0.001；****：P<0.0001。Figure 10B: Growth curves of bone marrow mononuclear cells (BMMNCs) from patients with defective erythroid regeneration after hematopoietic stem cell transplantation (EFAHSCT). *Represents pairwise comparisons between different treatment and control groups. GDC: GDC-0879; SB: SB-590885; ***: P<0.001; ****: P<0.0001.
图10C：EFAHSCT的BMMNC在第6天(左)和第16天(右)的红细胞发育的SB处理(下)和对照组(上)的红细胞表面标志物(CD71、CD235和Hoechst 33342)。Figure 10C: Red blood cell surface markers (CD71, CD235, and Hoechst 33342) in SB-treated (bottom) and control (top) erythrocyte development of BMMNCs from EFAHSCT at days 6 (left) and 16 (right).
图10D：从EFAHSCT的BMMNCs进行红系培养后获得的代表性吉姆萨-联苯胺染色红细胞。比例尺＝100μm。Figure 10D: Representative Giemsa-benzidine stained erythrocytes obtained after erythroid culture of BMMNCs from EFAHSCT. Scale bar = 100 μm.
图10E：50000个EFAHSCT的BMMNCs在H4435髓系甲基纤维素培养基中的代表性图像。条形刻度＝10mm。Figure 10E: Representative images of 50,000 EFAHSCT-derived BMMNCs in H4435 myeloid methylcellulose medium. Bar scale = 10mm.
图10F：50000个EFAHSCT的BMMNCs在H4435髓系甲基纤维素培养基中培养出的不同谱系的集落数量。Figure 10F: The number of colonies of different lineages cultured from 50,000 EFAHSCT BMMNCs in H4435 myeloid methylcellulose medium.
ns：无显著差异；***：p<0.001。数据显示为平均值±SD。使用未配对的学生t检验评估两组比较的统计显着性。ns: no significant difference; ***: p<0.001. Data are shown as mean ± SD. Statistical significance of two-group comparisons was assessed using an unpaired Student's t-test.
以上实施例证明，BRAF抑制剂可以挽救EFAHSCT患者的红系发育衰竭，极大促进红系的分化和发育进程，同时扩增细胞数量。The above examples prove that BRAF inhibitors can rescue erythroid developmental failure in patients with EFAHSCT, greatly promote the differentiation and development of erythroid, and expand the number of cells at the same time.
实施例11：SB-590885有效扩增DBA贫血患者外周单核细胞中的红系前体细胞Example 11: SB-590885 effectively expands erythroid precursor cells in peripheral mononuclear cells of patients with DBA anemia
在实验前一天复苏来源于DBA病人(RPL5和RPL11突变)患者的PBMC细胞，并在分化培养基中过夜，在第一天时按照每孔(35mm)300000个PBMC细胞接种于含EPO的甲基纤维素培养基Methocult H4330(StemCell,Vancouver,BC,CA,Cat H4330)中,向其中补充至10ng/ml IL3(StemCell,Vancouver,BC,CA,Cat 78042)和50ng/ml hSCF(StemCell,Vancouver,BC,CA,Cat 78062)，对给药组药物预混均匀，于培养箱中培养14天。在14天时使用Leica倒置相差显微镜对集落进行拍照和统计每孔的红系集落数量。PBMC cells derived from DBA patients (RPL5 and RPL11 mutations) were revived one day before the experiment, and overnight in the differentiation medium. On the first day, 300,000 PBMC cells per well (35 mm) were inoculated in the methyl group containing EPO. Cellulose medium Methocult H4330 (StemCell, Vancouver, BC, CA, Cat H4330), supplemented to 10ng/ml IL3 (StemCell, Vancouver, BC, CA, Cat 78042) and 50ng/ml hSCF (StemCell, Vancouver, BC, CA, Cat 78062), the medicines of the administration group were premixed evenly, and cultivated in the incubator for 14 days. At 14 days, the colonies were photographed using a Leica inverted phase-contrast microscope and the number of erythroid colonies in each well was counted.
图11显示在含EPO、IL3、SCF的甲基纤维素培养基中，1μM SB-590885处理不同种类DBA患者的外周单核细胞，每孔接种300000PBMC；图为14天时的红细胞集落形成结果。SB-590885在红系细胞培养的甲基纤维素培养基中培养14天的红细胞集落形成结果。用5×物镜下检测第14天红细胞特异性集落形成结果。在显微镜下，每个DBA患者PBMC体外培养形成的典型红细胞集落。处理组与对照组的集落面积有明显区别。RPL11Mut：指RPL11突变的DBA患者，本批次有DBA1和DBA2两个患者样本；RPL5Mut：指RPL5突变的DBA患者。Ctrl：未给药处理；Treated：仅在一开始给药1μM SB-590885。Figure 11 shows that in the methylcellulose medium containing EPO, IL3, and SCF, 1 μM SB-590885 treated peripheral mononuclear cells of different types of DBA patients, and inoculated 300,000 PBMCs per well; the figure shows the results of erythrocyte colony formation at 14 days. Erythrocyte colony formation results of SB-590885 cultured in methylcellulose medium for erythroid cell culture for 14 days. The results of erythrocyte-specific colony formation on day 14 were detected with a 5× objective lens. Under the microscope, typical erythrocyte colonies formed by in vitro culture of PBMC from each DBA patient. The colony area of the treatment group was significantly different from that of the control group. RPL11Mut: refers to DBA patients with RPL11 mutation, and there are two patient samples of DBA1 and DBA2 in this batch; RPL5Mut: refers to DBA patients with RPL5 mutation. Ctrl: treated without administration; Treated: only initially administered 1 μM SB-590885.
实施例12：SB-590885有效扩增DBA贫血患者外周单核细胞中的红系前体细胞的数据统计Example 12: Statistics of SB-590885 effectively amplifying erythroid precursor cells in peripheral mononuclear cells of patients with DBA anemia
图12显示对图11结果的数据统计。对图11中在甲基纤维素培养基上长出的红系集落照片进行拍照，并使用FIJI软件对单个红系细胞集落进行面积统计，并计算两个重复孔中的红系集落单个以及平均面积。对统计数据进行未配对学生t检验，并计算统计学意义是否显著。数据以平均值±SD表示；*：p值<0.05；**：p值<0.01。Figure 12 shows the statistics for the results of Figure 11. Take photos of the erythroid colonies grown on methylcellulose medium in Figure 11, and use FIJI software to count the area of a single erythroid colony, and calculate the individual and average erythroid colonies in two replicate wells area. Statistical data were subjected to unpaired Student's t-test and statistical significance was calculated. Data are presented as mean ± SD; *: p-value < 0.05; **: p-value < 0.01.
实施例11和12共同说明，BRAF抑制剂在体外可以促进DBA患者的红系增殖。Examples 11 and 12 together demonstrate that BRAF inhibitors can promote erythroid proliferation in DBA patients in vitro.
实施例13：BRAF抑制剂有效促进MDS患者细胞的红系分化和红系增殖Example 13: BRAF inhibitors effectively promote erythroid differentiation and erythroid proliferation of MDS patient cells
MDS为骨髓增生异常综合征的简称。主要由于患者血液祖细胞的不正常发育(癌变)，或者血液祖细胞因为种种原因在骨髓中，或在进入循环后死亡。进而导致不成熟的或有缺陷的细胞比健康的细胞数量，则出现健康的红细胞、健康的白细胞以及凝血血小板数量过少，但处于癌变前的血液祖细胞异常增多的现象。MDS is the abbreviation for Myelodysplastic Syndrome. It is mainly due to the abnormal development (cancerous) of the blood progenitor cells of the patient, or the blood progenitor cells die in the bone marrow for various reasons, or after entering the circulation. In turn, the number of immature or defective cells is lower than that of healthy cells, and the number of healthy red blood cells, healthy white blood cells, and platelets is too small, but the number of pre-cancerous blood progenitor cells is abnormally increased.
图13A：接种100000个MDS患者的BMMNC在H4435髓系细胞培养基中。比例尺＝10mm。Figure 13A: Inoculation of 100,000 BMMNCs of MDS patients in H4435 myeloid cell culture medium. Scale bar = 10mm.
图13B：上图中红系集落的代表形态图。比例尺＝500μm。Figure 13B: Representative morphology of erythroid colonies in the upper panel. Scale bar = 500 μm.
图13C：图13A中红系集落的面积统计。Figure 13C: Area statistics of the erythroid colonies in Figure 13A.
图13D：从MDS BMMNCs培养的代表性吉姆萨-联苯胺染色红细胞。比例尺＝100μm。Figure 13D: Representative Giemsa-benzidine stained erythrocytes cultured from MDS BMMNCs. Scale bar = 100 μm.
图13E：GDC治疗(下)和对照组(上)在第6天(左)和第9天(右)的MDS患者BMMNC的红系发育过程的红细胞表面标志物(CD71、CD235和Hoechst 33342)。Figure 13E: Erythrocyte surface markers (CD71, CD235 and Hoechst 33342) in the erythroid development process of BMMNC of MDS patients treated with GDC (lower) and control group (upper) on day 6 (left) and day 9 (right) .
实施例13证明，BRAF抑制剂可以挽救MDS患者的红系发育障碍，极大促进红系的分化和发育进程，同时扩增细胞数量。Example 13 proves that BRAF inhibitors can rescue erythroid dysplasia in MDS patients, greatly promote the differentiation and development of erythroid, and simultaneously expand the number of cells.
实施例14：BRAF抑制剂对造血干祖细胞(HSPC)的扩增作用Example 14: Effect of BRAF Inhibitor on Expansion of Hematopoietic Stem and Progenitor Cells (HSPC)
由于BRAF抑制剂对早期的红系前体细胞具有扩增效果，故也检测了BRAF对红系祖细胞的来源，即该药物对CD34+细胞的扩增作用。Since BRAF inhibitors have an expansion effect on early erythroid precursor cells, the source of BRAF on erythroid progenitor cells, that is, the drug's expansion effect on CD34+ cells, was also tested.
在起始Day0时对细胞进行计数，将CD34+细胞接种于造血干细胞/祖细胞无血清扩增培养基中，每孔10000个CD34+细胞，并在Day0时按照相应的浓度进行给药，换液时保持细胞密度在1000000/ml以下。每两天换液并给药，随后计数。A和B非同批次CD34+。A：UM171：UM171 35nM，SR1：SR1 500nM，GDC-0879：GDC-0879 1μM；B：GDC-0879：GDC-0879 1μM，G+U+S：GDC-0879 11μM+UM171 35nM+SR1 500nM。Count the cells at the beginning of Day0, inoculate CD34+ cells in hematopoietic stem cell/progenitor cell serum-free expansion medium, 10,000 CD34+ cells per well, and administer at the corresponding concentration on Day0, when changing the medium Keep the cell density below 1000000/ml. The medium was changed and administered every two days, followed by counting. A and B are not from the same batch of CD34+. A: UM171: UM171 35nM, SR1: SR1 500nM, GDC-0879: GDC-0879 1μM; B: GDC-0879: GDC-0879 1μM, G+U+S: GDC-0879 11μM+UM171 35nM+SR1 500nM.
此实施例说明，BRAF抑制剂对HSC依旧有促增殖作用，其矛盾激活效果也在HSC的自我更新上起效。This example shows that BRAF inhibitors still have pro-proliferation effects on HSCs, and their paradoxical activation effects also work on the self-renewal of HSCs.
实施例15：BRAF抑制剂促进小鼠体内造血1(正常情况)Example 15: BRAF inhibitors promote hematopoiesis in mice 1 (normal conditions)
A：体内施用BRAF抑制剂的示意图。C57BL/6N小鼠在6-8周时用溶剂或Encorafenib(5mg/kg、10mg/kg或25mg/kg)喂养3天，每天一次。在第3天采集并检查血样。红色箭头表示采集血样的日期。对照组n＝10，其他n＝5。A: Schematic representation of in vivo administration of BRAF inhibitors. C57BL/6N mice were fed vehicle or Encorafenib (5 mg/kg, 10 mg/kg or 25 mg/kg) once daily for 3 days at 6-8 weeks. Blood samples were collected and examined on day 3. Red arrows indicate the date the blood sample was collected. Control group n=10, others n=5.
B：在第3天测量红细胞(RBC)、血红蛋白(HGB)和平均红细胞体积(MCV)。每个点代表一只小鼠。B: Measurement of red blood cells (RBC), hemoglobin (HGB) and mean corpuscular volume (MCV) on day 3. Each point represents a mouse.
此实施例说明，BRAF抑制剂在正常情况下就可以促进体内的红系生成过程。This example demonstrates that BRAF inhibitors can promote erythropoiesis in vivo under normal conditions.
实施例16：BRAF抑制剂促进小鼠体内造血2(急性溶血模型1)Example 16: BRAF inhibitors promote hematopoiesis in mice 2 (acute hemolysis model 1)
PHZ(苯肼)可以诱导红细胞的溶血性贫血。通过对小鼠进行PHZ注射，可以诱导小鼠发生急性溶血性贫血，并可以以此做为一个急性贫血模型验证BRAF抑制剂的促进应激性造血的模型。PHZ (phenylhydrazine) can induce hemolytic anemia of red blood cells. By injecting PHZ into mice, acute hemolytic anemia can be induced in mice, which can be used as an acute anemia model to verify the stress hematopoiesis promotion model of BRAF inhibitors.
图16A：苯肼(PHZ)诱导的溶血性贫血模型中BRAF抑制剂的施用示意图。在第0天注射苯肼(PHZ)之前，用载体(对照组)，GDC-0879(30mg/kg)或Encorafenib(25mg/kg)在6-8周时预处理C57BL/6N小鼠10天(P.O.，B.D.，周一至周五3周)。在第0天，第2-5天和第7-11天收集并检查血液样本。在第12天处死小鼠，并在收集骨髓和脾脏样本时。红色箭头表示采集血液样本的天数。黄色箭头表示收集脾脏样本的天数。GDC-0879组n＝4，其他组n＝5。Figure 16A: Schematic representation of administration of BRAF inhibitors in the phenylhydrazine (PHZ)-induced hemolytic anemia model. C57BL/6N mice were pretreated with vehicle (control group), GDC-0879 (30 mg/kg) or Encorafenib (25 mg/kg) for 10 days at 6–8 weeks prior to injection of phenylhydrazine (PHZ) on day 0 ( P.O., B.D., Mon-Fri 3 weeks). Blood samples were collected and examined on Day 0, Days 2-5, and Days 7-11. Mice were sacrificed on day 12, when bone marrow and spleen samples were collected. Red arrows indicate the days on which blood samples were collected. Yellow arrows indicate days on which spleen samples were collected. GDC-0879 group n=4, other groups n=5.
图16B：第0天、第2～5天、第7～11天测定红细胞(RBC)(左图)和红细胞分布宽度CV(RDW-CV)(中图)。在第12天称量脾脏(右图)。误差线表示4或5个生物重复的平均±标准偏差。对两组(ns：P>0.05；*：P≤0.05，**：P≤0.01)进行不成对双尾学生t检验进行统计分析。Figure 16B: Determination of red blood cells (RBC) (left panel) and red blood cell distribution width CV (RDW-CV) (middle panel) on day 0, days 2-5, and days 7-11. Spleens were weighed on day 12 (right panel). Error bars represent mean±standard deviation of 4 or 5 biological replicates. The two groups (ns: P>0.05; *: P≤0.05, **: P≤0.01) were statistically analyzed by unpaired two-tailed Student's t test.
图16C：实施例16中体内实验里的血红蛋白(HGB)，血细胞比容(HCT)，平均红细胞血红蛋白(MCH)，血小板计数(PLT)，白细胞计数(WBC)和PHZ诱导的溶血性贫血期间的网织红细胞比值。Figure 16C: Hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), platelet count (PLT), white blood cell count (WBC) and PHZ-induced hemolytic anemia in the in vivo experiment in Example 16 Reticulocyte ratio.
图16D：实施例16中体内实验内的PHZ诱导后第12天脾脏中的非红细胞(CD71和Ter119双阴性)比率。Figure 16D: Non-erythroid (CD71 and Terl 19 double negative) ratio in the spleen at day 12 after PHZ induction in the in vivo experiment of Example 16.
图16E：实施例16中PHZ诱导后第12天骨髓中的CD117+祖细胞比例。FIG. 16E : CD117+ progenitor cell ratio in bone marrow on day 12 after PHZ induction in Example 16. FIG.
此实施例说明，BRAF抑制剂在急性溶血模型下，可以促进体内的应激性造血，尤其是髓外造血(脾脏造血)过程。This example illustrates that BRAF inhibitors can promote stress hematopoiesis in vivo, especially extramedullary hematopoiesis (splenic hematopoiesis) in an acute hemolysis model.
实施例17：BRAF抑制剂促进小鼠体内造血3(急性溶血模型2)Example 17: BRAF inhibitors promote hematopoiesis in mice 3 (acute hemolysis model 2)
图17A：在PHZ诱导的溶血性贫血模型中施用GDC-0879(50mg/kg，P.O.)和SB-590885(10mg/kg，IP)的21剂量(周一至周三Q.D.和周四至周五B.I.D.2周)的示意图。Figure 17A: 21 doses of GDC-0879 (50 mg/kg, P.O.) and SB-590885 (10 mg/kg, IP) were administered in the PHZ-induced hemolytic anemia model (Monday to Wednesday Q.D. and Thursday to Friday B.I.D. 2 week) diagram.
图17B：在第0-4天和第7-11天测量红细胞(RBC)，HGB，HCT和红细胞分布宽度CV(RDW-CV)。每组3只小鼠。Figure 17B: Measurement of red blood cell (RBC), HGB, HCT and red blood cell distribution width CV (RDW-CV) on days 0-4 and 7-11. 3 mice per group.
此实施例说明，BRAF抑制剂(SB-590885)在急性溶血模型下，同样可以促进体内的应激性造血。This example shows that the BRAF inhibitor (SB-590885) can also promote stress hematopoiesis in vivo in the acute hemolysis model.
实施例18：BRAF抑制剂在红系分化过程中诱导MAPK通路的矛盾激活Example 18: BRAF inhibitors induce paradoxical activation of the MAPK pathway during erythroid differentiation
BRAF抑制剂是ATP竞争性抑制剂，其竞争性结合到BRAF突变蛋白中ATP结合口袋中，然后它锁定R-Spine(αC-螺旋)和DFG结构域残基位置，进而阻断了R-Spine和DFG结构域与BRAF蛋白活性构象的形成，故而阻止了突变型BRAF蛋白的活化和下游MAPKs的激活。BRAF inhibitors are ATP-competitive inhibitors that competitively bind to the ATP-binding pocket in the BRAF mutant protein, and then it locks the R-Spine (αC-helix) and DFG domain residue positions, thereby blocking the R-Spine And the formation of the DFG domain and the active conformation of the BRAF protein, thus preventing the activation of the mutant BRAF protein and the activation of downstream MAPKs.
BRAFV600E突变的黑色素瘤中，BRAFV600E突变自激活MEK后直接介导下游的ERK激活，并最终导致成瘤。但在正常BRAF中(野生型)，加入BRAF抑制剂会导致BRAF处于类似于ATP结合的活化构象，此时BRAF丧失激酶活性，但构象变为激活状态，从而易于和CRAF的R-Spine对齐，形成R506盐桥，极大的促进了BRAF和CRAF组成异源二聚体的效率，BRAF-CRAF二聚体的形成是MAPK通路中RAS-RAF-MEK信号转导的关键，只有在二聚体存在的情况下，RAFs才能激活下游的MEK。但这一机制(MAPK的激活)存在一个前提，即BRAF和CRAF的激活都依赖于其蛋白上的Ras Binding Domain(RBD)。这意味着BRAF和CRAF的磷酸化依赖于RBD和上游活化的Ras-GTP结合，只有在RAS-GTP存在的前提下(即受到正常的激活信号，如细胞因子结合或GPCR的激活)，BRAF抑制剂诱导的和CRAF的二聚化才会被RAS-GTP磷酸化，此时CRAF在S338位点被磷酸化，继而实现经由剧烈的pCRAF激活而不是pBRAF引发下游的MAPK/ERK激活。而在红系 发育过程中，红系前体细胞的增殖和分化被两个重要的胞外细胞因子调控，即Stem Cell Factor(SCF)和Erythropoietin(EPO)。这两个细胞因子对红系前体细胞的增殖、分化和存活的一个重要通路就是经由MAPK/RAS/RAF/MEK/ERK来实现的。所以SCF和EPO均可以高效激活RAS，从而为BRAF抑制剂诱导的MAPK通路的矛盾激活打下了基础。In melanoma with BRAFV600E mutation, BRAFV600E mutation directly mediates downstream ERK activation after activating MEK, and finally leads to tumorigenesis. However, in normal BRAF (wild type), the addition of BRAF inhibitors will cause BRAF to be in an activated conformation similar to ATP binding. At this time, BRAF loses its kinase activity, but the conformation becomes activated, which makes it easy to align with the R-Spine of CRAF. The formation of the R506 salt bridge greatly promotes the efficiency of BRAF and CRAF to form heterodimers. The formation of BRAF-CRAF dimers is the key to RAS-RAF-MEK signal transduction in the MAPK pathway. Only in the presence of RAFs can the downstream MEK be activated. But there is a prerequisite for this mechanism (activation of MAPK), that is, the activation of BRAF and CRAF depends on the Ras Binding Domain (RBD) on their proteins. This means that the phosphorylation of BRAF and CRAF depends on RBD and upstream activated Ras-GTP binding, and only in the presence of RAS-GTP (i.e., by normal activation signals, such as cytokine binding or GPCR activation), BRAF inhibition Only drug-induced dimerization of CRAF can be phosphorylated by RAS-GTP, at which point CRAF is phosphorylated at S338, and then the downstream MAPK/ERK activation is triggered by intense pCRAF activation rather than pBRAF. In the process of erythroid development, the proliferation and differentiation of erythroid precursor cells are regulated by two important extracellular cytokines, namely Stem Cell Factor (SCF) and Erythropoietin (EPO). An important pathway for these two cytokines to the proliferation, differentiation and survival of erythroid precursor cells is achieved through MAPK/RAS/RAF/MEK/ERK. Therefore, both SCF and EPO can efficiently activate RAS, thus laying the foundation for the paradoxical activation of MAPK pathway induced by BRAF inhibitors.
本课题接着就验证了这一在其他细胞系中被阐明的BRAF抑制剂矛盾激活MAPKs通路原理是否即是BRAF抑制剂促进红系增殖的原因。在红系分化培养基培养到Day 9时，更换新的培养基后向培养基中进行BRAF抑制剂(SB-590885 1μM、GDC-0879 2μM和Encorafenib 500nM)给药30min，随后收样用Western Blot进行蛋白质定量检验，以验证BRAF抑制剂是否是通过MAPK通路的矛盾激活，从而促进红系前体细胞增殖的。结果表明，BRAF抑制剂SB-590885、GDC-0879和Encorafenib均可以激活pMEK和pERK实现MAPKs通路的矛盾激活(图18)。值得注意的是，SB-590885和GDC-0879具备更强的矛盾激活效果，不仅表现在pMEK和pERK的更强磷酸化上，对pCRAF S338的激活也侧面反应了SB-590885和GDC-0879是更强的MAPKs矛盾激活小分子。综上，基本可以判定BRAF抑制剂通过矛盾激活MAPK/ERK通路。This topic then verified whether the principle of paradoxical activation of the MAPKs pathway by BRAF inhibitors, which has been elucidated in other cell lines, is the reason why BRAF inhibitors promote erythroid proliferation. When the erythroid differentiation medium was cultured to Day 9, the medium was replaced with a new medium and BRAF inhibitors (SB-590885 1 μM, GDC-0879 2 μM and Encorafenib 500 nM) were administered for 30 minutes, and then collected for Western Blot Protein quantification was performed to verify whether BRAF inhibitors promote proliferation of erythroid precursors through paradoxical activation of the MAPK pathway. The results showed that BRAF inhibitors SB-590885, GDC-0879 and Encorafenib could all activate pMEK and pERK to achieve paradoxical activation of the MAPKs pathway (Figure 18). It is worth noting that SB-590885 and GDC-0879 have stronger contradictory activation effects, not only in the stronger phosphorylation of pMEK and pERK, but also in the activation of pCRAF S338. Stronger MAPKs paradoxically activate small molecules. In summary, it can be basically concluded that BRAF inhibitors activate the MAPK/ERK pathway through paradox.
图18：在Day 9时对红系分化培养基中的细胞收样并通过Western Blot进行蛋白质定量检验。Figure 18: Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot.
该实施例说明，在红系培养条件下，BRAF抑制剂确实在上游RAS激活的背景下(由EPO和SCF介导的)矛盾激活了MAPK通路，且这一矛盾激活是经由CRAF(pCRAF S338)的激活实现的。This example demonstrates that under erythroid culture conditions, BRAF inhibitors do paradoxically activate the MAPK pathway in the context of upstream RAS activation (mediated by EPO and SCF) and that this paradoxical activation is via CRAF (pCRAF S338) The activation is achieved.
实施例19：BRAF抑制剂矛盾激活MAPK/ERK通路存在浓度依赖效应Example 19: BRAF inhibitors paradoxically activate MAPK/ERK pathway and have concentration-dependent effects
图19：在Day 9时对红系分化培养基中的细胞收样并通过Western Blot进行蛋白质定量检验。Figure 19: Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot.
该实施例说明，在红系培养条件下，BRAF抑制剂中的Type 1抑制剂SB-590885和GDC-0879最显著的激活了MAPK通路。其他的BRAF抑制剂对MAPK的激活效率显然低于SB和GDC。值得一提的是，PLX-8394作为新一代避免矛盾激活的BRAF抑制剂，其基本不引发MAPK的矛盾激活。这说明BRAF抑制剂的结构确实决定了MAPK是否激活的最终结果。This example shows that, under erythroid culture conditions, Type 1 inhibitors SB-590885 and GDC-0879 among BRAF inhibitors most significantly activate the MAPK pathway. Other BRAF inhibitors were significantly less efficient at activating MAPK than SB and GDC. It is worth mentioning that PLX-8394, as a new generation of BRAF inhibitor that avoids paradoxical activation, basically does not cause paradoxical activation of MAPK. This shows that the structure of BRAF inhibitors does determine the final result of MAPK activation.
实施例20：BRAF抑制剂矛盾激活MAPK/ERK通路同时依赖于上游细胞因子信号和下游信号转导Example 20: Paradoxical activation of MAPK/ERK pathway by BRAF inhibitors depends on both upstream cytokine signaling and downstream signal transduction
为进一步研究BRAF抑制剂矛盾激活红系发育中的MAPK通路的原理是否依赖于上游RAS蛋白的激活，接下来本课题进行了两方面的探究。In order to further study whether the principle of paradoxical activation of the MAPK pathway in erythroid development by BRAF inhibitors is dependent on the activation of upstream RAS proteins, the following two aspects of this topic were explored.
第一，BRAF抑制剂的矛盾激活是否依赖于上游的信号通路的激活。这一点至关重要，一方面上游RAS的激活是RAF二聚化并最终导致矛盾激活的基础，如果不存在RAS的激活，RAF形成二聚化也不会导致MAPKs通路的激活和信号传导。另一方面，造血干细胞的造血发育至各谱系的过程中，造血干细胞首先需要从静止状态被激活为***状态，类似一种长期造血干细胞向短期造血干细胞的转换，这一转换过程伴随着细胞***和增殖的强烈激活，这一过程依赖于胞外信号对其进入***的刺激。而从造血干细胞向终末各谱系的血细胞发育过程中，也需要各种谱系相关的细胞因子刺激，这些细胞因子中一个比较普遍的就是干细胞因子(SCF)。SCF的受体即干细胞因子受体(KIT)广泛的分布于造血各谱系中具备高度增殖能力的前体细胞中，SCF和KIT的结合极大促进细胞增殖，其促进增殖的最主要的通路即是MAPKs通路。除SCF外，红系的***(EPO)也激活RAS。事实上，SCF和EPO均不同程度的激活RAS/MAPK通路从而促进红系祖细胞的增殖、存活和发育。所以，细胞因子带来的激活状态的RAS是正常造血和红系发育的结果，而这一结果是矛盾激活的基础。事实上，SCF和EPO均不同程度的激活RAS/MAPK通路从而促进红系祖细胞的增殖、存活和发育。所以，细胞因子带来的激活状态的RAS是正常造血和红系发育的结果，而这一结果是矛盾激活的基础。First, whether the paradoxical activation of BRAF inhibitors depends on the activation of upstream signaling pathways. This is crucial. On the one hand, the activation of upstream RAS is the basis for RAF dimerization and eventually lead to paradoxical activation. If there is no activation of RAS, RAF dimerization will not lead to the activation and signal transduction of MAPKs pathway. On the other hand, during the hematopoietic development of hematopoietic stem cells to each lineage, hematopoietic stem cells first need to be activated from a quiescent state to a dividing state, similar to a transition from a long-term hematopoietic stem cell to a short-term hematopoietic stem cell, which is accompanied by cell division and strong activation of proliferation, a process that relies on extracellular signals to stimulate its entry into division. The development of blood cells from hematopoietic stem cells to terminal lineages also requires the stimulation of various lineage-related cytokines. One of the more common cytokines is stem cell factor (SCF). The receptor of SCF, the stem cell factor receptor (KIT), is widely distributed in the precursor cells with high proliferative ability in various hematopoietic lineages. The combination of SCF and KIT greatly promotes cell proliferation. The most important pathway for promoting proliferation is It is the MAPKs pathway. In addition to SCF, the erythroid erythropoietin (EPO) also activates RAS. In fact, both SCF and EPO activate the RAS/MAPK pathway to varying degrees to promote the proliferation, survival and development of erythroid progenitor cells. Therefore, the activated RAS brought about by cytokines is the result of normal hematopoiesis and erythroid development, and this result is the basis of paradoxical activation. In fact, both SCF and EPO activate the RAS/MAPK pathway to varying degrees to promote the proliferation, survival and development of erythroid progenitor cells. Therefore, the activated RAS brought about by cytokines is the result of normal hematopoiesis and erythroid development, and this result is the basis of paradoxical activation.
第二，BRAF抑制剂是一种潜在促进细胞增殖的药物，所以势必需要考虑是否会带来超广谱的异常增殖或者潜在的致癌可能性。而对BRAF下游MEK蛋白磷酸化药理学抑制是一个常见的用于肿瘤治疗的方式。一方面，本课题需要证明BRAF抑制剂不会带来不依赖于RAS的增殖，不会有显著的致癌效果；另一方面，本课题也需证明对BRAF下游MEK/ERK的直接抑制是否可以直接阻断矛盾激活。Second, BRAF inhibitors are drugs that potentially promote cell proliferation, so it is necessary to consider whether it will bring about a broad spectrum of abnormal proliferation or potential carcinogenesis. Pharmacological inhibition of BRAF downstream MEK protein phosphorylation is a common approach for tumor therapy. On the one hand, this project needs to prove that BRAF inhibitors will not cause RAS-independent proliferation and will not have significant carcinogenic effects; on the other hand, this project also needs to prove whether the direct inhibition of BRAF downstream MEK/ERK can directly Block paradox activation.
所以接下来进行了两方面的实验验证。第一，在进行BRAF抑制剂给药的同时进行MEK抑制剂PD-0325901给药，以验证是否对MEK的抑制可以抑制BRAF抑制剂带来的矛盾激活(图20上)。第二，在完全剥夺细胞因子的情况下确定BRAF抑制剂还具备促进矛盾激活的能力。Therefore, two experimental verifications were carried out next. First, the MEK inhibitor PD-0325901 was administered at the same time as the BRAF inhibitor to verify whether MEK inhibition can suppress the paradoxical activation brought about by the BRAF inhibitor (Figure 20, top). Second, we determined that BRAF inhibitors also possess the ability to promote paradoxical activation in the event of complete cytokine deprivation.
图20：在Day 9时红系分化培养基中的细胞收样并通过Western Blot进行蛋白质定量检验。A：左边四道为正常培养基给药，右边四道为左边四种情况下另加1μM PD-0325901。B：左边四道为正常培养基给药，右边四道为在无SCF和EPO的无细胞因子培养基对的情况下进行给药。C：由左到右分别在含SCF和EPO，只含SCF，只含EPO的情况下进行给药。Figure 20: Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot. A: The four lanes on the left are normal medium administration, and the four lanes on the right are the addition of 1 μM PD-0325901 under the four conditions on the left. B: The four lanes on the left are normal medium administration, and the four lanes on the right are administration under the condition of cytokine-free medium pair without SCF and EPO. C: From left to right, the drug was administered under the condition of containing SCF and EPO, containing only SCF, and containing only EPO.
本实施例结果证明，在BRAF抑制剂存在的情况下，MEK抑制剂给药可以显著抑制下游的ERK激活。在培养基中无SCF和EPO的情况下，BRAF抑制剂给药完全不激活MAPK/ERK通路(图B)。最后，SCF似乎更强的促进了MAPK通路上游的RAS激活，使得细胞可以引发更强的矛盾激活(图C)。综上，BRAF抑制剂矛盾激活MAPK/ERK通路同时依赖于上游细胞因子信号对RAS的激活和下游MEK信号的转导。The results of this example demonstrate that in the presence of BRAF inhibitors, administration of MEK inhibitors can significantly inhibit downstream ERK activation. In the absence of SCF and EPO in the medium, BRAF inhibitor administration did not activate the MAPK/ERK pathway at all (Panel B). Finally, SCF appeared to more strongly promote RAS activation upstream of the MAPK pathway, allowing cells to elicit stronger paradoxical activation (Panel C). In summary, the paradoxical activation of MAPK/ERK pathway by BRAF inhibitors depends on the activation of RAS by upstream cytokine signals and the transduction of downstream MEK signals.
实施例21：BRAF抑制剂矛盾激活MAPK/ERK通路主要依赖于CRAF的激活Example 21: Paradoxical activation of MAPK/ERK pathway by BRAF inhibitors is mainly dependent on activation of CRAF
为研究BRAF抑制剂矛盾激活红系发育中的MAPK通路的机制是否如之前的报道类似，即是通过BRAF促进CRAF激活实现的，本课题接着着手构建RAF蛋白敲降的***来进行矛盾激活机制的验证。人的原代细胞普遍难以导入基因编辑***，从而很难进行基于CRISPR的敲除，故而本课题转而考虑使用慢病毒感染导入shRNA进行稳定敲降。由于RAF蛋白一共有3种变体：ARAF、BRAF和CRAF。故接下来通过构建分别靶向RAF三种单体的敲降质粒，通过慢病毒的方式对UCB-hCD34+接细胞进行感染和敲降。In order to study whether the mechanism of BRAF inhibitor paradoxically activating the MAPK pathway in erythroid development is similar to previous reports, that is, through BRAF promoting CRAF activation, this project then proceeded to construct a RAF protein knockdown system to carry out paradoxical activation mechanism verify. Human primary cells are generally difficult to introduce gene editing systems, making it difficult to perform CRISPR-based knockout. Therefore, this topic considers using lentiviral infection to introduce shRNA for stable knockdown. There are three variants of the RAF protein: ARAF, BRAF, and CRAF. Therefore, by constructing knockdown plasmids targeting the three monomers of RAF, the UCB-hCD34+ cells were infected and knocked down by lentivirus.
在完成构建慢病毒介导的敲降后，通过流式细胞仪分选出慢病毒感染上的Scramble、ARAF KD、BRAF KD、CRAF KD细胞株并进行为期七天的给药或正常情况下的培养，在第七天时比较各组进行给药导致的扩增细胞倍数。After the construction of lentivirus-mediated knockdown, Scramble, ARAF KD, BRAF KD, CRAF KD cell lines infected with lentivirus were sorted by flow cytometry and administered for seven days or cultured under normal conditions , on the seventh day, the amplified cell folds caused by administration of each group were compared.
如图21的A所示，本次针对每个RAF单体均构建了两条shRNA序列。在蛋白水平上均显示了较高的敲降效果。且针对RAF单体的敲降并不影响MAPK/ERK通路的转导。As shown in A of Figure 21, two shRNA sequences were constructed for each RAF monomer this time. All showed high knockdown effects at the protein level. And the knockdown of RAF monomer did not affect the transduction of MAPK/ERK pathway.
图21：在Day 9时红系分化培养基中的细胞收样并通过Western Blot进行蛋白质定量检验。A：各敲降组的蛋白表达情况。B：对照组和敲降组中BRAF抑制剂给药组相对DMSO处理组在Day 7时细胞数量倍增的比较。每个条件设置3个生物学重复，统计数据为平均值±SD，ns：无显著性差异，P>0.05；**：P<0.01。Figure 21: Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot. A: Protein expression of each knockdown group. B: Comparison of the doubling of the number of cells in the BRAF inhibitor administration group on Day 7 compared with the DMSO treatment group in the control group and the knockdown group. Three biological replicates were set for each condition, and the statistical data were mean ± SD, ns: no significant difference, P>0.05; **: P<0.01.
本实施例，尤其图21的B所示证明，仅有CRAF的敲降小幅度但显著降低给药后的BRAF抑制剂扩增效果，证明CRAF是BRAF抑制剂介导的红系发育过程种MAPK通路矛盾激活的关键效应因子。This example, especially as shown in B of Figure 21, proves that only the knockdown of CRAF is small but significantly reduces the amplification effect of BRAF inhibitors after administration, proving that CRAF is a MAPK in the erythroid development process mediated by BRAF inhibitors Key effectors of pathway paradoxical activation.
实施例22：BRAF抑制剂使红系发育在转录谱上呈现延迟但仅有限扰动转录谱Example 22: BRAF inhibitors delay but only limitedly perturb transcriptional profiles of erythroid development
BRAF抑制剂给药已知使得红系分化过程中表面的分化标志蛋白表达明显延迟；在生长曲线上，红系前体细胞在经历了6天的指数级扩增后，细胞数量增长的速度显著放缓，这是红系终末分化的特 征，即在最后阶段增殖和分化的平衡偏向分化。而BRAF抑制剂给药也的确延迟了细胞增殖数量减缓的时间，所以在细胞表面标志物上和细胞增殖上，均可以确定BRAF抑制剂导致红系发育明显延迟。而在BRAF抑制剂处理72h后，同样可以确定BRAF抑制剂也可在转录水平上延迟红细胞的分化。Administration of BRAF inhibitors is known to significantly delay the expression of surface differentiation marker proteins during erythroid differentiation; on the growth curve, after 6 days of exponential expansion of erythroid precursor cells, the cell number increased significantly slowing, which is characteristic of erythroid terminal differentiation, i.e., the balance of proliferation and differentiation is skewed toward differentiation during the final stages. The administration of BRAF inhibitors did delay the time of slowing down the number of cell proliferation, so it can be confirmed that BRAF inhibitors lead to a significant delay in erythroid development in terms of cell surface markers and cell proliferation. After 72 hours of BRAF inhibitor treatment, it can also be determined that BRAF inhibitor can also delay the differentiation of erythrocytes at the transcriptional level.
图22：分选红系前体细胞(CD71+，CD235-)的细胞，GDC-0879 2μM给药72h后进行转录谱测序。A：处理组(GDC)和对照组(DMSO)的特定基因的表达差异。B：对照组和BRAF抑制剂给药组的差异基因火山图(Foldchange>1.5；FDR<0.1)。C：对下调的基因(FDR<0.1)进行GSEA富集分析。Figure 22: Cells of erythroid precursor cells (CD71+, CD235-) were sorted, and transcriptional profiling was performed after 2 μM administration of GDC-0879 for 72 hours. A: Differences in the expression of specific genes between the treatment group (GDC) and the control group (DMSO). B: Volcano map of differential genes between the control group and the BRAF inhibitor-administered group (Foldchange>1.5; FDR<0.1). C: GSEA enrichment analysis of downregulated genes (FDR<0.1).
本实施例证明，BRAF抑制剂处理有限影响了转录谱，延迟了红系分化，展现了更多的早期干祖细胞特征。且从转录谱角度说明了BRAF抑制剂和糖皮质激素的协同效果。This example demonstrates that BRAF inhibitor treatment limitedly affected the transcriptional profile, delayed erythroid differentiation, and exhibited more early stem-progenitor characteristics. And it illustrates the synergistic effect of BRAF inhibitors and glucocorticoids from the perspective of transcriptional profiling.

Claims (96)

  1. 一种用于治疗原发性或继发性贫血的药物，其包含BRAF激酶抑制剂和一种或多种药学上可接受的赋形剂。A medicine for treating primary or secondary anemia, which comprises a BRAF kinase inhibitor and one or more pharmaceutically acceptable excipients.
  2. 权利要求1的药物，其中所述药物还包含TGF-β抑制剂和/或SMAD2/3抑制剂和/或糖皮质激素和/或***和/或干细胞因子。The medicine according to claim 1, wherein said medicine further comprises TGF-β inhibitor and/or SMAD2/3 inhibitor and/or glucocorticoid and/or erythropoietin and/or stem cell factor.
  3. 权利要求1或2的药物，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：The medicament of claim 1 or 2, wherein the BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, the BRAF kinase inhibitor promotes RAF protein dimerization; preferably, the BRAF kinase inhibitor induces αC in BRAF kinase - the IN conformation of at least one of the helix and the DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopes thereof derivative:
    Figure PCTCN2022122562-appb-100001
    Figure PCTCN2022122562-appb-100001
    Figure PCTCN2022122562-appb-100002
    Figure PCTCN2022122562-appb-100002
    Figure PCTCN2022122562-appb-100003
    Figure PCTCN2022122562-appb-100003
    Figure PCTCN2022122562-appb-100004
    Figure PCTCN2022122562-appb-100004
    优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、L-779450、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
    最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  4. 权利要求1或2的药物，其中所述BRAF激酶抑制剂为下式的化合物：The medicine of claim 1 or 2, wherein said BRAF kinase inhibitor is a compound of the following formula:
    Figure PCTCN2022122562-appb-100005
    Figure PCTCN2022122562-appb-100005
    其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、 -C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
    X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
    R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
    R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
    式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、- P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
    R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
    R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
    R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
    Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
    保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  5. 权利要求1或2的药物，其中所述BRAF激酶抑制剂为下式的化合物：The medicine of claim 1 or 2, wherein said BRAF kinase inhibitor is a compound of the following formula:
    Figure PCTCN2022122562-appb-100006
    Figure PCTCN2022122562-appb-100006
    其中，in,
    X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
    Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
    R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
    R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
    Ar为式a)或b)的基团：Ar is a group of formula a) or b):
    Figure PCTCN2022122562-appb-100007
    Figure PCTCN2022122562-appb-100007
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    R 15为O或N-OH； R 15 is O or N-OH;
    X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  6. 权利要求1或2的药物，其中所述BRAF激酶抑制剂为下式的化合物：The medicine of claim 1 or 2, wherein said BRAF kinase inhibitor is a compound of the following formula:
    Figure PCTCN2022122562-appb-100008
    Figure PCTCN2022122562-appb-100008
    其中，in,
    X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
    Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
    R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
    R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
    Ar是下式a)或b)：Ar is the following formula a) or b):
    Figure PCTCN2022122562-appb-100009
    Figure PCTCN2022122562-appb-100009
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  7. 权利要求2-6中任一项的药物，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：
    Figure PCTCN2022122562-appb-100010
    Figure PCTCN2022122562-appb-100011
    Figure PCTCN2022122562-appb-100012
    Figure PCTCN2022122562-appb-100013
    The medicine according to any one of claims 2-6, wherein said TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof:
    Figure PCTCN2022122562-appb-100010
    Figure PCTCN2022122562-appb-100011
    Figure PCTCN2022122562-appb-100012
    Figure PCTCN2022122562-appb-100013
    优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
    更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  8. 权利要求2-6中任一项的药物，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Luspatercept、Sotatercept、
    Figure PCTCN2022122562-appb-100014
    The medicine of any one of claims 2-6, wherein said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept,
    Figure PCTCN2022122562-appb-100014
  9. 权利要求1-8中任一项的药物，其中所述贫血为由红细胞生成减少或缺陷引起的贫血。The medicament according to any one of claims 1-8, wherein the anemia is anemia caused by decreased or defective erythropoiesis.
  10. 权利要求9的药物，其中所述由红细胞生成减少或缺陷引起的贫血为原发性的，包括原发性再生障碍性贫血、Diamond-Blackfan贫血(DBA)、Shwachman-Diamond综合征、先天性角化不良、Fanconi贫血、先天性红细胞生成障碍性贫血(CDA)、地中海型贫血、镰刀状细胞性贫血、骨髓增生异常综合征导致的贫血。The medicament of claim 9, wherein said anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome, congenital angular Anemia due to dysplasia, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, myelodysplastic syndrome.
  11. 权利要求9的药物，其中所述由红细胞生成减少或缺陷引起的贫血为继发性的，包括继发性骨髓增生异常综合征导致的贫血、继发性再生障碍贫血、维生素缺乏型贫血、缺铁性贫血、慢性炎症性贫血、内分泌疾病性贫血、肾功能衰竭导致EPO分泌不足的继发性贫血、造血干细胞移植后血液谱系重建不良；优选地，其中所述继发性再生障碍贫血由下列原因导致：自身免疫类疾病(如***性红斑狼疮、类风湿性关节炎、炎症性肠病等)、其他免疫机制导致的贫血(如ABO血型不合、干细胞移植、坏疽性脓皮病)、淋巴增生性疾病(如慢性淋巴细胞白血病、LGL白血病、霍奇金病、非霍奇淋巴瘤等)、其他血液***恶性肿瘤(如慢性粒细胞白血病、慢性粒单核细胞白血病)、实体瘤(如胸腺瘤、胃癌、乳腺癌、甲状腺癌、肾癌等)、病毒感染(如B19细小病毒、HIV、T细胞白血病淋巴瘤病毒等)、细菌感染(如结核菌、细菌性败血病)、药物和毒素导致的免疫反应(如外源人EPO诱导的抗体相关的纯红再障、铅/苯中毒)、对内/外源EPO抵抗所造成的贫血及各种原因导致的对***抵抗的贫血。The medicament of claim 9, wherein said anemia caused by decreased or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency anemia, deficiency Iron anemia, chronic inflammatory anemia, anemia of endocrine disease, secondary anemia caused by insufficient secretion of EPO due to renal failure, poor blood lineage reconstruction after hematopoietic stem cell transplantation; preferably, wherein said secondary aplastic anemia consists of the following Causes: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, pyoderma gangrenosum), lymphatic Proliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, etc.), other hematological malignancies (such as chronic myeloid leukemia, chronic myelomonocytic leukemia), solid tumors (such as Thymoma, gastric cancer, breast cancer, thyroid cancer, kidney cancer, etc.), viral infection (such as B19 parvovirus, HIV, T-cell leukemia lymphoma virus, etc.), bacterial infection (such as tuberculosis, bacterial septicemia), drugs Immune reactions caused by toxins (such as pure red aplastic anemia induced by exogenous human EPO antibodies, lead/benzene poisoning), anemia caused by endogenous/exogenous EPO resistance, and erythropoietin resistance caused by various reasons resistant anemia.
  12. 权利要求1-8中任一项的药物，其中所述贫血为由于红细胞破坏引起的贫血。The medicament according to any one of claims 1 to 8, wherein the anemia is anemia due to destruction of erythrocytes.
  13. 权利要求12的药物，其中所述由于红细胞破坏引起的贫血为原发性的，包括遗传性球形红细胞增多症、遗传性椭圆细胞增多症、无β脂蛋白血症、酶缺乏症导致的贫血(如丙酮酸激酶和己糖激酶缺陷导致糖酵解缺陷型贫血、葡萄糖6-磷酸脱氢酶缺乏症和谷胱甘肽合成酶缺乏症、氧化应激增加导致的贫血)。The medicine of claim 12, wherein said anemia caused by red blood cell destruction is primary, including hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, anemia caused by enzyme deficiency ( Such as pyruvate kinase and hexokinase deficiency leading to glycolysis deficient anemia, glucose 6-phosphate dehydrogenase deficiency and glutathione synthetase deficiency, anemia due to increased oxidative stress).
  14. 权利要求12的药物，其中所述由于红细胞破坏引起的贫血为继发性的，包括抗体介导的温性自身免疫性溶血性贫血、冷凝集素溶血性贫血、溶血性的疾病(如新生儿溶血症)、红细胞机械损伤(如微血管病性溶血性贫血，包括血栓性血小板减少性紫癜和弥散性血管内凝血)、感染导致的溶血性贫血(如疟疾感染)。The medicine of claim 12, wherein the anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic diseases (such as neonatal hemolytic disease), red blood cell mechanical injury (such as microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), hemolytic anemia caused by infection (such as malaria infection).
  15. 权利要求1-8中任一项的药物，其中所述贫血为失血过多引起的贫血。The medicament according to any one of claims 1 to 8, wherein the anemia is anemia caused by excessive blood loss.
  16. 权利要求15的药物，其中所述失血过多引起的贫血包括早产儿贫血(如实验室检测的频繁采血，并且任选地合并红细胞生成不足)、外/内伤(各种创伤或手术导致的急性失血)、胃肠道病变导致的急性出血(如静脉曲张病变、消化性溃疡)或慢性失血(如血管发育不良)、妇科疾病导致的慢性失血、癌症(包括结肠直肠癌和膀胱癌导致的急性或慢性失血，尤其是在晚期)、以血液为食的肠道线虫感染(如钩虫和鞭虫导致失血性贫血)、医源性贫血、反复抽血和医疗程序造成的失血。The medicine of claim 15, wherein said anemia caused by excessive blood loss comprises premature infant anemia (such as laboratory detection of frequent blood sampling, and optionally combined with insufficient erythropoiesis), external/internal injury (acute blood loss), acute bleeding from gastrointestinal lesions (such as varicose lesions, peptic ulcers) or chronic blood loss (such as angiodysplasia), chronic blood loss from gynecological diseases, acute bleeding from cancer (including colorectal cancer and bladder cancer) or chronic blood loss, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
  17. 权利要求9-16中任一项的药物，其中所述疾病对***或糖皮质激素或其他现有治疗贫血的药物具有耐药性。The medicament according to any one of claims 9-16, wherein the disease is resistant to erythropoietin or glucocorticoids or other existing drugs for the treatment of anemia.
  18. 一种试剂盒，其包括：A test kit comprising:
    a)BRAF激酶抑制剂，和任选地，a) a BRAF kinase inhibitor, and optionally,
    b)***。b) Erythropoietin.
  19. 权利要求18的试剂盒，其还包括：The kit of claim 18, further comprising:
    c)TGF-β抑制剂和/或SMAD2/3抑制剂和/或糖皮质激素和/或干细胞因子。c) TGF-beta inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or stem cell factor.
  20. 权利要求18或19的试剂盒，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC- 螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、BRAF抑制剂1(化合物13)、RAF709、L-779450、LY3009120、Belvarafenib(HM95573)、RO5126766(CH5126766)、TAK-632、PLX-4720、Agerafenib(RXDX-105)、Regorafenib(BAY 73-4506)、Sorafenib(BAY 43-9006)、Donafenib(Sorafenib D3)、Lifirafenib(BGB-283)、BRAF IN 1、Vemurafenib(PLX4032)、RAF265(chir265)、AZ 628、AZ304、CCT196969、Doramapimod(BIRB796)、Encorafenib(LGX818)、Naporafenib(LXH254)、BMS-908662、BGB659、GW5074、MLN2480(TAK580)、ARQ-736或ZM336372；The kit of claim 18 or 19, wherein the BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, the BRAF kinase inhibitor promotes RAF protein dimerization; preferably, the BRAF kinase inhibitor induces dimerization of the BRAF kinase The IN conformation of at least one of the αC-helix and the DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and Isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX -4720, Agerafenib (RXDX-105), Regorafenib (BAY 73-4506), Sorafenib (BAY 43-9006), Donafenib (Sorafenib D3), Lifirafenib (BGB-283), BRAF IN 1, Vemurafenib (PLX4032), RAF265 ( chir265), AZ 628, AZ304, CCT196969, Doramapimod (BIRB796), Encorafenib (LGX818), Naporafenib (LXH254), BMS-908662, BGB659, GW5074, MLN2480 (TAK580), ARQ-736, or ZM336372;
    优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、L-779450、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
    最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  21. 权利要求18或19的试剂盒，其中所述BRAF激酶抑制剂为下式的化合物：The kit of claim 18 or 19, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100015
    Figure PCTCN2022122562-appb-100015
    其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、 -S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
    X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
    R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
    R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
    式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环 基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
    R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
    R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
    R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
    Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
    保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  22. 权利要求18或19的试剂盒，其中所述BRAF激酶抑制剂为下式的化合物：The kit of claim 18 or 19, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100016
    Figure PCTCN2022122562-appb-100016
    其中，in,
    X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
    Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
    R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
    R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
    Ar为式a)或b)的基团：Ar is a group of formula a) or b):
    Figure PCTCN2022122562-appb-100017
    Figure PCTCN2022122562-appb-100017
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    R 15为O或N-OH； R 15 is O or N-OH;
    X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  23. 权利要求18或19的试剂盒，其中所述BRAF激酶抑制剂为下式的化合物：The kit of claim 18 or 19, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100018
    Figure PCTCN2022122562-appb-100018
    其中，in,
    X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
    Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
    R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
    R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
    Ar是下式a)或b)：Ar is the following formula a) or b):
    Figure PCTCN2022122562-appb-100019
    Figure PCTCN2022122562-appb-100019
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  24. 权利要求19-23中任一项的试剂盒，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761、Galunisertib(LY2157299)、LY364947、SB431542、LDN-193189、SB525334、SB505124、GW788388、RepSox(E-616452)、K02288、BIBF-0775、TP0427736、A-83-01、LDN-214117、SD-208、Vactosertib(TEW-7197)、LDN-212854、Dorsomorphin(Compound C)或LY3200882；The kit according to any one of claims 19-23, wherein said TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299), LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214118, SDact -7197), LDN-212854, Dorsomorphin (Compound C) or LY3200882;
    优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
    更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  25. 权利要求19-24中任一项的试剂盒，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Luspatercept、Sotatercept、SIS3 HCl、Alantolactone、Halofuginone和AUDA。The kit according to any one of claims 19-24, wherein the SMAD2/3 inhibitor is selected from the group consisting of the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives: Luspatercept, Sotatercept , SIS3 HCl, Alantolactone, Halofuginone, and AUDA.
  26. BRAF激酶抑制剂，或者BRAF激酶抑制剂和***和/或干细胞因子，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合在制备用于扩增红系前体细胞或成红细胞的药物中的用途。BRAF kinase inhibitor, or BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or BRAF kinase inhibitor and TGF-beta inhibitor, or BRAF kinase inhibitor and SMAD2/3 inhibitor, or BRAF kinase inhibitor , TGF-beta inhibitors and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of a medicament for expanding erythroid precursor cells or erythroblasts.
  27. 权利要求26的用途，其中所述BRAF激酶抑制剂延缓红细胞的终末期分化进程。The use of claim 26, wherein said BRAF kinase inhibitor delays the progression of terminal differentiation of erythrocytes.
  28. 权利要求26或27的用途，其中所述BRAF激酶抑制剂促使成红细胞具有更多的自我更新。The use of claim 26 or 27, wherein said BRAF kinase inhibitor promotes more self-renewal of erythroblasts.
  29. 权利要求26-28中任一项的用途，其中所述红系前体细胞扩增后产生成熟脱核的红细胞。The use of any one of claims 26-28, wherein the erythroid precursor cells are expanded to produce mature denucleated erythrocytes.
  30. 权利要求26-29中任一项的用途，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、BRAF抑制剂1(化合物13)、RAF709、L-779450、LY3009120、Belvarafenib(HM95573)、RO5126766(CH5126766)、TAK-632、PLX-4720、Agerafenib(RXDX-105)、Regorafenib(BAY 73-4506)、Sorafenib(BAY 43-9006)、Donafenib(Sorafenib D3)、Lifirafenib(BGB-283)、BRAF IN 1、Vemurafenib(PLX4032)、RAF265(chir265)、AZ 628、AZ304、CCT196969、Doramapimod(BIRB 796)、Encorafenib(LGX818)、Naporafenib(LXH254)、BMS-908662、BGB659、GW5074、MLN2480(TAK580)、ARQ-736或ZM336372；The use of any one of claims 26-29, wherein the BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, the BRAF kinase inhibitor promotes RAF protein dimerization; preferably, the BRAF kinase inhibitor induces IN conformation of at least one of the αC-helix and DFG domain in BRAF kinase, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, poly Crystal forms and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK- 632, PLX-4720, Agerafenib (RXDX-105), Regorafenib (BAY 73-4506), Sorafenib (BAY 43-9006), Donafenib (Sorafenib D3), Lifirafenib (BGB-283), BRAF IN 1, Vemurafenib (PLX4032) , RAF265 (chir265), AZ 628, AZ304, CCT196969, Doramapimod (BIRB 796), Encorafenib (LGX818), Naporafenib (LXH254), BMS-908662, BGB659, GW5074, MLN2480 (TAK580), ARQ-736, or ZM3;3
    优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同 位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、L-779450、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
    最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  31. 权利要求26-29中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 26-29, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100020
    Figure PCTCN2022122562-appb-100020
    其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
    X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环 基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
    R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
    R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
    式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
    R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
    R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
    R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
    Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
    保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  32. 权利要求26-29中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 26-29, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100021
    Figure PCTCN2022122562-appb-100021
    其中，in,
    X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
    Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
    R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
    R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
    Ar为式a)或b)的基团：Ar is a group of formula a) or b):
    Figure PCTCN2022122562-appb-100022
    Figure PCTCN2022122562-appb-100022
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    R 15为O或N-OH； R 15 is O or N-OH;
    X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  33. 权利要求26-29中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 26-29, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100023
    Figure PCTCN2022122562-appb-100023
    其中，in,
    X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
    Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
    R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
    R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
    Ar是下式a)或b)：Ar is the following formula a) or b):
    Figure PCTCN2022122562-appb-100024
    Figure PCTCN2022122562-appb-100024
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  34. 权利要求26-33中任一项的用途，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761、Galunisertib(LY2157299)、LY364947、SB431542、LDN-193189、SB525334、SB505124、GW788388、RepSox(E-616452)、K02288、BIBF-0775、TP0427736、A-83-01、LDN-214117、SD-208、Vactosertib(TEW-7197)、LDN-212854、Dorsomorphin(Compound C)或LY3200882；The use of any one of claims 26-33, wherein the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761, Galunisertib ( LY2157299), LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214018, Vact-2 7197), LDN-212854, Dorsomorphin (Compound C) or LY3200882;
    优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
    更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  35. 权利要求26-34中任一项的用途，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Luspatercept、Sotatercept、SIS3 HCl、Alantolactone、Halofuginone和AUDA。The use of any one of claims 26-34, wherein the SMAD2/3 inhibitor is selected from the group consisting of the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives: Luspatercept, Sotatercept, SIS3 HCl, Alantolactone, Halofuginone, and AUDA.
  36. BRAF激酶抑制剂，或者BRAF激酶抑制剂和***和/或干细胞因子，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合在制备用于治疗治疗原发性或继发性贫血的药物中的用途。BRAF kinase inhibitor, or BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or BRAF kinase inhibitor and TGF-beta inhibitor, or BRAF kinase inhibitor and SMAD2/3 inhibitor, or BRAF kinase inhibitor , TGF-β inhibitors and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of medicines for the treatment of primary or secondary anemia.
  37. 权利要求36的用途，其中所述贫血为由红细胞生成减少或缺陷引起的贫血。The use of claim 36, wherein said anemia is anemia caused by decreased or defective erythropoiesis.
  38. 权利要求37的用途，其中所述由红细胞生成减少或缺陷引起的贫血为原发性的，包括原发性再生障碍性贫血、Diamond-Blackfan贫血(DBA)、Shwachman-Diamond综合征、先天性角化不良、Fanconi贫血、先天性红细胞生成障碍性贫血(CDA)、地中海型贫血、镰刀状细胞性贫血、骨髓增生异常综合征导致的贫血。The purposes of claim 37, wherein said anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome, congenital angular Anemia due to dysplasia, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, myelodysplastic syndrome.
  39. 权利要求37的用途，其中所述由红细胞生成减少或缺陷引起的贫血为继发性的，包括继发性骨髓增生异常综合征导致的贫血、继发性再生障碍贫血、维生素缺乏型贫血、缺铁性贫血、慢性炎症性贫血、内分泌疾病性贫血、肾功能衰竭导致EPO分泌不足的继发性贫血、造血干细胞移植后血液谱系重建不良；优选地，其中所述继发性再生障碍贫血由下列原因导致：自身免疫类疾病(如***性红斑狼疮、类风湿性关节炎、炎症性肠病等)、其他免疫机制导致的贫血(如ABO血型不合、干细胞移植、坏疽性脓皮病)、淋巴增生性疾病(如慢性淋巴细胞白血病、LGL白血病、霍奇金病、非霍奇淋巴瘤等)、其他血液***恶性肿瘤(如慢性粒细胞白血病、慢性粒单核细胞白血病)、实体瘤(如胸腺瘤、胃癌、乳腺癌、甲状腺癌、肾癌等)、病毒感染(如B19细小病毒、HIV、T细胞白血病淋巴瘤病毒等)、细菌感染(如结核菌、细菌性败血病)、药物和毒素导致的免疫反应(如外源人EPO诱导的抗体相关的纯红再障、铅/苯中毒)、对内/外源EPO抵抗所造成的贫血及各种原因导致的对***抵抗的贫血。The use of claim 37, wherein said anemia caused by reduced or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency anemia, deficiency Iron anemia, chronic inflammatory anemia, anemia of endocrine disease, secondary anemia caused by insufficient secretion of EPO due to renal failure, poor blood lineage reconstruction after hematopoietic stem cell transplantation; preferably, wherein said secondary aplastic anemia consists of the following Causes: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, pyoderma gangrenosum), lymphatic Proliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, etc.), other hematological malignancies (such as chronic myeloid leukemia, chronic myelomonocytic leukemia), solid tumors (such as Thymoma, gastric cancer, breast cancer, thyroid cancer, kidney cancer, etc.), viral infection (such as B19 parvovirus, HIV, T-cell leukemia lymphoma virus, etc.), bacterial infection (such as tuberculosis, bacterial septicemia), drugs Immune reactions caused by toxins (such as pure red aplastic anemia induced by exogenous human EPO antibodies, lead/benzene poisoning), anemia caused by endogenous/exogenous EPO resistance, and erythropoietin resistance caused by various reasons resistant anemia.
  40. 权利要求36的用途，其中所述贫血为由于红细胞破坏引起的贫血。The use of claim 36, wherein the anemia is anemia due to destruction of red blood cells.
  41. 权利要求40的用途，其中所述由于红细胞破坏引起的贫血为原发性的，包括遗传性球形红细胞增多症、遗传性椭圆细胞增多症、无β脂蛋白血症、酶缺乏症导致的贫血(如丙酮酸激酶和己糖激酶缺陷导致糖酵解缺陷型贫血、葡萄糖6-磷酸脱氢酶缺乏症和谷胱甘肽合成酶缺乏症、氧化应激增加导致的贫血)。The purposes of claim 40, wherein said anemia caused by red blood cell destruction is primary, including hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, anemia caused by enzyme deficiency ( Such as pyruvate kinase and hexokinase deficiency leading to glycolysis deficient anemia, glucose 6-phosphate dehydrogenase deficiency and glutathione synthetase deficiency, anemia due to increased oxidative stress).
  42. 权利要求40的用途，其中所述由于红细胞破坏引起的贫血为继发性的，包括抗体介导的温性自身免疫性溶血性贫血、冷凝集素溶血性贫血、溶血性的疾病(如新生儿溶血症)、红细胞机械损伤(如微血管病性溶血性贫血，包括血栓性血小板减少性紫癜和弥散性血管内凝血)、感染导致的溶血性贫血(如疟疾感染)。The purposes of claim 40, wherein said anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic diseases (such as neonatal hemolytic disease), red blood cell mechanical injury (such as microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), hemolytic anemia caused by infection (such as malaria infection).
  43. 权利要求36的用途，其中所述贫血为失血过多引起的贫血。The use of claim 36, wherein the anemia is anemia caused by excessive blood loss.
  44. 权利要求43的用途，其中所述失血过多引起的贫血包括早产儿贫血(如实验室检测的频繁采血，并且任选地合并红细胞生成不足)、外/内伤(各种创伤或手术导致的急性失血)、胃肠道病变导致的急性出血(如静脉曲张病变、消化性溃疡)或慢性失血(如血管发育不良)、妇科疾病导致的慢性失血、癌 症(包括结肠直肠癌和膀胱癌导致的急性或慢性失血，尤其是在晚期)、以血液为食的肠道线虫感染(如钩虫和鞭虫导致失血性贫血)、医源性贫血、反复抽血和医疗程序造成的失血。The purposes of claim 43, wherein said anemia caused by excessive blood loss comprises anemia of premature infants (such as frequent blood sampling in laboratory testing, and optionally combined with insufficient erythropoiesis), external/internal injury (acute blood loss), acute bleeding from gastrointestinal lesions (such as varicose lesions, peptic ulcers) or chronic blood loss (such as angiodysplasia), chronic blood loss from gynecological diseases, acute bleeding from cancer (including colorectal cancer and bladder cancer) or chronic blood loss, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
  45. 权利要求36-44中任一项的用途，其中所述疾病对***、糖皮质激素或其他现有治疗贫血药物具有耐药性。The use according to any one of claims 36-44, wherein the disease is resistant to erythropoietin, glucocorticoids or other existing drugs for the treatment of anemia.
  46. 权利要求36-45中任一项的用途，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、BRAF抑制剂1(化合物13)、RAF709、L-779450、LY3009120、Belvarafenib(HM95573)、RO5126766(CH5126766)、TAK-632、PLX-4720、Agerafenib(RXDX-105)、Regorafenib(BAY 73-4506)、Sorafenib(BAY 43-9006)、Donafenib(Sorafenib D3)、Lifirafenib(BGB-283)、BRAF IN 1、Vemurafenib(PLX4032)、RAF265(chir265)、AZ 628、AZ304、CCT196969、Doramapimod(BIRB 796)、Encorafenib(LGX818)、Naporafenib(LXH254)、BMS-908662、BGB659、GW5074、MLN2480(TAK580)、ARQ-736或ZM336372；The use of any one of claims 36-45, wherein the BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, the BRAF kinase inhibitor promotes RAF protein dimerization; preferably, the BRAF kinase inhibitor induces IN conformation of at least one of the αC-helix and DFG domain in BRAF kinase, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, poly Crystal forms and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK- 632, PLX-4720, Agerafenib (RXDX-105), Regorafenib (BAY 73-4506), Sorafenib (BAY 43-9006), Donafenib (Sorafenib D3), Lifirafenib (BGB-283), BRAF IN 1, Vemurafenib (PLX4032) , RAF265 (chir265), AZ 628, AZ304, CCT196969, Doramapimod (BIRB 796), Encorafenib (LGX818), Naporafenib (LXH254), BMS-908662, BGB659, GW5074, MLN2480 (TAK580), ARQ-736, or ZM3;3
    优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、L-779450、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
    最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  47. 权利要求36-45中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 36-45, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100025
    Figure PCTCN2022122562-appb-100025
    其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、 -S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
    X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
    R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
    R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
    式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
    R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
    R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
    R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
    Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
    保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  48. 权利要求36-45中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 36-45, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100026
    Figure PCTCN2022122562-appb-100026
    其中，in,
    X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
    Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
    R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
    R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
    Ar为式a)或b)的基团：Ar is a group of formula a) or b):
    Figure PCTCN2022122562-appb-100027
    Figure PCTCN2022122562-appb-100027
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    R 15为O或N-OH； R 15 is O or N-OH;
    X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  49. 权利要求36-45中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 36-45, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100028
    Figure PCTCN2022122562-appb-100028
    其中，in,
    X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
    Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
    R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
    R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
    Ar是下式a)或b)：Ar is the following formula a) or b):
    Figure PCTCN2022122562-appb-100029
    Figure PCTCN2022122562-appb-100029
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  50. 权利要求36-49中任一项的用途，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761、Galunisertib(LY2157299)、LY364947、SB431542、LDN-193189、SB525334、SB505124、GW788388、RepSox(E-616452)、K02288、BIBF-0775、TP0427736、A-83-01、LDN-214117、SD-208、Vactosertib(TEW-7197)、LDN-212854、Dorsomorphin(Compound C)或LY3200882；The use of any one of claims 36-49, wherein the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761, Galunisertib ( LY2157299), LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214018, Vact-2 7197), LDN-212854, Dorsomorphin (Compound C) or LY3200882;
    优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
    更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  51. 权利要求36-50中任一项的用途，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Luspatercept、Sotatercept、SIS3 HCl、Alantolactone、Halofuginone和AUDA。The use of any one of claims 36-50, wherein the SMAD2/3 inhibitor is selected from the group consisting of the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives: Luspatercept, Sotatercept, SIS3 HCl, Alantolactone, Halofuginone, and AUDA.
  52. BRAF激酶抑制剂，或者BRAF激酶抑制剂和***和/或干细胞因子，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合在扩增红系前体细胞或成红细胞中的用途。BRAF kinase inhibitor, or BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or BRAF kinase inhibitor and TGF-beta inhibitor, or BRAF kinase inhibitor and SMAD2/3 inhibitor, or BRAF kinase inhibitor , TGF-beta inhibitors and SMAD2/3 inhibitors, and their use in combination with glucocorticoids for expanding erythroid precursor cells or erythroblasts.
  53. 权利要求52的用途，其中所述BRAF激酶抑制剂延缓了红细胞的终末期分化进程。The use of claim 52, wherein said BRAF kinase inhibitor delays the progression of terminal phase differentiation of erythrocytes.
  54. 权利要求52或53的用途，其中所述BRAF激酶抑制剂促进了成红细胞具有更多的自我更新。The use of claim 52 or 53, wherein said BRAF kinase inhibitor promotes greater self-renewal of erythroblasts.
  55. 权利要求52-54中任一项的用途，其中所述红系前体细胞扩增后产生成熟脱核的红细胞。The use of any one of claims 52-54, wherein the erythroid precursor cells are expanded to produce mature denucleated erythrocytes.
  56. 权利要求52-55中任一项的用途，其中所述红系前体细胞选自红细胞集落形成单位(CFU-E)、爆发形成单位红细胞祖细胞(BFU-E)和原成红细胞。The use of any one of claims 52-55, wherein the erythroid precursor cells are selected from the group consisting of erythroid colony forming units (CFU-E), burst forming unit erythroid progenitors (BFU-E) and proerythroblasts.
  57. 权利要求52-56中任一项的用途，其中所述红系前体细胞为基因改造的红系前体细胞或可搭 载药物的红系前体细胞。The use according to any one of claims 52-56, wherein the erythroid precursor cells are genetically modified erythroid precursor cells or drug-loadable erythroid precursor cells.
  58. 权利要求57的用途，其中所述基因改造的红系前体细胞能够治疗遗传性疾病，如苯丙酮尿症和癌症。The use of claim 57, wherein said genetically modified erythroid precursor cells are capable of treating genetic diseases such as phenylketonuria and cancer.
  59. 权利要求52-58中任一项的用途，其中所述红系前体细胞或成红细胞来自血液来源，包括哺乳动物例如小鼠、大鼠、人类等的血液和血液制品。The use according to any one of claims 52-58, wherein said erythroid precursor cells or erythroblasts are derived from blood sources, including blood and blood products of mammals such as mice, rats, humans and the like.
  60. 权利要求52-59中任一项的用途，其中所述红系前体细胞或成红细胞为从骨髓、脐带血或胚胎/诱导多能性干细胞(iPSC/ESC)获得的CD34+来源的细胞。The use according to any one of claims 52-59, wherein the erythroid precursor cells or erythroblasts are CD34+ derived cells obtained from bone marrow, umbilical cord blood or embryonic/induced pluripotent stem cells (iPSC/ESC).
  61. 权利要求52-60中任一项的用途，其中所述红系前体细胞或成红细胞来自外周血单核细胞(PBMC)的体外分化和扩增的人红系前体细胞和干细胞，或者来自PBMC中的干细胞。The use of any one of claims 52-60, wherein the erythroid precursor cells or erythroblasts are derived from in vitro differentiated and expanded human erythroid precursor cells and stem cells from peripheral blood mononuclear cells (PBMC), or from Stem cells in PBMCs.
  62. 权利要求61的用途，其中所述干细胞为造血干细胞。The use of claim 61, wherein said stem cells are hematopoietic stem cells.
  63. 权利要求62的用途，其中所述造血干细胞来源于骨髓或脐带血。The use of claim 62, wherein said hematopoietic stem cells are derived from bone marrow or umbilical cord blood.
  64. 权利要求63的用途，其中所述干细胞在培养之前或培养期间进行基因改造。The use of claim 63, wherein said stem cells are genetically modified before or during culture.
  65. 权利要求52-64中任一项的用途，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、BRAF抑制剂1(化合物13)、RAF709、L-779450、LY3009120、Belvarafenib(HM95573)、RO5126766(CH5126766)、TAK-632、PLX-4720、Agerafenib(RXDX-105)、Regorafenib(BAY 73-4506)、Sorafenib(BAY 43-9006)、Donafenib(Sorafenib D3)、Lifirafenib(BGB-283)、BRAF IN 1、Vemurafenib(PLX4032)、RAF265(chir265)、AZ 628、AZ304、CCT196969、Doramapimod(BIRB 796)、Encorafenib(LGX818)、Naporafenib(LXH254)、BMS-908662、BGB659、GW5074、MLN2480(TAK580)、ARQ-736或ZM336372；The use of any one of claims 52-64, wherein the BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, the BRAF kinase inhibitor promotes RAF protein dimerization; preferably, the BRAF kinase inhibitor induces IN conformation of at least one of the αC-helix and DFG domain in BRAF kinase, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, poly Crystal forms and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK- 632, PLX-4720, Agerafenib (RXDX-105), Regorafenib (BAY 73-4506), Sorafenib (BAY 43-9006), Donafenib (Sorafenib D3), Lifirafenib (BGB-283), BRAF IN 1, Vemurafenib (PLX4032) , RAF265 (chir265), AZ 628, AZ304, CCT196969, Doramapimod (BIRB 796), Encorafenib (LGX818), Naporafenib (LXH254), BMS-908662, BGB659, GW5074, MLN2480 (TAK580), ARQ-736, or ZM3;3
    优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、L-779450、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
    最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  66. 权利要求52-64中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 52-64, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100030
    Figure PCTCN2022122562-appb-100030
    其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
    X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
    R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
    R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、 -SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
    式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
    R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
    R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
    R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
    Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
    保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  67. 权利要求52-64中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 52-64, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100031
    Figure PCTCN2022122562-appb-100031
    其中，in,
    X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
    Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
    R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
    R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
    Ar为式a)或b)的基团：Ar is a group of formula a) or b):
    Figure PCTCN2022122562-appb-100032
    Figure PCTCN2022122562-appb-100032
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    R 15为O或N-OH； R 15 is O or N-OH;
    X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  68. 权利要求52-64中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 52-64, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100033
    Figure PCTCN2022122562-appb-100033
    其中，in,
    X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
    Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
    R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
    R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
    Ar是下式a)或b)：Ar is the following formula a) or b):
    Figure PCTCN2022122562-appb-100034
    Figure PCTCN2022122562-appb-100034
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  69. 权利要求52-68中任一项的用途，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761、Galunisertib(LY2157299)、LY364947、SB431542、LDN-193189、SB525334、SB505124、GW788388、RepSox(E-616452)、K02288、BIBF-0775、TP0427736、A-83-01、LDN-214117、SD-208、Vactosertib(TEW-7197)、LDN-212854、Dorsomorphin(Compound C)或LY3200882；The use of any one of claims 52-68, wherein the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761, Galunisertib ( LY2157299), LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214018, Vact-2 7197), LDN-212854, Dorsomorphin (Compound C) or LY3200882;
    优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
    更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  70. 权利要求52-69中任一项的用途，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Luspatercept、Sotatercept、SIS3 HCl、Alantolactone、Halofuginone和AUDA。The use of any one of claims 52-69, wherein the SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3 HCl, Alantolactone, Halofuginone, and AUDA.
  71. 权利要求65-68中任一项的用途，其中所述BRAF激酶抑制剂以1nM至100μM，优选100nM至10μM的浓度、更优选1μM至10μM的浓度使用。The use according to any one of claims 65-68, wherein the BRAF kinase inhibitor is used at a concentration of 1 nM to 100 μM, preferably 100 nM to 10 μM, more preferably 1 μM to 10 μM.
  72. 权利要求52-71中任一项的用途，其中还同时使用***。The use according to any one of claims 52-71, wherein erythropoietin is also used concomitantly.
  73. 一种扩增红系前体细胞或成红细胞的方法，其包括向受试者给药BRAF激酶抑制剂，或者BRAF激酶抑制剂和***和/或干细胞因子，或者BRAF激酶抑制剂和TGF-β抑制剂，或者BRAF激酶抑制剂和SMAD2/3抑制剂，或者BRAF激酶抑制剂、TGF-β抑制剂和SMAD2/3抑制剂，以及他们与糖皮质激素的组合。A method of expanding erythroid precursor cells or erythroblasts comprising administering to a subject a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and TGF-beta inhibitors, or BRAF kinase inhibitors and SMAD2/3 inhibitors, or BRAF kinase inhibitors, TGF-beta inhibitors and SMAD2/3 inhibitors, and combinations thereof with glucocorticoids.
  74. 权利要求73的方法，其中所述方法延缓了红细胞的终末期分化进程。73. The method of claim 73, wherein said method retards the progression of terminal differentiation of erythrocytes.
  75. 权利要求73或74的方法，其中所述方法促进了成红细胞具有更多的自我更新。73. The method of claim 73 or 74, wherein said method promotes greater self-renewal of erythroblasts.
  76. 权利要求73-75中任一项的方法，其中所述方法产生成熟脱核的红细胞。73. The method of any one of claims 73-75, wherein the method produces mature denucleated red blood cells.
  77. 权利要求73-76中任一项的方法，其中所述红系前体细胞选自红细胞集落形成单位(CFU-E)、爆发形成单位红细胞祖细胞(BFU-E)和原成红细胞。73. The method of any one of claims 73-76, wherein the erythroid precursor cells are selected from the group consisting of erythroid colony forming units (CFU-E), burst forming unit erythroid progenitors (BFU-E), and proerythroblasts.
  78. 权利要求73-77中任一项的方法，其中所述受试者为哺乳动物，例如小鼠、大鼠、人类等。The method of any one of claims 73-77, wherein the subject is a mammal, such as a mouse, a rat, a human, or the like.
  79. 权利要求73-78中任一项的方法，其中所述方法用于治疗原发性或继发性贫血。The method of any one of claims 73-78, wherein the method is for the treatment of primary or secondary anemia.
  80. 权利要求79的方法，其中所述贫血为由红细胞生成减少或缺陷引起的贫血。79. The method of claim 79, wherein said anemia is anemia caused by decreased or defective erythropoiesis.
  81. 权利要求80的方法，其中所述由红细胞生成减少或缺陷引起的贫血为原发性的，包括原发性再生障碍性贫血、Diamond-Blackfan贫血(DBA)、Shwachman-Diamond综合征、先天性角化不良、Fanconi贫血、先天性红细胞生成障碍性贫血(CDA)、地中海型贫血、镰刀状细胞性贫血、骨髓增生异常综合征导致的贫血。The method of claim 80, wherein said anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome, congenital angular Anemia due to dysplasia, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, myelodysplastic syndrome.
  82. 权利要求80的方法，其中所述由红细胞生成减少或缺陷引起的贫血为继发性的，包括继发性骨髓增生异常综合征导致的贫血、继发性再生障碍贫血、维生素缺乏型贫血、缺铁性贫血、慢性炎症性贫血、内分泌疾病性贫血、肾功能衰竭导致EPO分泌不足的继发性贫血、造血干细胞移植后血液谱系重建不良；优选地，其中所述继发性再生障碍贫血由下列原因导致：自身免疫类疾病(如***性红斑狼疮、类风湿性关节炎、炎症性肠病等)、其他免疫机制导致的贫血(如ABO血型不合、干细胞移植、坏疽性脓皮病)、淋巴增生性疾病(如慢性淋巴细胞白血病、LGL白血病、霍奇金病、非霍奇淋巴瘤等)、其他血液***恶性肿瘤(如慢性粒细胞白血病、慢性粒单核细胞白血病)、实体瘤(如胸腺瘤、胃癌、乳腺癌、甲状腺癌、肾癌等)、病毒感染(如B19细小病毒、HIV、T细胞白血病淋巴瘤病毒等)、细菌感染(如结核菌、细菌性败血病)、药物和毒素导致的免疫反应(如外源人EPO诱导的抗体相关的纯红再障、铅/苯中毒)、对内/外源EPO抵抗所造成的贫血及各种原因导致的对***抵抗的贫血。The method of claim 80, wherein said anemia caused by decreased or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency anemia, deficiency Iron anemia, chronic inflammatory anemia, anemia of endocrine disease, secondary anemia caused by insufficient secretion of EPO due to renal failure, poor blood lineage reconstruction after hematopoietic stem cell transplantation; preferably, wherein said secondary aplastic anemia consists of the following Causes: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, pyoderma gangrenosum), lymphatic Proliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, etc.), other hematological malignancies (such as chronic myeloid leukemia, chronic myelomonocytic leukemia), solid tumors (such as Thymoma, gastric cancer, breast cancer, thyroid cancer, kidney cancer, etc.), viral infection (such as B19 parvovirus, HIV, T-cell leukemia lymphoma virus, etc.), bacterial infection (such as tuberculosis, bacterial septicemia), drugs Immune reactions caused by toxins (such as pure red aplastic anemia induced by exogenous human EPO antibodies, lead/benzene poisoning), anemia caused by endogenous/exogenous EPO resistance, and erythropoietin resistance caused by various reasons resistant anemia.
  83. 权利要求79的方法，其中所述贫血为由于红细胞破坏引起的贫血。79. The method of claim 79, wherein said anemia is anemia due to destruction of red blood cells.
  84. 权利要求83的方法，其中所述由于红细胞破坏引起的贫血为原发性的，包括遗传性球形红细胞增多症、遗传性椭圆细胞增多症、无β脂蛋白血症、酶缺乏症导致的贫血(如丙酮酸激酶和己糖激酶缺陷导致糖酵解缺陷型贫血、葡萄糖6-磷酸脱氢酶缺乏症和谷胱甘肽合成酶缺乏症、氧化应激增加导致的贫血)。The method of claim 83, wherein the anemia caused by destruction of red blood cells is primary, including anemia caused by hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, enzyme deficiency ( Such as pyruvate kinase and hexokinase deficiency leading to glycolysis deficient anemia, glucose 6-phosphate dehydrogenase deficiency and glutathione synthetase deficiency, anemia due to increased oxidative stress).
  85. 权利要求83的方法，其中所述由于红细胞破坏引起的贫血为继发性的，包括抗体介导的温性自身免疫性溶血性贫血、冷凝集素溶血性贫血、溶血性的疾病(如新生儿溶血症)、红细胞机械损伤(如 微血管病性溶血性贫血，包括血栓性血小板减少性紫癜和弥散性血管内凝血)、感染导致的溶血性贫血(如疟疾感染)。The method of claim 83, wherein the anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic diseases (such as neonatal hemolytic disease), red blood cell mechanical injury (such as microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), hemolytic anemia caused by infection (such as malaria infection).
  86. 权利要求79的方法，其中所述贫血为失血过多引起的贫血。79. The method of claim 79, wherein said anemia is anemia caused by excessive blood loss.
  87. 权利要求86的方法，其中所述失血过多引起的贫血包括早产儿贫血(如实验室检测的频繁采血，并且任选地合并红细胞生成不足)、外/内伤(各种创伤或手术导致的急性失血)、胃肠道病变导致的急性出血(如静脉曲张病变、消化性溃疡)或慢性失血(如血管发育不良)、妇科疾病导致的慢性失血、癌症(包括结肠直肠癌和膀胱癌导致的急性或慢性失血，尤其是在晚期)、以血液为食的肠道线虫感染(如钩虫和鞭虫导致失血性贫血)、医源性贫血、反复抽血和医疗程序造成的失血。The method according to claim 86, wherein the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood sampling in laboratory tests, and optionally combined with insufficient erythropoiesis), external/internal injuries (acute blood loss), acute bleeding from gastrointestinal lesions (such as varicose lesions, peptic ulcers) or chronic blood loss (such as angiodysplasia), chronic blood loss from gynecological diseases, acute bleeding from cancer (including colorectal cancer and bladder cancer) or chronic blood loss, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
  88. 权利要求79-87中任一项的方法，其中所述疾病对***或糖皮质激素或其他现有治疗贫血药物具有耐药性。The method of any one of claims 79-87, wherein the disease is resistant to erythropoietin or glucocorticoids or other existing drugs for the treatment of anemia.
  89. 权利要求73-88中任一项的方法，其中所述BRAF激酶抑制剂诱发MAPK矛盾激活；优选地，所述BRAF激酶抑制剂促进RAF蛋白二聚化；优选地，所述BRAF激酶抑制剂诱导BRAF激酶中αC-螺旋和DFG域其中至少一个的IN构象，以及R506的IN构象；优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、BRAF抑制剂1(化合物13)、RAF709、L-779450、LY3009120、Belvarafenib(HM95573)、RO5126766(CH5126766)、TAK-632、PLX-4720、Agerafenib(RXDX-105)、Regorafenib(BAY 73-4506)、Sorafenib(BAY 43-9006)、Donafenib(Sorafenib D3)、Lifirafenib(BGB-283)、BRAF IN 1、Vemurafenib(PLX4032)、RAF265(chir265)、AZ 628、AZ304、CCT196969、Doramapimod(BIRB 796)、Encorafenib(LGX818)、Naporafenib(LXH254)、BMS-908662、BGB659、GW5074、MLN2480(TAK580)、ARQ-736或ZM336372；The method of any one of claims 73-88, wherein the BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, the BRAF kinase inhibitor promotes RAF protein dimerization; preferably, the BRAF kinase inhibitor induces IN conformation of at least one of the αC-helix and DFG domain in BRAF kinase, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, poly Crystal forms and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK- 632, PLX-4720, Agerafenib (RXDX-105), Regorafenib (BAY 73-4506), Sorafenib (BAY 43-9006), Donafenib (Sorafenib D3), Lifirafenib (BGB-283), BRAF IN 1, Vemurafenib (PLX4032) , RAF265 (chir265), AZ 628, AZ304, CCT196969, Doramapimod (BIRB 796), Encorafenib (LGX818), Naporafenib (LXH254), BMS-908662, BGB659, GW5074, MLN2480 (TAK580), ARQ-736, or ZM3;3
    优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330、Dabrafenib、PLX-4720、Sorafenib(BAY 43-9006)、BRAF抑制剂1(化合物13)、Vemurafenib(PLX4032)、Doramapimod(BIRB 796)、Encorafenib(LGX818)、BGB659、TAK-632、LY3009120、GW5074、L-779450、Regorafenib或ZM336372；Preferably, the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885、SB-682330或BMS-908662；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
    更优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879、SB-590885或SB-682330；More preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
    最优选地，所述BRAF激酶抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：GDC-0879或SB-590885。Most preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  90. 权利要求73-88中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 73-88, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100035
    Figure PCTCN2022122562-appb-100035
    其中，A环为：(i)5或6元杂环，其具有一个或两个独立地选自O、N和S的杂原子，(ii)5或6元碳环，其任选地与5或6元杂环稠合，或(iii)苯环，其中所述杂环、碳环和苯环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、＝NOR 20、＝NR 20、＝N +(O)OR 20、＝NNR 20R 21、＝O、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、＝S、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基，和保护基，其中所述烷基、烯基、炔基、芳基、碳环基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基、C 2-C 20杂环基； Wherein, ring A is: (i) 5 or 6 membered heterocyclic rings, which have one or two heteroatoms independently selected from O, N and S, (ii) 5 or 6 membered carbocyclic rings, which are optionally combined with 5- or 6-membered heterocycle fused, or (iii) benzene ring, wherein said heterocycle, carbocycle and benzene ring are optionally substituted by one or more groups independently selected from: F, Cl, Br , I, -C(=Y)R 20 , -C(=Y)OR 20 , C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , =NOR 20 , =NR 20 , =N + (O)OR 20 , =NNR 20 R 21 , =O, -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , =S, - SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 - C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl, and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl, Carbocyclyl and heterocyclyl are optionally and independently substituted with one or more groups independently selected from: F, Cl, Br, I, -C(=Y)R 20 , -C(=Y )OR 20 , -C(=Y)NR 20 R 21 , NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O ) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , C 1 -C 8 alkyl, C 2 -C 8 alkene Base, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl, C 2 -C 20 heterocyclyl;
    X选自C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基； X is selected from C 2 -C 20 heterocyclyl, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP( OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered cyclic lactam, 5-7-membered cyclic lactone, 5-7-membered cyclic sulphonamide, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein the alkyl , alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl;
    R 1选自H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、(C 1-C 8烷基)NR 20R 21、C 2-C 20杂环基、C 3-C 12碳环基和C 6-C 20芳基，其中所述烷基、烯基、炔基、杂环基、碳环基和芳基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、OR 20、CN、C(＝O)NR 20R 21、C(＝O)OR 20、C 1-C 8烷基、(C 1-C 8烷基)NR 20R 21和C 2-C 20杂环基； R 1 is selected from H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, (C 1 -C 8 alkyl) NR 20 R 21 , C 2 -C 20 hetero Cyclic group, C 3 -C 12 carbocyclyl and C 6 -C 20 aryl, wherein the alkyl, alkenyl, alkynyl, heterocyclyl, carbocyclyl and aryl are optionally replaced by one or more Substitution with a group independently selected from F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , OR 20 , CN, C(=O)NR 20 R 21 , C(=O)OR 20 , C 1 -C 8 alkyl, (C 1 -C 8 alkyl)NR 20 R 21 and C 2 -C 20 heterocyclyl;
    R 2选自H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基，或 R 2 is selected from H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR)NR 20 R 21 , -SR 20 , -S(O )R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(=Y )R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7 membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring sulphonolactam, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, wherein The alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl, or
    式Ia的R 1和R 2以及它们连接的原子任选地形成饱和的、部分不饱和的或芳香的5或6元稠合杂环，其具有至少两个独立地选自O、N和S的杂原子，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、- OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R and R of formula Ia and the atoms to which they are attached optionally form a saturated, partially unsaturated or aromatic 5- or 6-membered fused heterocyclic ring having at least two independently selected from O, N and S wherein the heterocycle is optionally substituted by one or more groups independently selected from the group consisting of F, Cl, Br, I, -C(=Y)R 20 , -C(=Y) OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y) NR 20 R 21 , -OR 20 , -OC(=Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), - OP(=Y)(OR 20 )(OR 21 ), -OP(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 ) NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S( O) 2 (OR 20 ), -SC(=Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring Lactone, 5-7 membered ring sulphonyl, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 Aryl and C 2 -C 20 heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally selected from one or more of the following groups independently Substitution: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 3、R 4和R 5独立地选自：H、F、Cl、Br、I、-C(＝Y)R 20、-C(＝Y)OR 20、-C(＝Y)NR 20R 21、-NR 20R 21、-NR 20C(＝Y)R 21、-NR 20C(＝Y)OR 21、-NR 23C(＝Y)NR 20R 21、-OR 20、-OC(＝Y)R 20、-OC(＝Y)OR 20、-OC(＝Y)NR 20R 21、-OS(O) 2(OR 20)、-OP(＝Y)(OR 20)(OR 21)、-OP(OR 20)(OR 21)、-P(＝Y)(OR 20)(OR 21)、-P(＝Y)(OR 23)NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、-S(O) 2NR 20R 21、-S(O)(OR 20)、-S(O) 2(OR 20)、-SC(＝Y)R 20、-SC(＝Y)OR 20、-SC(＝Y)NR 20R 21、5-7元环内酰胺、5-7元环内脂、5-7元环磺内酰胺、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 3-C 12碳环基、C 6-C 20芳基和C 2-C 20杂环基，其中所述烷基、烯基、炔基、碳环基、芳基和杂环基任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、OR 20、NR 20R 21、-SR 20、-S(O)R 20、-S(O) 2R 20、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 3-C 12碳环基和C 2-C 20杂环基； R 3 , R 4 and R 5 are independently selected from: H, F, Cl, Br, I, -C(=Y)R 20 , -C(=Y)OR 20 , -C(=Y)NR 20 R 21 , -NR 20 R 21 , -NR 20 C(=Y)R 21 , -NR 20 C(=Y)OR 21 , -NR 23 C(=Y)NR 20 R 21 , -OR 20 , -OC( =Y)R 20 , -OC(=Y)OR 20 , -OC(=Y)NR 20 R 21 , -OS(O) 2 (OR 20 ), -OP(=Y)(OR 20 )(OR 21 ), -OP(OR 20 )(OR 21 ), -P(=Y)(OR 20 )(OR 21 ), -P(=Y)(OR 23 )NR 20 R 21 , -SR 20 , -S( O)R 20 , -S(O) 2 R 20 , -S(O) 2 NR 20 R 21 , -S(O)(OR 20 ), -S(O) 2 (OR 20 ), -SC(= Y)R 20 , -SC(=Y)OR 20 , -SC(=Y)NR 20 R 21 , 5-7-membered ring lactam, 5-7-membered ring lactone, 5-7-membered ring sultone, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl and C 2 -C 20 heterocyclyl, Wherein said alkyl, alkenyl, alkynyl, carbocyclyl, aryl and heterocyclyl are optionally substituted by one or more groups independently selected from the following groups: F, Cl, Br, I, OR 20 , NR 20 R 21 , -SR 20 , -S(O)R 20 , -S(O) 2 R 20 , C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl , C 6 -C 20 aryl, C 3 -C 12 carbocyclyl and C 2 -C 20 heterocyclyl;
    R 20和R 21独立地选自：H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基和保护基，其中所述烷基、烯基、炔基、芳基和杂环基任选地和独立地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、-C(＝Y)R a、-C(＝Y)OR a、-C(＝Y)NR aR b、-OR a、-OC(＝Y)R a、-OC(＝Y)OR a、-OC(＝Y)NR aR b、-OS(O) 2(OR a)、-OP(＝Y)(OR a)(OR b)、-OP(OR a)(OR b)、-P(＝Y)(OR a)(OR b)、-P(＝Y)(OR)NR aR b、-SR a、-S(O)R a、-S(O) 2R a、-S(O) 2NR aR b、-S(O)(OR a)、-S(O) 2(OR a)、-SC(＝Y)R a、-SC(＝Y)OR a和-SC(＝Y)NR aR b，或者 R 20 and R 21 are independently selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 hetero Cyclic groups and protecting groups, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally and independently substituted by one or more groups independently selected from the group consisting of F, Cl, Br , I, -C(=Y)R a , -C(=Y)OR a , -C(=Y)NR a R b , -OR a , -OC(=Y)R a , -OC(=Y )OR a , -OC(=Y)NR a R b , -OS(O) 2 (OR a ), -OP(=Y)(OR a )(OR b ), -OP(OR a )(OR b ), -P(=Y)(OR a )(OR b ), -P(=Y)(OR)NR a R b , -SR a , -S(O)R a , -S(O) 2 R a , -S(O) 2 NR a R b , -S(O)(OR a ), -S(O) 2 (OR a ), -SC(=Y)R a , -SC(=Y)OR a and -SC(=Y)NR a R b , or
    R 20和R 21以及它们连接的原子形成杂环，其中所述杂环任选地被一个或多个独立地选自以下的基团取代：F、Cl、Br、I、C 1-C 8烷基、C 2-C 8烯基和C 2-C 8炔基； R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
    R 23为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基、C 2-C 20杂环基或保护基； R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
    R a和R b独立地为H、C 1-C 8烷基、C 2-C 8烯基、C 2-C 8炔基、C 6-C 20芳基或C 2-C 20杂环基； R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
    Y独立地为O、S、NR 20、 +N(O)R 20、N(OR 20)、 +N(O)(OR 20)或N-NR 20R 21；以及 Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ; and
    保护基选自三烷基甲硅烷基、二烷基苯基甲硅烷基、苯甲酰氧基、苄基、苄氧基甲基、甲基、甲氧基甲基、三芳基甲基、苯二甲酰亚氨基、叔丁氧基羰基(BOC)、苄氧基羰基(CBz)、9-芴基甲基氧基羰基(Fmoc)和四氢吡喃基，或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。The protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  91. 权利要求73-88中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 73-88, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100036
    Figure PCTCN2022122562-appb-100036
    其中，in,
    X为O、CH 2、CO、S或NH，或者基团X-R 1为氢； X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
    Y 1和Y 2独立地为N或CH； Y 1 and Y 2 are independently N or CH;
    R l为氢、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，其中任一可以任选地被取代；此外，当X为CH 2时，则R l可以为羟基或C 1-6烷氧基，其可以任选地被取代； R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
    R 2为H、C 1-6烷基、C 2-6烯基、C 3-7环烷基、C 5-7环烯基、杂环基、芳基或杂芳基，其中任一可以任选地被取代； R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
    Ar为式a)或b)的基团：Ar is a group of formula a) or b):
    Figure PCTCN2022122562-appb-100037
    Figure PCTCN2022122562-appb-100037
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    R 15为O或N-OH； R 15 is O or N-OH;
    X l和X 2之一为N，另一个为NR 6，其中R 6为氢或C 1-6烷基； One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  92. 权利要求73-88中任一项的用途，其中所述BRAF激酶抑制剂为下式的化合物：The use of any one of claims 73-88, wherein the BRAF kinase inhibitor is a compound of the formula:
    Figure PCTCN2022122562-appb-100038
    Figure PCTCN2022122562-appb-100038
    其中，in,
    X是O、CH 2、CO、S或NH，或X-R 1是H； X is O, CH2 , CO, S or NH, or XR1 is H;
    Y 1和Y 2独立地选自CH或N； Y1 and Y2 are independently selected from CH or N;
    R 1是H、C 1-6烷基、C 3-7环烷基、芳基、芳基C 1-6烷基、杂环基、杂环基C 1-6烷基、杂芳基或杂芳基C 1-6烷基，除H外，其可被任选地取代； R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
    R 2是H，或任选地取代的芳基或杂芳基； R is H, or optionally substituted aryl or heteroaryl;
    Ar是下式a)或b)：Ar is the following formula a) or b):
    Figure PCTCN2022122562-appb-100039
    Figure PCTCN2022122562-appb-100039
    其中A表示稠合的5-至7-元环，任选地包含最多两个选自O、S和NR 5的杂原子，其中R 5为氢或C 1-6烷基，所述环任选地被最多两个选自以下的取代基取代：卤素、C 1-6烷基、羟基、C 1-6烷氧基或酮基； wherein A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
    R 3和R 4独立地选自：氢、卤素、C 1-6烷基、芳基、芳基C 1-6烷基、C 1-6烷氧基、C 1-6烷氧基C 1-6烷基、卤代C 1-6烷基、芳基C 1-6烷氧基、羟基、硝基、氰基、叠氮基、氨基、单取代或二取代-N-C 1-6烷基氨基、酰基氨基、芳基羰基氨基、酰基氧基、羧基、羧基盐、羧基酯、氨基磺酰基、单取代或二取代-N-C 1-6烷基氨基磺酰基、C 1-6烷氧基羰基、芳基氧基羰基、脲基、胍基、C 1-6烷基胍基、脒基、C 1-6烷基脒基、磺酰基氨基、氨基磺酰基、C 1-6烷硫基、C 1-6烷基亚磺酰基或C 1-6烷基磺酰基； R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfonylamino, aminosulfonyl, C 1-6 alkylthio, C 1-6 alkylsulfinyl or C 1-6 alkylsulfonyl;
    X l和X 2之一选自O、S或NR 11，另一个为CH，其中R 11为氢、C 1-6烷基、芳基或芳基C 1-6烷基； One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
    或其立体异构体、互变异构体、可药用盐、溶剂合物、水合物、多晶型和同位素衍生物。Or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof.
  93. 权利要求73-92中任一项的方法，其中所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761、Galunisertib(LY2157299)、LY364947、SB431542、LDN-193189、SB525334、SB505124、GW788388、RepSox(E-616452)、K02288、BIBF-0775、TP0427736、A-83-01、LDN-214117、SD-208、Vactosertib(TEW-7197)、LDN-212854、Dorsomorphin(Compound C)或LY3200882；The method of any one of claims 73-92, wherein the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761, Galunisertib ( LY2157299), LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214018, Vact-2 7197), LDN-212854, Dorsomorphin (Compound C) or LY3200882;
    优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：LY2109761或Galunisertib(LY2157299)；Preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
    更优选地，所述TGF-β抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Galunisertib(LY2157299)。More preferably, the TGF-β inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  94. 权利要求73-93中任一项的方法，其中所述SMAD2/3抑制剂选自以下化合物或其可药用盐、溶剂合物、水合物、多晶型和同位素衍生物：Luspatercept、Sotatercept、SIS3 HCl、Alantolactone、Halofuginone和AUDA。The method of any one of claims 73-93, wherein the SMAD2/3 inhibitor is selected from the group consisting of the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives: Luspatercept, Sotatercept, SIS3 HCl, Alantolactone, Halofuginone, and AUDA.
  95. 权利要求89-92中任一项的方法，其中所述BRAF激酶抑制剂以1nM至100μM，优选100nM至10μM的浓度、更优选1μM至10μM的浓度使用。The method of any one of claims 89-92, wherein the BRAF kinase inhibitor is used at a concentration of 1 nM to 100 μM, preferably 100 nM to 10 μM, more preferably 1 μM to 10 μM.
  96. 权利要求73-95中任一项的方法，其中还同时使用***。The method of any one of claims 73-95, wherein erythropoietin is also administered concomitantly.
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CN116570599A (en) * 2023-07-04 2023-08-11 四川大学华西医院 Application of VS6766 combined with LXH254 and pharmaceutical composition
CN116570599B (en) * 2023-07-04 2023-10-20 四川大学华西医院 Application of VS6766 in combination with LY3009120 and pharmaceutical composition

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