WO2021148043A1 - Nitrophenyl ether compound, preparation method therefor, pharmaceutical composition thereof, and use thereof - Google Patents

Nitrophenyl ether compound, preparation method therefor, pharmaceutical composition thereof, and use thereof Download PDF

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WO2021148043A1
WO2021148043A1 PCT/CN2021/074875 CN2021074875W WO2021148043A1 WO 2021148043 A1 WO2021148043 A1 WO 2021148043A1 CN 2021074875 W CN2021074875 W CN 2021074875W WO 2021148043 A1 WO2021148043 A1 WO 2021148043A1
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methyl
methoxy
biphenyl
nitro
benzyl
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PCT/CN2021/074875
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French (fr)
Chinese (zh)
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赖宜生
欧阳宜强
赵磊
徐强
郭文洁
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中国药科大学
南京中澳转化医学研究院有限公司
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Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a class of nitrophenyl ether compounds or their stereoisomers, pharmaceutically acceptable salts, crystals, and solvents as inhibitors of PD-1/PD-L1 protein-protein interaction Compounds or prodrugs, their preparation methods, pharmaceutical compositions containing these compounds, and the use of these compounds or compositions in the treatment of diseases related to PD-1/PD-L1 mediated immunosuppression, such as cancer.
  • Immune escape is a basic feature of malignant tumors. Under normal physiological conditions, the body's immune system can recognize foreign molecules and eliminate them in time. But for tumor patients, due to the low immunity of the body and the special biological characteristics of tumor cells, tumor cells can escape the recognition and killing of the immune system through various mechanisms, and ultimately survive and develop in the body. Tumor immune escape is a complex pathological process, and the escape mechanism mediated by immune checkpoints has attracted much attention.
  • Immune checkpoints are the regulators of the immune system in the human body, composed of a series of co-stimulatory molecules and co-suppressive molecules, and play an important regulatory role in the body's immune system.
  • the costimulatory molecules of immune checkpoints mainly include CD27, CD40, OX40, GITR, CD137, OX40 and ICOS, etc.
  • the co-inhibitory molecules are mainly CTLA-4, PD-1, PD-L1, PD-L2, TIM-3 , VISTA and IDO etc.
  • costimulatory molecules can enhance the immune response of the body, thereby helping immune cells to eliminate foreign molecules, while co-suppressive molecules play a negative regulatory role on the immune response, thereby maintaining the immune homeostasis of the body, and avoiding excessive immunity from causing the host Damage to normal tissues.
  • tumor cells can use immune checkpoints to achieve immune evasion.
  • the common escape mechanism is that tumor cells inhibit the activation of T lymphocytes by inducing themselves, antigen presenting cells (APC) and T lymphocytes and other surfaces to overexpress co-inhibitory molecules.
  • PD-1 programmed death receptor 1
  • PD-L1/2 programmed death receptor 1
  • PD-1/PD-L1 is currently used as a target for tumor immunotherapy The point has been fully confirmed.
  • PD-1 can also be expressed at low levels in CD4 - CD8 - T cells, B cells, dendritic cells (DC) and natural killer (NK) cells in the thymus.
  • PD-1 has two ligands, of which PD-L1 is mainly expressed in mature T cells, B cells and some non-hematopoietic cells, but PD-L1 can be expressed in inflammatory factors (such as IFN- ⁇ , TNF- ⁇ and VEGF) Under the induction of expression on a variety of cells.
  • PD-L2 expression range is relatively narrow, mainly expressed in macrophages and DC cells.
  • the negative regulation of immunity by PD-1/PD-L is mainly by inhibiting the PI3K-AKT and RAS signaling pathways, blocking the activation of transcription factors that play an important role in T cell activation, proliferation, function and survival, such as activator protein- 1 (AP-1), nuclear factor of activated T cells (NFAT) and NF- ⁇ B.
  • transcription factors that play an important role in T cell activation, proliferation, function and survival, such as activator protein- 1 (AP-1), nuclear factor of activated T cells (NFAT) and NF- ⁇ B.
  • AP-1 activator protein- 1
  • NFAT nuclear factor of activated T cells
  • NF- ⁇ B NF- ⁇ B
  • the PD-1/PD-L signaling pathway can induce and maintain the tolerance of peripheral tissues during immune responses to prevent excessive immune responses in the tissues.
  • excessive activation of the PD-1/PD-L signaling pathway will inhibit the secretion of immune stimulating factors such as IFN- ⁇ , TNF- ⁇ and IL-2 and the expression of survivin.
  • the abnormality of the PD-1/PD-L signaling pathway is closely related to the occurrence, development and poor prognosis of many human tumors.
  • tumor cells can survive through anti-apoptotic signals and inhibit the activity of antigen-specific T lymphocytes.
  • the use of PD-1 or PD-L1 antibodies to block the PD-1/PD-L signaling pathway can inhibit the growth of tumor cells.
  • T lymphocytes It is mainly by reversing the effect on T lymphocyte signal transduction, reactivating T lymphocytes, promoting the generation of effector T lymphocytes and memory T lymphocytes, and inhibiting the differentiation of regulatory T lymphocytes, and ultimately enhancing the tumor microenvironment
  • the immune killing ability of T lymphocytes can achieve the purpose of treating tumors.
  • PD-1/PD-L1 monoclonal antibody drugs such as Keytruda, Opdivo, Imfinzi, Tecentriq and Bavencio have been marketed worldwide, which are widely used in the clinical treatment of malignant melanoma, non-small cell lung cancer, gastric cancer, liver cancer, kidney cancer, Various solid tumors such as bladder cancer and blood cancer have significant clinical treatment effects, and the indications are still increasing.
  • clinical trials of PD-1/PD-L1 monoclonal antibody combined with other drugs to treat a variety of malignant tumors are also actively being carried out.
  • PD-1/PD-L1 monoclonal antibody drugs also have obvious shortcomings. For example, they can easily lead to excessive activation of T cells and cause immune-related side effects. In addition, they cannot be administered orally, have poor compliance, and are difficult to prepare and purify, resulting in price Expensive etc. Therefore, the development of small molecule inhibitors of PD-1/PD-L1 has important application value.
  • the present invention provides a novel nitrophenyl ether compound.
  • the nitrophenyl ether compounds of the present invention have significant inhibitory activity on the PD-1/PD-L1 protein-protein interaction, so they can be used in the preparation of inhibitors that inhibit PD-1/PD-L1 activity, and can be used as Immune checkpoint inhibitors are used in tumor immunotherapy.
  • the invention also provides a preparation method, pharmaceutical composition and application of the nitrophenyl ether compound.
  • the present invention provides a nitrophenyl ether compound with the structural characteristics of general formula (I), its stereoisomers, metabolites, metabolic precursors, prodrugs, solvates, crystals or Its pharmaceutically acceptable salt:
  • R 1 is selected from: methyl or bromine
  • R 2 is selected from: hydrogen, C 1 -C 4 alkoxy or -O(CH 2 ) n Ar; wherein, n is selected from an integer of 0-4; Ar is selected from an aryl group or an aromatic heterocyclic ring;
  • the heterocycle may optionally contain one or more heteroatoms selected from O, S or N; the aryl or aromatic heterocycle may optionally be substituted by one or more W groups;
  • W is selected from: hydrogen, Halogen, cyano, hydroxyl, mercapto, carboxy, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino or C 1 -C 6 haloalkyl;
  • R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen atom to which R 3 and R 4 are connected Together to form a 5-7 membered heterocyclic ring; the alkyl, alkoxy or cycloalkyl group may be optionally substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, mercapto, and methylmercapto , Carboxyl, amino, furanyl, dimethylamino, hydroxy-substituted isopropylamino, diethylamino, tetrahydropyrrolyl, morpholinyl, N-methylpiperazinyl or -NHCOR 5 ; where R 5 is selected From C 1 -C 8 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
  • R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl, or the nitrogen atom to which R 6 and R 7 are connected Together to form a 5-7 membered heterocyclic ring; the alkyl, alkoxy, cycloalkyl or heterocyclic ring may be optionally substituted by one or more Y groups; Y is selected from: hydrogen, halogen, hydroxyl, mercapto , Carboxyl, amino or acetamide group;
  • the alkyl group represents a straight chain alkyl group, a branched chain alkyl group or a cyclic alkyl group
  • the alkoxy group represents a straight chain alkoxy group, a branched chain alkoxy group or a cyclic alkoxy group
  • Amino represents linear alkylamino, branched alkylamino or cyclic alkylamino
  • the aryl group represents phenyl, naphthyl, acenaphthyl or tetrahydronaphthyl;
  • the aromatic heterocycle represents pyrrolyl, pyrazolyl, imidazolyl, furyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridyl Monocyclic heterocyclic ring of azinyl; or quinolinyl, quinoxalinyl, indolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl , Benzofuranyl, benzothienyl, 2,3-dihydrobenzo[1,4]dioxenyl, or benzo[1,3]dioxolyl bicyclic hetero ring;
  • the halogenated alkyl group is a linear or branched saturated hydrocarbon group, or a cyclic saturated hydrocarbon group, or a cyclic saturated hydrocarbon group connected to a linear or branched saturated hydrocarbon group; wherein one or more hydrogen atoms are replaced by one or more halogens. Atom substitution.
  • R 1 is selected from: methyl or bromine
  • R 2 is selected from: hydrogen or -OCH 2 Ar; wherein, Ar is selected from an aryl group or an aromatic heterocyclic ring; the aromatic heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; The aryl or aromatic heterocycle may be optionally substituted by one or more W groups; W is selected from: hydrogen, halogen, cyano, hydroxyl, mercapto, carboxy, C 1 -C 4 alkyl or C 1- C 4 alkoxy;
  • R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen atom to which R 3 and R 4 are connected Together to form a 5-7 membered heterocyclic ring; the alkyl, alkoxy or cycloalkyl group may be optionally substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, mercapto, and methylmercapto , Carboxyl, amino, furanyl, dimethylamino, hydroxy-substituted isopropylamino, diethylamino, tetrahydropyrrolyl, morpholinyl, N-methylpiperazinyl or -NHCOR 5 ; where R 5 is selected From C 1 -C 8 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
  • R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen to which R 6 and R 7 are connected Atoms together form a 5-7 membered heterocyclic ring; wherein the alkyl, alkoxy, cycloalkyl or heterocyclic ring may be optionally substituted by one or more Y groups; wherein Y is selected from: hydrogen, halogen , Hydroxyl, mercapto, carboxyl, amino or acetamide.
  • R 1 is selected from: methyl or bromine
  • R 2 is selected from: hydrogen or -OCH 2 Ar; wherein, Ar is selected from aryl or aromatic heterocycle; said aromatic heterocycle may optionally contain one or more N atoms; said aryl or aromatic heterocycle The ring may be optionally substituted with one or more W groups; W is selected from: hydrogen or cyano;
  • R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen atom to which R 3 and R 4 are connected Together to form a 5-6 membered heterocycle; the alkyl group or alkoxy group may be optionally substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, carboxyl, methylmercapto, amino, and furan , Dimethylamino, hydroxy-substituted isopropylamino or -NHCOR 5 ; wherein R 5 is selected from C 1 -C 4 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
  • R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or R 6 and R 7 together with the nitrogen atom to which they are connected to form a 5-6 membered heterocyclic ring ; Wherein the alkyl group, alkoxy group or heterocyclic ring may be optionally substituted by one or more Y groups; wherein Y is selected from: hydrogen, halogen, hydroxyl, carboxyl, amino or acetamido.
  • R 1 is selected from: methyl or bromine
  • R 2 is selected from: hydrogen or -OCH 2 Ar; wherein, Ar is selected from an aryl group or a 5-6 membered aromatic heterocyclic ring; the aromatic heterocyclic ring may optionally contain one or more N atoms; the aromatic The group or aromatic heterocyclic ring may be optionally substituted by one or more W groups; W is selected from: hydrogen or cyano;
  • R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl or R 3 and R 4 together with the nitrogen atom to which they are attached form a 5-6 membered heterocyclic ring; the alkyl group may optionally Substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, carboxyl, methylmercapto, amino, furanyl, dimethylamino, hydroxy-substituted dimethylamino or -NHCOR 5 ; wherein R 5 is selected from C 1 -C 4 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
  • R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 6 alkyl or R 6 and R 7 together with the nitrogen atom to which they are connected to form a 5-6 membered heterocyclic ring; wherein the alkyl or hetero The ring may be optionally substituted with one or more Y groups; wherein Y is selected from: hydrogen, halogen, hydroxyl, carboxyl, amino, or acetamido.
  • the indole compound is any one of the following compounds:
  • Another object of the present invention is to provide a preparation method of the compound represented by general formula (I), the preparation method is as follows:
  • R 1 , R 2 , R 3 and R 4 are as defined above.
  • the "method of one or more of the condensation reaction and the hydrolysis reaction” includes only condensation reaction or hydrolysis reaction; or first hydrolysis reaction, then condensation reaction; or first hydrolysis reaction, then condensation reaction, and then hydrolysis Reaction, wait.
  • the pharmaceutically acceptable salt of the compound of general formula (I) can be synthesized by general chemical methods.
  • the salt can be prepared by reacting a free base or acid with an equivalent or excess acid (inorganic acid or organic acid) or base (inorganic base or organic base) in a suitable solvent or solvent composition.
  • the present invention also provides a pharmaceutical composition, which is mainly composed of a therapeutically effective amount of active components and pharmaceutically acceptable excipients; the active components include a compound of general formula (I) or its pharmaceutically acceptable One or more of the accepted salt.
  • the adjuvant includes a pharmaceutically acceptable carrier, diluent and/or excipient.
  • the pharmaceutical composition can be made into various types of dosage unit dosage forms, such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories and injections (solutions and suspensions), etc. Preference is given to tablets, capsules, liquids, suspensions and injections (solutions and suspensions).
  • any excipient known and widely used in the art can be used.
  • the solution or suspension can be sterilized (preferably by adding an appropriate amount of sodium chloride, glucose or glycerin) to prepare an injection that is isotonic with blood.
  • any carriers commonly used in the art can also be used.
  • common dissolving agents and buffering agents can also be added.
  • the content of the composition of the present invention in the pharmaceutical composition is not particularly limited, and can be selected in a wide range, usually 5-95% by mass, preferably 30-85% by mass.
  • the method of administration of the pharmaceutical composition of the present invention is not particularly limited. Various dosage forms can be selected for administration according to the age, sex, and other conditions and symptoms of the patient.
  • the present invention also provides the application of the compound of general formula (I), its pharmaceutically acceptable salt or the said pharmaceutical composition in the preparation of PD-1/PD-L1 inhibitor drugs.
  • the PD-1/PD-L1 inhibitor is used to treat PD-1/PD-L1 mediated immune suppression related diseases, and the related diseases include cancer.
  • the present invention also provides the use of the compound of general formula (I), its pharmaceutically acceptable salt or the pharmaceutical composition in the preparation of a medicine, which is used for the treatment of cancer.
  • the cancers include but are not limited to: malignant melanoma, lung cancer, breast cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, lymphoma, leukemia, prostate cancer, testicular cancer, kidney cancer, brain cancer, head and neck cancer, ovarian cancer
  • malignant melanoma lung cancer, breast cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, lymphoma, leukemia, prostate cancer, testicular cancer, kidney cancer, brain cancer, head and neck cancer, ovarian cancer
  • One or more of cancer, cervical cancer, endometrial cancer, mesothelial cancer, thyroid tumor, liver cancer, and esophageal cancer are examples of cancer, cervical cancer, endometrial cancer, mesothelial cancer, thyroid tumor, liver cancer, and esophageal cancer.
  • the present invention also provides that the compound of general formula (I), its pharmaceutically acceptable salt or the pharmaceutical composition can be combined with one or more other types of therapeutic agents and/or therapeutic methods for the treatment of PD -L/PD-L1 mediated related diseases.
  • therapeutic agents and/or treatment methods include, but are not limited to: chemotherapeutics, targeted antitumor drugs, immune checkpoint inhibitors, immune checkpoint agonists, antitumor vaccines, antiviral agents, antiviral vaccines, Cytokine therapy, adoptive cellular immunotherapy or radiation therapy.
  • chemotherapeutic agents include but are not limited to: alkylating agents, tubulin inhibitors, topozyme inhibitors, platinum drugs, antimetabolites or hormone antitumor drugs.
  • the targeted anti-tumor drugs include, but are not limited to: protein kinase inhibitors, proteasome inhibitors, isocitrate dehydrogenase inhibitors, anti-tumor drugs based on epigenetics, or cell cycle signal pathway inhibitors.
  • the immune checkpoint inhibitors include, but are not limited to: CTLA-4 inhibitors, TIM-3 inhibitors, VISTA inhibitors, LAG3 inhibitors, TIGIT inhibitors, A2AR inhibitors or VTCN1 inhibitors.
  • the immune checkpoint agonists include but are not limited to: STING agonists, 4-1BB agonists, OX40 agonists, ROR ⁇ agonists or ICOS agonists.
  • the nitrophenyl ether compound of the present invention has significant inhibitory activity on the interaction of PD-1/PD-L1, and can effectively block the immunosuppressive effect mediated by PD-1/PD-L1.
  • the in vivo pharmacodynamic evaluation results show that the compound of the present invention can significantly inhibit the growth of transplanted tumors in mice, and significantly promote the infiltration of lymphocytes in tumor tissues, increase the level of INF- ⁇ , reduce the expression of PCNA protein, and affect the immune system. Defective mice transplanted tumors have no effect, indicating that the compound of the present invention can play an anti-tumor effect by activating the host immune response.
  • Figure 1 shows that the compound of the present invention reverses the effect of PD-1/PD-L1 on inhibiting the secretion of INF- ⁇ from PBMC in a dose-dependent manner
  • Figure 2 shows the effect of the compound of the present invention on the viability of Lewis lung cancer cells at different concentrations
  • Figure 3 shows that the compound of the present invention inhibits the growth of transplanted tumors in mice with Lewis lung cancer in a dose-dependent manner
  • Figure 4 shows the effect of the compound of the present invention on the infiltration of T lymphocytes in transplanted tumors in mice, in which: A) CD45 + cells; B) CD45 + CD3 + cells; C) CD4 + CD45 + CD3 + cells; D) CD8 + CD45 + CD3 + cells.
  • Reagents and materials The reagents required for the experiment are all commercially available chemical or analytical products without special instructions.
  • Dissolve 1C (2.0g, 10.1mmol) in 30mL of anhydrous DCM, cool in an ice bath, slowly drip 1mL of phosphorus tribromide (2.7g, 10.1mmol), react for 1h, remove the ice bath, react at room temperature for 8h, spin off The solvent was cooled in an ice bath, 10 mL of water was slowly added dropwise, extracted with ethyl acetate, and concentrated to obtain 2.3 g of a white solid with a yield of 87%.
  • Dissolve 1F in 5mL methanol add LiOH (50mg), react for 5h, spin off the solvent, add 10mL saturated brine, cool in an ice bath, adjust pH to 2 with 6M hydrochloric acid, filter with suction, and dry to obtain a white solid of 65mg, yield 54%.
  • Dissolve 33B (0.8g, 2.2mmol), m-cyanobenzyl bromide (0.5g, 2.4mmol) and K 2 CO 3 (0.6g, 4.4mmol) in 10mL DMF, react at room temperature for 1 hour, add 50mL saturated brine , Suction filtration, drying, to obtain 0.7 g of white solid, with a yield of 67%.
  • Dissolve 33C (0.22g, 0.46mmol) in 5mL DMF, add L-threonine methyl ester hydrochloride (0.14g, 0.92mmol), TEA (0.12g, 1.38mmol), glacial acetic acid (0.14g, 2.3mmol) and sodium cyanoborohydride (0.14g, 2.3mmol), react at room temperature for 24h, add 10mL water, extract with ethyl acetate, concentrate under reduced pressure, and directly put into the next reaction.
  • the purchased kit contains the reagents needed for experiments such as PD-1, PD-L1, Anti-tag1-Eu, Anti-tag2-XL665, Dilute Buffer and Detection Buffer ; Positive drug BMS-1018; M1000 Multifunctional Microplate Reader; Echo520 Ultrasonic Micro Liquid Transfer System (Labcyte).
  • the inhibitory activity of the compounds of the examples on the PD-1/PD-L1 protein-protein interaction is shown in Table 1. The results show that the compound of the present invention can significantly inhibit the interaction of PD-1/PD-L1.
  • BMS-1018 is the No. 1018 compound in patent WO 2015160641 A2.
  • Cytokines are a class of unique proteins with dual effects and regulation. They play an important role in immune regulation in the response of lymphocytes. Activated human peripheral blood mononuclear cells (PBMC) can release cytokines such as IFN- ⁇ , IL-2 and TNF- ⁇ , and when PD-1 expressed on the PBMC membrane binds to its ligand PD-L1, it will inhibit the cell Factor release.
  • PBMC peripheral blood mononuclear cells
  • the purpose of this experiment is to test the ability of the compound of the present invention to reverse PD-1/PD-L1's inhibition of PBMC secretion of INF- ⁇ .
  • the level of INF- ⁇ can be significantly increased at a concentration of 100 nM, which indicates that the compound of the present invention can block the inhibitory effect of PD-1/PD-L1 on PBMC, thereby promoting the secretion of INF- ⁇ .
  • the MTT method is used to detect the effect of the compound of the present invention on the activity of Lewis lung cancer cells, and the purpose is to investigate whether the compound of the present invention has cytotoxicity.
  • PCNA Proliferating cell nuclear antigen
  • mice select female mice aged 7-8 weeks and raise them in an SPF-class animal breeding room for one week. Each mouse weighs about 18-20g.
  • Treatment of tumor cells Collect tumor cells in logarithmic growth phase, centrifuge at 180g for 5 min (4°C), wash twice with pre-cooled PBS, pipette evenly, and make a final cell concentration of 1 ⁇ 10 7 /mL, ice bath for later use.
  • Tumor cell transplantation The tumor cell suspension was inoculated subcutaneously into the right axilla of the mouse, and the number of tumor cells inoculated was 1 ⁇ 10 6 per mouse. The tumor size of the mouse was measured with a vernier caliper every two days, and the mouse weight was weighed once. When the average tumor volume reaches about 40mm 3 , the drug is started.
  • mice When the tumor volume reached a certain size, the animal experiment was ended. Weigh the weight of the mice, take blood from their eyeballs, and euthanize the mice, strip the tumor tissues, weigh the tumor tissues and take pictures. At the same time, some tissues were placed in 10% neutral fixative, samples were sent for paraffin-embedded tissues, paraffin tissue sections were made, and H&E staining, TUNEL and immunohistochemical analysis were performed. Refer to the test kit instructions for experimental operations.
  • mice transplanted with Lewis lung cancer were divided into 4 groups with 6 mice in each group.
  • Model group (solvent: PBS+2% Tween 20+2% DMSO, intragastric administration, once a day), positive control group (BMS-1018, intragastric administration, once a day, dose: 5mg/kg),
  • Drug treatment group 1 compound 33, intragastric administration, once a day, dose: 2 mg/kg
  • drug treatment group 2 compound 33, intragastric administration, once a day, dose: 5 mg/kg.
  • Model group (solvent: PBS+2% Tween 20+2% DMSO, intragastric administration, once a day), positive control group (BMS-1018, intragastric administration, once a day, dose: 5mg/kg), Drug treatment group 1 (compound 33, intragastric administration, once a day, dose: 2 mg/kg), drug treatment group 2 (compound 33, intragastric administration, once a day, dose: 5 mg/kg).
  • mice transplanted with CT26 colorectal tumors were divided into 5 groups with 6 mice in each group.
  • Model group (solvent: PBS+2% Tween 20+2% DMSO, intragastric administration, once a day), positive control group-1 (5-FU, dose: 25mg/kg, intraperitoneal injection, once every two days ), positive control group-2 (BMS-1018, intragastric administration, once a day, dose: 5mg/kg), drug treatment group (compound 33, intragastric administration, once a day, dose: 5mg/kg), combined Administration group (5-FU, dose: 25 mg/kg, intraperitoneal injection, once every two days; compound 33, intragastric administration, once a day, dose: 5 mg/kg).
  • mice transplanted with CT26 colorectal tumors were divided into 4 groups with 6 mice in each group.
  • Model group (solvent: PBS+2% Tween 20+2% DMSO, intragastric administration, once a day), positive control group-1 (5-FU, dose: 25mg/kg, intraperitoneal injection, once every two days ), positive control group-2 (BMS-1018, intragastric administration, once a day, dose: 5mg/kg), drug treatment group (compound 33, intragastric administration, once a day, dose: 5mg/kg).
  • Model group (solvent: PBS+2% Tween 20+2% DMSO, intragastric administration, once a day), positive control group (BMS-1018, intragastric administration, once a day, dose: 5mg/kg), Drug treatment group (Compound 33, intragastric administration, once a day, dose: 5mg/kg).
  • Example Compound 33 can significantly inhibit the growth of Lewis lung cancer transplanted tumors in mice at 2 mg/kg and 5 mg/kg, showing a dose-dependent, and no Affect the weight of mice.
  • the inhibitory activity of compound 33 on transplanted tumors was significantly better than that of the control group BMS-1018 (tumor inhibition rate 19.6%).
  • Example Compound 33 can significantly promote the infiltration of T lymphocytes in tumor tissues at the doses of 2 mg/kg and 5 mg/kg, and increase the level of IFN- ⁇ , Reduce the expression of PCNA protein, and at a dose of 5 mg/kg, compound 33 reverses PD-1/PD-L1 mediated immunosuppression stronger than the BMS-1018 control group.
  • Example Compound 33 showed a dose-dependent inhibition of the growth of B16F1 melanoma transplanted tumors in mice at the doses of 2 mg/kg and 5 mg/kg, and had no effect on the body weight of the mice. At the dose (5 mg/kg), the inhibitory activity of compound 33 on transplanted tumors was better than that of the BMS-1018 control group.
  • the results of immunohistochemistry and TUNEL experiments show that the compound 33 administration group of Example can also significantly promote the infiltration of T lymphocytes in tumor tissues, increase the level of IFN- ⁇ , reduce the expression of PCNA protein, and the effect is stronger than that at the same dose. BMS-1018.
  • Example Compound 33 can inhibit the growth of CT26 colorectal cancer BALB/c mice transplanted tumors, and the combination with 5-FU can increase the tumor inhibition rate of the latter.
  • Example Compound 33 can effectively inhibit the growth of PAN02 pancreatic cancer transplanted tumors in mice at a dose of 5 mg/kg.
  • Immunohistochemistry and TUNEL experiments show that compound 33 can block the immunosuppressive effect mediated by PD-1/PD-L1.
  • T lymphocytes are the core executors of the human immune system and play an important role in tumor immune response.
  • Tumor infiltrating lymphocytes are those white blood cells that leave the bloodstream and enter the tumor. When there are a large number of tumor-infiltrating lymphocytes in the tumor microenvironment, it indicates that the body has initiated an immune response against tumors.
  • the activation of the PD-1/PD-L1 signaling pathway will inhibit the anti-tumor immune microenvironment, resulting in a decrease in lymphocyte infiltration.
  • the purpose of this experiment is to analyze the effect of the compound of the present invention on the infiltration of T lymphocytes in the tumor microenvironment.
  • Example Compound 33 can significantly promote CD45 + white blood cells, CD45 + CD3 + T lymphocytes, CD8 + CD45 + CD3 + cells at the doses of 2 mg/kg and 5 mg/kg.
  • the infiltration of toxic T cells is dose-dependent, while the effect on CD4 + CD45 + CD3 + regulatory T lymphocytes is weak.
  • the ability of Example Compound 33 to promote lymphocyte infiltration is stronger than that of BMS-1018, especially the increase in CD45 + CD3 + CD8 + cytotoxic T lymphocyte infiltration.

Abstract

The present invention relates to the field of drugs, and particularly relates to a nitrophenyl ether compound having structural characteristics of a general formula (I), a stereoisomer, a metabolite, a metabolic precursor, a prodrug, a solvate, a crystal, or a pharmaceutically acceptable salt thereof, a preparation method therefor, and use thereof as PD-1/PD-L1 inhibitors. Pharmacological experiment results show that the compound of the present invention has remarkable inhibition activity on PD-1/PD-L1 protein-protein interaction and can effectively inhibit PD-1/PD-L1 mediated tumor immune escape, so that the compound can be used for preparing an inhibitor drug having the activity of inhibiting PD-1/PD-L1 and can be used as an immune checkpoint inhibitor drug for immunotherapy of tumors.

Description

硝基苯醚类化合物、其制备方法和药物组合物与用途Nitrophenyl ether compound, its preparation method, pharmaceutical composition and use 技术领域Technical field
本发明属于生物医药领域，具体涉及一类作为PD-1/PD-L1蛋白-蛋白相互作用抑制剂的硝基苯醚类化合物或其立体异构体、药学上可以接受的盐、结晶、溶剂化物或前药、它们的制备方法、含有这些化合物的药物组合物、以及这些化合物或组合物在治疗与PD-1/PD-L1介导的免疫抑制有关的疾病如癌症的用途。The invention belongs to the field of biomedicine, and specifically relates to a class of nitrophenyl ether compounds or their stereoisomers, pharmaceutically acceptable salts, crystals, and solvents as inhibitors of PD-1/PD-L1 protein-protein interaction Compounds or prodrugs, their preparation methods, pharmaceutical compositions containing these compounds, and the use of these compounds or compositions in the treatment of diseases related to PD-1/PD-L1 mediated immunosuppression, such as cancer.
背景技术Background technique
免疫逃逸是恶性肿瘤的一种基本特征。在正常生理条件下，人体的免疫***能够识别出异己分子并及时进行清除。但对于肿瘤患者而言，由于机体免疫能力低下和肿瘤细胞特殊的生物学特征，使得肿瘤细胞可以通过各种不同的机制逃避免疫***的识别和杀灭，最终得以在体内生存与发展。肿瘤免疫逃逸是一个复杂的病理过程，其中由免疫检查点介导的逃逸机制深受人们关注。Immune escape is a basic feature of malignant tumors. Under normal physiological conditions, the body's immune system can recognize foreign molecules and eliminate them in time. But for tumor patients, due to the low immunity of the body and the special biological characteristics of tumor cells, tumor cells can escape the recognition and killing of the immune system through various mechanisms, and ultimately survive and develop in the body. Tumor immune escape is a complex pathological process, and the escape mechanism mediated by immune checkpoints has attracted much attention.
免疫检查点是人体内免疫***的调节器，由一系列共刺激分子和共抑制分子组成，在机体免疫***中起重要的调控作用。免疫检查点的共刺激分子主要包括CD27、CD40、OX40、GITR、CD137、OX40和ICOS等，而共抑制分子则主要为CTLA-4、PD-1、PD-L1、PD-L2、TIM-3、VISTA和IDO等。其中，共刺激分子可以增强机体的免疫应答，从而有利于免疫细胞清除异己分子，而共抑制分子则对免疫应答起着负性调节作用，从而维护机体的免疫稳态，避免过度免疫而造成宿主正常组织的损伤。然而，肿瘤细胞却能够利用免疫检查点而实现免疫逃避。其中，常见的逃避机制便是肿瘤细胞通过诱导自身、抗原呈递细胞(APC)和T淋巴细胞等表面过度表达共抑制分子，从而抑制T淋巴细胞的激活。其中，程序性死亡受体1(PD-1)及其配体PD-L1/2作为免疫检查点中重要的共抑制分子受到广泛关注，目前PD-1/PD-L1作为肿瘤免疫治疗的靶点已得到充分的确证。Immune checkpoints are the regulators of the immune system in the human body, composed of a series of co-stimulatory molecules and co-suppressive molecules, and play an important regulatory role in the body's immune system. The costimulatory molecules of immune checkpoints mainly include CD27, CD40, OX40, GITR, CD137, OX40 and ICOS, etc., while the co-inhibitory molecules are mainly CTLA-4, PD-1, PD-L1, PD-L2, TIM-3 , VISTA and IDO etc. Among them, costimulatory molecules can enhance the immune response of the body, thereby helping immune cells to eliminate foreign molecules, while co-suppressive molecules play a negative regulatory role on the immune response, thereby maintaining the immune homeostasis of the body, and avoiding excessive immunity from causing the host Damage to normal tissues. However, tumor cells can use immune checkpoints to achieve immune evasion. Among them, the common escape mechanism is that tumor cells inhibit the activation of T lymphocytes by inducing themselves, antigen presenting cells (APC) and T lymphocytes and other surfaces to overexpress co-inhibitory molecules. Among them, programmed death receptor 1 (PD-1) and its ligand PD-L1/2 have received widespread attention as important co-suppressive molecules in immune checkpoints, and PD-1/PD-L1 is currently used as a target for tumor immunotherapy The point has been fully confirmed.
PD-1除了表达在成熟的T细胞外，还可以低水平表达在胸腺内的CD4 -CD8 -T细胞、B细胞、树突状细胞(DC)和自然杀伤(NK)细胞。PD-1有两个配体，其中PD-L1主要表达在成熟的T细胞、B细胞以及一些非造血类型细胞，但PD-L1可以在炎症因子(如IFN-γ、TNF-α和VEGF)的诱导下于多种细胞上表达。PD-L2表达范围相对较窄，主要是在巨噬细胞和DC细胞中表达。当PD-1与其配体结合后会引起胞质区ITSM结构域中的酪氨酸发生磷酸化，从而通过招募TCR附近的SHP-2磷酸酶，抑制TCR近端激酶的活化，导致TCR-CD3分子和Lck介导的ZAP-70磷酸化水平减弱，进而激活其下游信号通路。PD-1/PD-L对免疫的负调控主要是通过抑制PI3K-AKT和RAS信号通路，阻断对T细胞激活、增殖、功能和生存等具有重要作用的转录因子的活化，如激活蛋白-1(AP-1)、活化T细胞的核因子(NFAT)和NF-κB。另外，还可以通过上调转录因子BATF的表达来抑制T细胞功能。 In addition to being expressed on mature T cells, PD-1 can also be expressed at low levels in CD4 - CD8 - T cells, B cells, dendritic cells (DC) and natural killer (NK) cells in the thymus. PD-1 has two ligands, of which PD-L1 is mainly expressed in mature T cells, B cells and some non-hematopoietic cells, but PD-L1 can be expressed in inflammatory factors (such as IFN-γ, TNF-α and VEGF) Under the induction of expression on a variety of cells. PD-L2 expression range is relatively narrow, mainly expressed in macrophages and DC cells. When PD-1 binds to its ligand, it will cause phosphorylation of the tyrosine in the ITSM domain of the cytoplasm, thereby recruiting SHP-2 phosphatase near the TCR to inhibit the activation of the TCR proximal kinase, leading to TCR-CD3 The molecular and Lck-mediated phosphorylation of ZAP-70 is weakened, which in turn activates its downstream signaling pathways. The negative regulation of immunity by PD-1/PD-L is mainly by inhibiting the PI3K-AKT and RAS signaling pathways, blocking the activation of transcription factors that play an important role in T cell activation, proliferation, function and survival, such as activator protein- 1 (AP-1), nuclear factor of activated T cells (NFAT) and NF-κB. In addition, T cell function can also be suppressed by up-regulating the expression of the transcription factor BATF.
在正常生理条件下，PD-1/PD-L信号通路可以诱导并维持外周组织在免疫反应时的耐受性，以防止组织发生过度免疫应答。当机体处于病理状态下，PD-1/PD-L信号通路过度激活会抑制免疫刺激因子如IFN-γ、TNF-α和IL-2的分泌与存活蛋白的表达。大量研究表明，PD-1/PD-L信号通路的异常与病毒感染、糖尿病、神经变性疾病、器官移植排斥和自身免疫性疾病等密切相关。Under normal physiological conditions, the PD-1/PD-L signaling pathway can induce and maintain the tolerance of peripheral tissues during immune responses to prevent excessive immune responses in the tissues. When the body is in a pathological state, excessive activation of the PD-1/PD-L signaling pathway will inhibit the secretion of immune stimulating factors such as IFN-γ, TNF-α and IL-2 and the expression of survivin. A large number of studies have shown that abnormalities in the PD-1/PD-L signaling pathway are closely related to viral infections, diabetes, neurodegenerative diseases, organ transplant rejection, and autoimmune diseases.
此外，众多研究表明，PD-1/PD-L信号通路的异常与人类多种肿瘤的发生、发展和预后不良存在密切的关系。在肿瘤微环境中，当PD-1/PD-L信号通路被过度激活后，肿瘤细胞可以通过抗凋亡信号以及抑制抗原特异性T淋巴细胞的活性而获得生存。另外，使用PD-1或PD-L1抗体阻断PD-1/PD-L信号通路则能够抑制肿瘤细胞的生长。其主要是通过逆转对T淋巴细胞信号转导的影响，重新激活T淋 巴细胞，同时促进效应T淋巴细胞和记忆T淋巴细胞的生成以及抑制调节性T淋巴细胞的分化，最终增强肿瘤微环境内T淋巴细胞的免疫杀伤能力，从而达到***的目的。目前全球已经有Keytruda、Opdivo、Imfinzi、Tecentriq和Bavencio等多款PD-1/PD-L1单克隆抗体药物上市，广泛应用于临床治疗恶性黑色素瘤、非小细胞肺癌、胃癌、肝癌、肾癌、膀胱癌等多种实体肿瘤和血液癌症，临床治疗效果显著，并且目前适应症还在不断地增加。与此同时，PD-1/PD-L1单抗药物与其它药物联用治疗多种恶性肿瘤的临床试验也在积极的开展中。In addition, numerous studies have shown that the abnormality of the PD-1/PD-L signaling pathway is closely related to the occurrence, development and poor prognosis of many human tumors. In the tumor microenvironment, when the PD-1/PD-L signaling pathway is over-activated, tumor cells can survive through anti-apoptotic signals and inhibit the activity of antigen-specific T lymphocytes. In addition, the use of PD-1 or PD-L1 antibodies to block the PD-1/PD-L signaling pathway can inhibit the growth of tumor cells. It is mainly by reversing the effect on T lymphocyte signal transduction, reactivating T lymphocytes, promoting the generation of effector T lymphocytes and memory T lymphocytes, and inhibiting the differentiation of regulatory T lymphocytes, and ultimately enhancing the tumor microenvironment The immune killing ability of T lymphocytes can achieve the purpose of treating tumors. At present, a variety of PD-1/PD-L1 monoclonal antibody drugs such as Keytruda, Opdivo, Imfinzi, Tecentriq and Bavencio have been marketed worldwide, which are widely used in the clinical treatment of malignant melanoma, non-small cell lung cancer, gastric cancer, liver cancer, kidney cancer, Various solid tumors such as bladder cancer and blood cancer have significant clinical treatment effects, and the indications are still increasing. At the same time, clinical trials of PD-1/PD-L1 monoclonal antibody combined with other drugs to treat a variety of malignant tumors are also actively being carried out.
然而，PD-1/PD-L1单抗药物也存在明显的不足，如易导致T细胞过度激活，引发免疫相关副作用，加上不能口服给药，依从性差，且制备和纯化难度大，造成价格昂贵等。因此研发PD-1/PD-L1小分子抑制剂具有重要应用价值。However, PD-1/PD-L1 monoclonal antibody drugs also have obvious shortcomings. For example, they can easily lead to excessive activation of T cells and cause immune-related side effects. In addition, they cannot be administered orally, have poor compliance, and are difficult to prepare and purify, resulting in price Expensive etc. Therefore, the development of small molecule inhibitors of PD-1/PD-L1 has important application value.
发明内容Summary of the invention
发明目的：针对现有技术存在的问题，本发明提供一种新型的硝基苯醚类化合物。本发明的硝基苯醚类化合物对PD-1/PD-L1蛋白-蛋白相互作用具有显著的抑制活性，因此可以应用在制备具有抑制PD-1/PD-L1活性的抑制剂，并且可以作为免疫检查点抑制剂应用于肿瘤的免疫治疗。Objective of the invention: In view of the problems in the prior art, the present invention provides a novel nitrophenyl ether compound. The nitrophenyl ether compounds of the present invention have significant inhibitory activity on the PD-1/PD-L1 protein-protein interaction, so they can be used in the preparation of inhibitors that inhibit PD-1/PD-L1 activity, and can be used as Immune checkpoint inhibitors are used in tumor immunotherapy.
本发明还提供所述硝基苯醚类化合物的制备方法、药物组合物及其用途。The invention also provides a preparation method, pharmaceutical composition and application of the nitrophenyl ether compound.
技术方案：为了实现上述目的，本发明提供一种具有通式(I)结构特征的硝基苯醚类化合物、其立体异构体、代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐：Technical solution: In order to achieve the above object, the present invention provides a nitrophenyl ether compound with the structural characteristics of general formula (I), its stereoisomers, metabolites, metabolic precursors, prodrugs, solvates, crystals or Its pharmaceutically acceptable salt:
Figure PCTCN2021074875-appb-000001
Figure PCTCN2021074875-appb-000001
其中：in:
R 1选自：甲基或溴； R 1 is selected from: methyl or bromine;
R 2选自：氢、C 1-C 4烷氧基或-O(CH 2) nAr；其中，n选自0-4的整数；Ar选自芳基或芳杂环；所述的芳杂环可任选地包含一个或多个选自O、S或N杂原子；所述的芳基或芳杂环可任选地被一个或多个W基团取代；W选自：氢、卤素、氰基、羟基、巯基、羧基、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氨基或C 1-C 6卤代烷基； R 2 is selected from: hydrogen, C 1 -C 4 alkoxy or -O(CH 2 ) n Ar; wherein, n is selected from an integer of 0-4; Ar is selected from an aryl group or an aromatic heterocyclic ring; The heterocycle may optionally contain one or more heteroatoms selected from O, S or N; the aryl or aromatic heterocycle may optionally be substituted by one or more W groups; W is selected from: hydrogen, Halogen, cyano, hydroxyl, mercapto, carboxy, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino or C 1 -C 6 haloalkyl;
R 3和R 4各自独立地选自：氢、C 1-C 8烷基、C 1-C 8烷氧基、C 3-C 8环烷基或R 3和R 4跟它们连接的氮原子一起形成5-7元杂环；所述的烷基、烷氧基或环烷基可任选地被一个或多个X基团取代；X选自：氢、卤素、羟基、巯基、甲巯基、羧基、氨基、呋喃基、二甲胺基、羟基取代的异丙胺基、二乙胺基、四氢吡咯基、吗啉基、N-甲基哌嗪基或-NHCOR 5；其中R 5选自C 1-C 8烷基；所述的杂环可被羧基或-CONR 6R 7取代； R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen atom to which R 3 and R 4 are connected Together to form a 5-7 membered heterocyclic ring; the alkyl, alkoxy or cycloalkyl group may be optionally substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, mercapto, and methylmercapto , Carboxyl, amino, furanyl, dimethylamino, hydroxy-substituted isopropylamino, diethylamino, tetrahydropyrrolyl, morpholinyl, N-methylpiperazinyl or -NHCOR 5 ; where R 5 is selected From C 1 -C 8 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
R 6和R 7各自独立地选自：氢、C 1-C 8烷基、C 1-C 8烷氧基、C 3-C 8环烷基或R 6和R 7跟它们连接的氮原子一起形成5-7元杂环；所述的烷基、烷氧基、环烷基或杂环可任选地被一个或多个Y基团取代；Y选自：氢、卤素、羟基、巯基、羧基、氨基或乙酰胺基； R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl, or the nitrogen atom to which R 6 and R 7 are connected Together to form a 5-7 membered heterocyclic ring; the alkyl, alkoxy, cycloalkyl or heterocyclic ring may be optionally substituted by one or more Y groups; Y is selected from: hydrogen, halogen, hydroxyl, mercapto , Carboxyl, amino or acetamide group;
所述的烷基代表直链烷基、支链烷基或环状烷基；所述的烷氧基代表直链烷氧基、支链烷氧基或环状烷氧基；所述的烷氨基代表直链烷氨基、支链烷氨基或环状烷氨基；The alkyl group represents a straight chain alkyl group, a branched chain alkyl group or a cyclic alkyl group; the alkoxy group represents a straight chain alkoxy group, a branched chain alkoxy group or a cyclic alkoxy group; Amino represents linear alkylamino, branched alkylamino or cyclic alkylamino;
所述的芳基代表苯基、萘基、苊基或四氢萘基；The aryl group represents phenyl, naphthyl, acenaphthyl or tetrahydronaphthyl;
所述的芳杂环代表吡咯基、吡唑基、咪唑基、呋喃基、噻吩基、噁唑基、异噁唑基、噻唑基、异噻 唑基、吡啶基、嘧啶基、吡嗪基或哒嗪基的单环杂环；或喹啉基、喹喔啉基、吲哚基、苯并咪唑基、苯并噁唑基、苯并异噁唑基、苯并噻唑基、苯并异噻唑基、苯并呋喃基、苯并噻吩基、2,3-二氢苯并[1,4]二氧杂环己烯基、或苯并[1,3]二氧杂环戊烯基的双环杂环；The aromatic heterocycle represents pyrrolyl, pyrazolyl, imidazolyl, furyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridyl Monocyclic heterocyclic ring of azinyl; or quinolinyl, quinoxalinyl, indolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl , Benzofuranyl, benzothienyl, 2,3-dihydrobenzo[1,4]dioxenyl, or benzo[1,3]dioxolyl bicyclic hetero ring;
所述的卤代烷基为直链或支链饱和烃基，或为环状饱和烃基，或为连接直链或支链饱和烃基的环状饱和烃基；其中一个或多个氢原子被一个或多个卤原子取代。The halogenated alkyl group is a linear or branched saturated hydrocarbon group, or a cyclic saturated hydrocarbon group, or a cyclic saturated hydrocarbon group connected to a linear or branched saturated hydrocarbon group; wherein one or more hydrogen atoms are replaced by one or more halogens. Atom substitution.
优选，所述硝基苯醚类化合物结构中：Preferably, in the structure of the nitrophenyl ether compound:
R 1选自：甲基或溴； R 1 is selected from: methyl or bromine;
R 2选自：氢或-OCH 2Ar；其中，Ar选自芳基或芳杂环；所述的芳杂环可任选地包含一个或多个选自O、S或N杂原子；所述的芳基或芳杂环可任选地被一个或多个W基团取代；W选自：氢、卤素、氰基、羟基、巯基、羧基、C 1-C 4烷基或C 1-C 4烷氧基； R 2 is selected from: hydrogen or -OCH 2 Ar; wherein, Ar is selected from an aryl group or an aromatic heterocyclic ring; the aromatic heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; The aryl or aromatic heterocycle may be optionally substituted by one or more W groups; W is selected from: hydrogen, halogen, cyano, hydroxyl, mercapto, carboxy, C 1 -C 4 alkyl or C 1- C 4 alkoxy;
R 3和R 4各自独立地选自：氢、C 1-C 8烷基、C 1-C 8烷氧基、C 3-C 8环烷基或R 3和R 4跟它们连接的氮原子一起形成5-7元杂环；所述的烷基、烷氧基或环烷基可任选地被一个或多个X基团取代；X选自：氢、卤素、羟基、巯基、甲巯基、羧基、氨基、呋喃基、二甲胺基、羟基取代的异丙胺基、二乙胺基、四氢吡咯基、吗啉基、N-甲基哌嗪基或-NHCOR 5；其中R 5选自C 1-C 8烷基；所述的杂环可被羧基或-CONR 6R 7取代； R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen atom to which R 3 and R 4 are connected Together to form a 5-7 membered heterocyclic ring; the alkyl, alkoxy or cycloalkyl group may be optionally substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, mercapto, and methylmercapto , Carboxyl, amino, furanyl, dimethylamino, hydroxy-substituted isopropylamino, diethylamino, tetrahydropyrrolyl, morpholinyl, N-methylpiperazinyl or -NHCOR 5 ; where R 5 is selected From C 1 -C 8 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
其中R 6和R 7各自独立地选自：氢、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 8环烷基或R 6和R 7跟它们连接的氮原子一起形成5-7元杂环；其中所述的烷基、烷氧基、环烷基或杂环可任选地被一个或多个Y基团取代；其中的Y选自：氢、卤素、羟基、巯基、羧基、氨基或乙酰胺基。 Wherein R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen to which R 6 and R 7 are connected Atoms together form a 5-7 membered heterocyclic ring; wherein the alkyl, alkoxy, cycloalkyl or heterocyclic ring may be optionally substituted by one or more Y groups; wherein Y is selected from: hydrogen, halogen , Hydroxyl, mercapto, carboxyl, amino or acetamide.
优选，所述硝基苯醚类化合物结构中：Preferably, in the structure of the nitrophenyl ether compound:
R 1选自：甲基或溴； R 1 is selected from: methyl or bromine;
R 2选自：氢或-OCH 2Ar；其中，Ar选自芳基或芳杂环；所述的芳杂环可任选地包含一个或多个N原子；所述的芳基或芳杂环可任选地被一个或多个W基团取代；W选自：氢或氰基； R 2 is selected from: hydrogen or -OCH 2 Ar; wherein, Ar is selected from aryl or aromatic heterocycle; said aromatic heterocycle may optionally contain one or more N atoms; said aryl or aromatic heterocycle The ring may be optionally substituted with one or more W groups; W is selected from: hydrogen or cyano;
R 3和R 4各自独立地选自：氢、C 1-C 8烷基、C 1-C 8烷氧基、C 3-C 8环烷基或R 3和R 4跟它们连接的氮原子一起形成5-6元杂环；所述的烷基或烷氧基可任选地被一个或多个X基团取代；X选自：氢、卤素、羟基、羧基、甲巯基、氨基、呋喃基、二甲胺基、羟基取代的异丙胺基或-NHCOR 5；其中R 5选自C 1-C 4烷基；所述的杂环可被羧基或-CONR 6R 7取代； R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen atom to which R 3 and R 4 are connected Together to form a 5-6 membered heterocycle; the alkyl group or alkoxy group may be optionally substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, carboxyl, methylmercapto, amino, and furan , Dimethylamino, hydroxy-substituted isopropylamino or -NHCOR 5 ; wherein R 5 is selected from C 1 -C 4 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
其中R 6和R 7各自独立地选自：氢、C 1-C 6烷基、C 1-C 6烷氧基或R 6和R 7跟它们连接的氮原子一起形成5-6元杂环；其中所述的烷基、烷氧基或杂环可任选地被一个或多个Y基团取代；其中的Y选自：氢、卤素、羟基、羧基、氨基或乙酰胺基。 Wherein R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or R 6 and R 7 together with the nitrogen atom to which they are connected to form a 5-6 membered heterocyclic ring ; Wherein the alkyl group, alkoxy group or heterocyclic ring may be optionally substituted by one or more Y groups; wherein Y is selected from: hydrogen, halogen, hydroxyl, carboxyl, amino or acetamido.
优选，所述硝基苯醚类化合物结构中：Preferably, in the structure of the nitrophenyl ether compound:
R 1选自：甲基或溴； R 1 is selected from: methyl or bromine;
R 2选自：氢或-OCH 2Ar；其中，Ar选自芳基或5-6元芳杂环；所述的芳杂环可任选地包含一个或多个N原子；所述的芳基或芳杂环可任选地被一个或多个W基团取代；W选自：氢或氰基； R 2 is selected from: hydrogen or -OCH 2 Ar; wherein, Ar is selected from an aryl group or a 5-6 membered aromatic heterocyclic ring; the aromatic heterocyclic ring may optionally contain one or more N atoms; the aromatic The group or aromatic heterocyclic ring may be optionally substituted by one or more W groups; W is selected from: hydrogen or cyano;
R 3和R 4各自独立地选自：氢、C 1-C 8烷基或R 3和R 4跟它们连接的氮原子一起形成5-6元杂环；所述的烷基可任选地被一个或多个X基团取代；X选自：氢、卤素、羟基、羧基、甲基巯基、氨基、呋喃基、二甲胺基、羟基取代的二甲胺基或-NHCOR 5；其中R 5选自C 1-C 4烷基；所述的杂环可被羧基或-CONR 6R 7取代； R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl or R 3 and R 4 together with the nitrogen atom to which they are attached form a 5-6 membered heterocyclic ring; the alkyl group may optionally Substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, carboxyl, methylmercapto, amino, furanyl, dimethylamino, hydroxy-substituted dimethylamino or -NHCOR 5 ; wherein R 5 is selected from C 1 -C 4 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
其中R 6和R 7各自独立地选自：氢、C 1-C 6烷基或R 6和R 7跟它们连接的氮原子一起形成5-6元杂环；其中所述的烷基或杂环可任选地被一个或多个Y基团取代；其中的Y选自：氢、卤素、羟基、羧基、氨基或乙酰胺基。 Wherein R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 6 alkyl or R 6 and R 7 together with the nitrogen atom to which they are connected to form a 5-6 membered heterocyclic ring; wherein the alkyl or hetero The ring may be optionally substituted with one or more Y groups; wherein Y is selected from: hydrogen, halogen, hydroxyl, carboxyl, amino, or acetamido.
更具体地，所述吲哚类化合物为以下任一化合物：More specifically, the indole compound is any one of the following compounds:
Figure PCTCN2021074875-appb-000002
Figure PCTCN2021074875-appb-000002
本发明的另一目的在于提供通式(I)所示化合物的制备方法，所述制备方法如下：Another object of the present invention is to provide a preparation method of the compound represented by general formula (I), the preparation method is as follows:
以a为原料，与取代苯甲醛b或c分别得到中间体i和ii；i与胺类化合物HNR 3R 4经还原胺化制得通式(I)化合物，或再进一步经缩合反应和水解反应中的一种或几种组合的方法制得通式(I)化合物；ii与溴代物经醚化反应制得中间体iii，iii与胺类化合物HNR 3R 4经还原胺化制得通式(I)化合物，或再进一步经缩合反应和水解反应中的一种或几种组合的方法制得通式(I)化合物； Using a as a raw material, and substituted benzaldehyde b or c to obtain intermediates i and ii, respectively; i and amine compound HNR 3 R 4 are reductively amination to obtain a compound of general formula (I), or further subjected to condensation reaction and hydrolysis One or a combination of methods in the reaction to obtain the compound of general formula (I); ii and bromide are etherified to obtain intermediate iii, iii and amine compound HNR 3 R 4 are reductively amination to obtain the intermediate The compound of formula (I), or the compound of general formula (I) can be prepared by one or more methods of condensation reaction and hydrolysis reaction;
Figure PCTCN2021074875-appb-000003
Figure PCTCN2021074875-appb-000003
其中，R 1、R 2、R 3和R 4的定义如前所述。 Wherein, R 1 , R 2 , R 3 and R 4 are as defined above.
其中，所述“缩合反应和水解反应中的一种或几种组合的方法”，包括仅缩合反应或水解反应；或者先水解反应，再缩合反应；或者先水解反应，再缩合反应，再水解反应，等等。Wherein, the "method of one or more of the condensation reaction and the hydrolysis reaction" includes only condensation reaction or hydrolysis reaction; or first hydrolysis reaction, then condensation reaction; or first hydrolysis reaction, then condensation reaction, and then hydrolysis Reaction, wait.
所述通式(I)化合物的药学上可接受的盐可通过一般的化学方法合成。The pharmaceutically acceptable salt of the compound of general formula (I) can be synthesized by general chemical methods.
一般情况下，盐的制备可以通过游离碱或酸与等化学当量或过量酸(无机酸或有机酸)或碱(无机碱或有机碱)在合适的溶剂或溶剂组合物中反应制得。In general, the salt can be prepared by reacting a free base or acid with an equivalent or excess acid (inorganic acid or organic acid) or base (inorganic base or organic base) in a suitable solvent or solvent composition.
本发明还提供了一种药物组合物，其主要由在治疗上有效量的活性组分和药学上可接受的辅料组成；所述的活性组分包括通式(I)化合物或其药学上可接受的盐的一种或多种。所述药物组合物中，所述的辅料包括药学上可接受的载体、稀释剂和/或赋形剂。The present invention also provides a pharmaceutical composition, which is mainly composed of a therapeutically effective amount of active components and pharmaceutically acceptable excipients; the active components include a compound of general formula (I) or its pharmaceutically acceptable One or more of the accepted salt. In the pharmaceutical composition, the adjuvant includes a pharmaceutically acceptable carrier, diluent and/or excipient.
根据治疗目的可将药物组合物制成各种类型的给药单位剂型，如片剂、丸剂、粉剂、液体、悬浮液、乳液、颗粒剂、胶囊、栓剂和针剂(溶液和悬浮液)等，优选片剂、胶囊、液体、悬浮液和针剂(溶液和悬浮液)。According to the purpose of treatment, the pharmaceutical composition can be made into various types of dosage unit dosage forms, such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories and injections (solutions and suspensions), etc. Preference is given to tablets, capsules, liquids, suspensions and injections (solutions and suspensions).
为了使片剂、丸剂或栓剂形式的药物组合物成形，可使用本领域任何已知并广泛使用的赋形剂。In order to shape the pharmaceutical composition in the form of tablets, pills or suppositories, any excipient known and widely used in the art can be used.
为了制备针剂形式的药物组合物，可将溶液或悬浮液消毒后(最好加入适量的氯化钠，葡萄糖或甘油)，制成与血液等渗压的针剂。在制备针剂时，也可以使用本领域内任何常用的载体。例如：水、乙醇、丙二醇、乙氧基化的异硬脂醇、聚乙氧基化的异硬脂醇和聚乙烯脱水山梨醇的脂肪酸酯等。此外，还可以加入通常溶解剂和缓冲剂等。In order to prepare a pharmaceutical composition in the form of an injection, the solution or suspension can be sterilized (preferably by adding an appropriate amount of sodium chloride, glucose or glycerin) to prepare an injection that is isotonic with blood. When preparing injections, any carriers commonly used in the art can also be used. For example: water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyethoxylated isostearyl alcohol and fatty acid esters of polyethylene sorbitan, etc. In addition, common dissolving agents and buffering agents can also be added.
本发明所述的组合物在药物组合物中的含量无特殊限制，可在很宽的范围内进行选择，通常可为质量百分比的5～95％，优先为质量百分比的30～85％。The content of the composition of the present invention in the pharmaceutical composition is not particularly limited, and can be selected in a wide range, usually 5-95% by mass, preferably 30-85% by mass.
本发明所述的药物组合物的给药方法没有特殊限制。可根据患者年龄、性别和其它条件及症状，选择各种剂型的制剂给药。The method of administration of the pharmaceutical composition of the present invention is not particularly limited. Various dosage forms can be selected for administration according to the age, sex, and other conditions and symptoms of the patient.
本发明还提供了所述通式(I)化合物、其药学上可接受的盐或所述的药物组合物在制备PD-1/PD-L1抑制剂药物中的应用。所述的PD-1/PD-L1抑制剂用于治疗PD-1/PD-L1介导的免疫抑制的相关疾病，所述的相关疾病包括癌症。The present invention also provides the application of the compound of general formula (I), its pharmaceutically acceptable salt or the said pharmaceutical composition in the preparation of PD-1/PD-L1 inhibitor drugs. The PD-1/PD-L1 inhibitor is used to treat PD-1/PD-L1 mediated immune suppression related diseases, and the related diseases include cancer.
本发明还提供了所述通式(I)化合物、其药学上可接受的盐或所述药物组合物在制备药物中的用途，所述药物用于治疗癌症。The present invention also provides the use of the compound of general formula (I), its pharmaceutically acceptable salt or the pharmaceutical composition in the preparation of a medicine, which is used for the treatment of cancer.
所述的癌症包括但不限于：恶性黑色瘤、肺癌、乳腺癌、胃癌、结肠癌、膀胱癌、胰腺癌、淋巴癌、白血病、***癌、睾丸癌、肾癌、脑癌、头颈癌、卵巢癌、***、子宫内膜癌、间皮癌、甲状腺瘤、肝癌和食管癌中的一种或多种。The cancers include but are not limited to: malignant melanoma, lung cancer, breast cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, lymphoma, leukemia, prostate cancer, testicular cancer, kidney cancer, brain cancer, head and neck cancer, ovarian cancer One or more of cancer, cervical cancer, endometrial cancer, mesothelial cancer, thyroid tumor, liver cancer, and esophageal cancer.
本发明还提供了所述通式(I)化合物、其药学上可接受的盐或所述药物组合物可以与一种或多种其他种类的治疗剂和/或治疗方法联合用于治疗由PD-L/PD-L1介导的相关疾病。The present invention also provides that the compound of general formula (I), its pharmaceutically acceptable salt or the pharmaceutical composition can be combined with one or more other types of therapeutic agents and/or therapeutic methods for the treatment of PD -L/PD-L1 mediated related diseases.
所述其他种类的治疗剂和/或治疗方法包括但不限于：化疗剂、靶向抗肿瘤药物、免疫检查点抑制剂、免疫检查点激动剂、抗肿瘤疫苗、抗病毒剂、抗病毒疫苗、细胞因子疗法、过继性细胞免疫治疗或放射治疗。The other types of therapeutic agents and/or treatment methods include, but are not limited to: chemotherapeutics, targeted antitumor drugs, immune checkpoint inhibitors, immune checkpoint agonists, antitumor vaccines, antiviral agents, antiviral vaccines, Cytokine therapy, adoptive cellular immunotherapy or radiation therapy.
所述的化疗剂包括但不限于：烷化剂、微管蛋白抑制剂、拓扑酶抑制剂、铂类药物、抗代谢类药物或激素类抗肿瘤药物。The chemotherapeutic agents include but are not limited to: alkylating agents, tubulin inhibitors, topozyme inhibitors, platinum drugs, antimetabolites or hormone antitumor drugs.
所述的靶向抗肿瘤药物包括但不限于：蛋白激酶抑制剂、蛋白酶体抑制剂、异柠檬酸脱氢酶抑制剂、基于表观遗传学的抗肿瘤药物或细胞周期信号通路抑制剂。The targeted anti-tumor drugs include, but are not limited to: protein kinase inhibitors, proteasome inhibitors, isocitrate dehydrogenase inhibitors, anti-tumor drugs based on epigenetics, or cell cycle signal pathway inhibitors.
所述的免疫检查点抑制剂包括但不限于：CTLA-4抑制剂、TIM-3抑制剂、VISTA抑制剂、LAG3抑制剂、TIGIT抑制剂、A2AR抑制剂或VTCN1抑制剂。The immune checkpoint inhibitors include, but are not limited to: CTLA-4 inhibitors, TIM-3 inhibitors, VISTA inhibitors, LAG3 inhibitors, TIGIT inhibitors, A2AR inhibitors or VTCN1 inhibitors.
所述的免疫检查点激动剂包括但不限于：STING激动剂、4-1BB激动剂、OX40激动剂、RORγ激动剂或ICOS激动剂。The immune checkpoint agonists include but are not limited to: STING agonists, 4-1BB agonists, OX40 agonists, RORγ agonists or ICOS agonists.
有益效果：与现有技术相比，本发明具有如下显著优点：Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:
本发明的硝基苯醚类化合物对PD-1/PD-L1相互作用具有显著的抑制活性，能够有效阻断PD-1/PD-L1介导的免疫抑制作用。体内药效学评价结果表明，本发明的化合物能够显著抑制小鼠移植瘤的生长，并且显著促进肿瘤组织中淋巴细胞的浸润，提高INF-γ的水平，降低PCNA蛋白的表达，而对免疫***缺陷的小鼠移植瘤的则无影响，说明本发明的化合物能够通过激活宿主免疫应答而起抗肿瘤作用。The nitrophenyl ether compound of the present invention has significant inhibitory activity on the interaction of PD-1/PD-L1, and can effectively block the immunosuppressive effect mediated by PD-1/PD-L1. The in vivo pharmacodynamic evaluation results show that the compound of the present invention can significantly inhibit the growth of transplanted tumors in mice, and significantly promote the infiltration of lymphocytes in tumor tissues, increase the level of INF-γ, reduce the expression of PCNA protein, and affect the immune system. Defective mice transplanted tumors have no effect, indicating that the compound of the present invention can play an anti-tumor effect by activating the host immune response.
附图说明Description of the drawings
图1为本发明化合物呈剂量依赖性逆转PD-1/PD-L1抑制PBMC分泌INF-γ的作用；Figure 1 shows that the compound of the present invention reverses the effect of PD-1/PD-L1 on inhibiting the secretion of INF-γ from PBMC in a dose-dependent manner;
图2为本发明化合物在不同浓度下对Lewis肺癌细胞活力的影响；Figure 2 shows the effect of the compound of the present invention on the viability of Lewis lung cancer cells at different concentrations;
图3为本发明化合物呈剂量依赖性抑制Lewis肺癌小鼠移植瘤的生长；Figure 3 shows that the compound of the present invention inhibits the growth of transplanted tumors in mice with Lewis lung cancer in a dose-dependent manner;
图4为本发明化合物对小鼠移植瘤T淋巴细胞浸润的影响，其中：A)CD45 +细胞；B)CD45 +CD3 +细胞；C)CD4 +CD45 +CD3 +细胞；D)CD8 +CD45 +CD3 +细胞。 Figure 4 shows the effect of the compound of the present invention on the infiltration of T lymphocytes in transplanted tumors in mice, in which: A) CD45 + cells; B) CD45 + CD3 + cells; C) CD4 + CD45 + CD3 + cells; D) CD8 + CD45 + CD3 + cells.
具体实施方式Detailed ways
下面结合实施例对本发明的技术方案作进一步说明。The technical solution of the present invention will be further described below in conjunction with embodiments.
试剂与材料：实验所需要的试剂未经特别说明均为市售化学纯或分析纯产品。Reagents and materials: The reagents required for the experiment are all commercially available chemical or analytical products without special instructions.
仪器： 1HNMR用Bruker AV-300或400MHz型核磁共振仪测定，耦合常数(J)值以Hz为单位，TMS为内标。质谱分析仪器为岛津LCMS-2020型质谱仪测定。薄层层析使用青岛海洋化学有限公司生产HG/T2354-92型GF254薄层层析硅胶。ZF7型三用紫外分析仪254nm显色。 Instrument: 1 HNMR is measured with Bruker AV-300 or 400MHz nuclear magnetic resonance instrument, the coupling constant (J) value is in Hz, and TMS is the internal standard. The mass spectrometer is determined by Shimadzu LCMS-2020 mass spectrometer. Thin layer chromatography uses HG/T2354-92 GF254 thin layer chromatography silica gel produced by Qingdao Ocean Chemical Co., Ltd. ZF7 type three-purpose ultraviolet analyzer 254nm color development.
实施例1：(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)-L-丝氨酸(1)的合成Example 1: Synthesis of (3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-serine (1)
Figure PCTCN2021074875-appb-000004
Figure PCTCN2021074875-appb-000004
(1)2-甲基-3-溴苯甲酸乙酯(1A)的合成(1) Synthesis of ethyl 2-methyl-3-bromobenzoate (1A)
将2-甲基-3-溴苯甲酸(10.0g,46.5mmol)溶于乙醇(50mL)中，冰浴下缓慢滴入20mL SOCl 2，70℃下搅拌2小时，加入200mL水，乙酸乙酯萃取，减压浓缩，得1A，直接投入下一步反应。 Dissolve 2-methyl-3-bromobenzoic acid (10.0g, 46.5mmol) in ethanol (50mL), slowly drip 20mL SOCl 2 in an ice bath, stir at 70℃ for 2 hours, add 200mL water, ethyl acetate Extract and concentrate under reduced pressure to obtain 1A, which is directly put into the next reaction.
(2)[1,1'-联苯]-2-甲基-3-羧酸乙酯(1B)的合成(2) Synthesis of [1,1'-biphenyl]-2-methyl-3-carboxylic acid ethyl ester (1B)
将1A(4.0g,16.5mmol)、苯硼酸(3.0g,24.7mmol)、醋酸钾(4.8g,49.4mmol)和Pd(dppf)Cl 2(1.2g,1.7mmol)加入30mL DMF和8mL水中，氮气保护下于90℃反应12h，抽滤，乙酸乙酯萃取，无水硫酸镁干燥，柱层析纯化[石油醚:乙酸乙酯＝20:1(V:V)]，得白色固体3.5g，收率88％。MS(EI)m/z239[M-H] -；H NMR(300MHz,DMSO-d 6):δ(ppm)7.74-7.67(m,1H),7.50-7.38(m,3H),7.38-7.34(m,2H),7.33-7.28(m,2H),4.33-4.26(m,2H),2.29(s,3H),1.31(t,J＝7.5Hz,3H). Add 1A (4.0g, 16.5mmol), phenylboronic acid (3.0g, 24.7mmol), potassium acetate (4.8g, 49.4mmol) and Pd(dppf)Cl 2 (1.2g, 1.7mmol) into 30mL DMF and 8mL water, Under nitrogen protection, react at 90°C for 12 hours, filter with suction, extract with ethyl acetate, dry with anhydrous magnesium sulfate, and purify by column chromatography [petroleum ether: ethyl acetate = 20:1 (V: V)] to obtain 3.5 g of white solid , The yield is 88%. MS(EI)m/z239[MH] - ;H NMR(300MHz,DMSO-d 6 ):δ(ppm)7.74-7.67(m,1H),7.50-7.38(m,3H),7.38-7.34(m ,2H),7.33-7.28(m,2H),4.33-4.26(m,2H),2.29(s,3H),1.31(t,J=7.5Hz,3H).
(3)([1,1'-联苯]-2-甲基-3-基)甲醇(1C)的合成(3) Synthesis of ([1,1'-biphenyl]-2-methyl-3-yl)methanol (1C)
将1B(5.0g,20.8mmol)加入50mL无水THF中，冰浴冷却，缓慢加入氢化铝锂(1.6g,41.6mmol)，反应1h，撤去冰浴，室温反应4h，冰浴下缓慢滴加10mL水，抽滤，浓缩滤液，得白色固体3.9g，收率94％。MS(EI)m/z 197[M-H] -； 1H NMR(300MHz,DMSO-d 6):δ(ppm)7.45-7.32(m,4H),7.3-7.15(m,3H),7.07(d,J＝7.5Hz,1H),5.14(q,J＝4.8,4.1Hz,1H),4.54(d,J＝5.3Hz,2H),2.10(s,3H). Add 1B (5.0g, 20.8mmol) to 50mL of anhydrous THF, cool in an ice bath, slowly add lithium aluminum hydride (1.6g, 41.6mmol), react for 1h, remove the ice bath, react at room temperature for 4h, slowly add dropwise under ice bath 10 mL of water was filtered with suction, and the filtrate was concentrated to obtain 3.9 g of white solid with a yield of 94%. MS(EI)m/z 197[MH] - ; 1 H NMR(300MHz,DMSO-d 6 ):δ(ppm)7.45-7.32(m,4H),7.3-7.15(m,3H),7.07(d ,J=7.5Hz,1H), 5.14(q,J=4.8,4.1Hz,1H), 4.54(d,J=5.3Hz,2H), 2.10(s,3H).
(4)3-(溴甲基)-2-甲基-1,1'-联苯(1D)的合成(4) Synthesis of 3-(bromomethyl)-2-methyl-1,1'-biphenyl (1D)
将1C(2.0g,10.1mmol)溶于30mL无水DCM中，冰浴冷却，缓慢滴入1mL三溴化磷(2.7g,10.1mmol)，反应1h，撤去冰浴，室温反应8h，旋除溶剂，冰浴冷却，缓慢滴入10mL水，乙酸乙酯萃取，浓缩，得白色固体2.3g，收率87％。MS(EI)m/z 259[M-H] -； 1H NMR(300MHz,DMSO-d 6):δ(ppm)7.45-7.39(m,3H),7.39-7.33(m,1H),7.29-7.25(m,2H),7.21(d,J＝7.6Hz,1H),7.18-7.09(m,1H),4.77(s,2H),2.21(s,3H). Dissolve 1C (2.0g, 10.1mmol) in 30mL of anhydrous DCM, cool in an ice bath, slowly drip 1mL of phosphorus tribromide (2.7g, 10.1mmol), react for 1h, remove the ice bath, react at room temperature for 8h, spin off The solvent was cooled in an ice bath, 10 mL of water was slowly added dropwise, extracted with ethyl acetate, and concentrated to obtain 2.3 g of a white solid with a yield of 87%. MS(EI) m/z 259[MH] - ; 1 H NMR(300MHz, DMSO-d 6 ): δ(ppm)7.45-7.39(m,3H), 7.39-7.33(m,1H), 7.29-7.25 (m, 2H), 7.21 (d, J = 7.6 Hz, 1H), 7.18-7.09 (m, 1H), 4.77 (s, 2H), 2.21 (s, 3H).
(5)3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苯甲醛(1E)的合成(5) Synthesis of 3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzaldehyde (1E)
将3-硝基-4-羟基苯甲醛(1.2g,7.2mmol)和NaHCO 3(1.5g,18.1mmol)加入20mL乙腈中，搅拌30min，加入1D(2.1g,7.9mmol)，回流12h，旋除溶剂，乙酸乙酯萃取，柱层析纯化[石油醚:乙酸乙酯＝4:1(V:V)]，得白色固体1.06g，收率65％。MS(EI)m/z 346[M-H] -； 1H NMR(300MHz,DMSO-d 6)δ10.15(d,J＝12.9Hz,1H),8.34(s,1H),7.55(d,J＝7.1Hz,1H),7.45(d,J＝6.9Hz,2H),7.40(d,J＝6.6Hz,1H),7.32(t,J＝7.0Hz,3H),7.24(d,J＝7.5Hz,1H),6.95(d,J＝7.8Hz,1H),5.40(s,2H),2.21(s,3H). Add 3-nitro-4-hydroxybenzaldehyde (1.2g, 7.2mmol) and NaHCO 3 (1.5g, 18.1mmol) to 20mL of acetonitrile, stir for 30min, add 1D (2.1g, 7.9mmol), reflux for 12h, spin Remove the solvent, extract with ethyl acetate, and purify by column chromatography [petroleum ether: ethyl acetate=4:1 (V: V)] to obtain 1.06 g of white solid with a yield of 65%. MS(EI)m/z 346[MH] - ; 1 H NMR(300MHz,DMSO-d 6 )δ10.15(d,J=12.9Hz,1H), 8.34(s,1H),7.55(d,J ＝7.1Hz,1H),7.45(d,J＝6.9Hz,2H),7.40(d,J＝6.6Hz,1H),7.32(t,J＝7.0Hz,3H),7.24(d,J＝7.5 Hz, 1H), 6.95 (d, J = 7.8 Hz, 1H), 5.40 (s, 2H), 2.21 (s, 3H).
(6)(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)-L-丝氨酸甲酯(1F)的合成(6) Synthesis of (3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-serine methyl ester (1F)
将1E(0.16g,0.46mmol)、L-丝氨酸甲酯盐酸盐(0.14g,0.92mmol)、TEA(0.12g,1.38mmol)、冰醋酸(0.14g,2.3mmol)和氰基硼氢化钠(0.14g,2.3mmol)加入5mL DMF中，室温反应24h，乙 酸乙酯萃取，浓缩，直接投入下一步反应。Combine 1E (0.16g, 0.46mmol), L-serine methyl ester hydrochloride (0.14g, 0.92mmol), TEA (0.12g, 1.38mmol), glacial acetic acid (0.14g, 2.3mmol) and sodium cyanoborohydride (0.14g, 2.3mmol) was added to 5mL DMF, reacted at room temperature for 24h, extracted with ethyl acetate, concentrated, and directly put into the next reaction.
(7)(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)-L-丝氨酸(1)的合成(7) Synthesis of (3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-serine (1)
将1F溶于5mL甲醇，加入LiOH(50mg)，反应5h，旋除溶剂，加入10mL饱和食盐水，冰浴冷却，6M盐酸调pH值至2，抽滤，干燥，得白色固体65mg，收率54％。MS(EI)m/z 435[M-H] -； 1H NMR(300MHz,DMSO-d 6)δ8.12(s,1H),7.81-7.76(m,1H),7.61(s,1H),7.47(s,3H),7.42-7.37(m,1H),7.31(s,3H),7.23(s,1H),5.39(s,2H),4.18(s,1H),3.85(d,J＝36.5Hz,3H),2.21(s,3H). Dissolve 1F in 5mL methanol, add LiOH (50mg), react for 5h, spin off the solvent, add 10mL saturated brine, cool in an ice bath, adjust pH to 2 with 6M hydrochloric acid, filter with suction, and dry to obtain a white solid of 65mg, yield 54%. MS(EI)m/z 435[MH] - ; 1 H NMR(300MHz,DMSO-d 6 )δ8.12(s,1H),7.81-7.76(m,1H),7.61(s,1H),7.47 (s,3H),7.42-7.37(m,1H),7.31(s,3H),7.23(s,1H),5.39(s,2H),4.18(s,1H),3.85(d,J=36.5 Hz, 3H), 2.21 (s, 3H).
化合物2～17的制备参照实施例1。Refer to Example 1 for the preparation of compounds 2-17.
Figure PCTCN2021074875-appb-000005
Figure PCTCN2021074875-appb-000005
Figure PCTCN2021074875-appb-000006
Figure PCTCN2021074875-appb-000006
实施例2：2-(3-硝基-4-((2-溴-[1,1'-联苯]-3-基)甲氧基)苄基)-L-苏氨酸(18)的合成Example 2: 2-(3-nitro-4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-threonine (18) Synthesis
参照实施例1的方法，由2-溴-3-溴甲基-1,1'-联苯制得白色固体，收率54％。MS(EI)m/z 514[M-H] -； 1H NMR(300MHz,DMSO-d 6)δ7.98(s,1H),7.72(t,J＝7.8Hz,2H),7.66(d,J＝7.2Hz,2H),7.61(d,J＝8.4Hz,1H),7.51-7.45(m,3H),7.42-7.36(m,2H),5.34(s,2H),3.97(s,1H),3.92(s,1H),3.83-3.76(m,2H),2.91-2.87(m,1H),1.09(d,J＝6.0Hz,3H). With reference to the method of Example 1, a white solid was prepared from 2-bromo-3-bromomethyl-1,1'-biphenyl with a yield of 54%. MS(EI)m/z 514[MH] - ; 1 H NMR(300MHz,DMSO-d 6 )δ7.98(s,1H),7.72(t,J=7.8Hz,2H),7.66(d,J =7.2Hz,2H),7.61(d,J=8.4Hz,1H),7.51-7.45(m,3H),7.42-7.36(m,2H),5.34(s,2H),3.97(s,1H) ,3.92(s,1H),3.83-3.76(m,2H),2.91-2.87(m,1H),1.09(d,J=6.0Hz,3H).
实施例3：(S)-N-(1-羟基-2-(羟甲基)丁-2-基)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)吡咯烷-2-羧酰胺(19)的合成Example 3: (S)-N-(1-hydroxy-2-(hydroxymethyl)butan-2-yl)-1-(3-nitro-4-((2-methyl-[1,1 Synthesis of'-Biphenyl)-3-yl)methoxy)benzyl)pyrrolidine-2-carboxamide (19)
将化合物3(0.5mmol)和N,N-羰基二咪唑(0.5mmol)加入10mL DMF中，80℃反应1小时，再将2-氨基-2-乙基-1,3-丙二醇(0.5mmol)加入反应液，继续反应1小时，柱层析分离，得产品103mg，收率47％。MS(EI)m/z 546[M-H] -； 1H NMR(300MHz,DMSO-d 6)δ7.88(d,J＝1.9Hz,1H),7.72-7.65(m,2H),7.54-7.43(m,4H),7.40(d,J＝7.2Hz,1H),7.34-7.27(m,3H),7.22(d,J＝6.6Hz,1H),5.35(s,2H),4.99(dd,J＝13.0,5.5Hz,2H),3.86(d,J＝12.9Hz,1H),3.48-3.40(m,4H),3.38(s,1H),3.02(dd,J＝9.6,4.7Hz,1H),2.91-2.84(m,1H),2.31(dd,J＝15.7,8.9Hz,1H),2.21(s,3H),2.17-2.07(m,1H),1.67(ddd,J＝36.0,13.7,7.5Hz,5H),1.07-1.00(m,1H),0.73(t,J＝7.4Hz,3H). Compound 3 (0.5mmol) and N,N-carbonyldiimidazole (0.5mmol) were added to 10mL DMF, reacted at 80°C for 1 hour, and then 2-amino-2-ethyl-1,3-propanediol (0.5mmol) The reaction solution was added, the reaction was continued for 1 hour, and the column chromatography was separated to obtain 103 mg of the product with a yield of 47%. MS(EI)m/z 546[MH] - ; 1 H NMR(300MHz,DMSO-d 6 )δ7.88(d,J=1.9Hz,1H),7.72-7.65(m,2H),7.54-7.43 (m,4H),7.40(d,J=7.2Hz,1H),7.34-7.27(m,3H),7.22(d,J=6.6Hz,1H),5.35(s,2H),4.99(dd, J = 13.0, 5.5 Hz, 2H), 3.86 (d, J = 12.9 Hz, 1H), 3.48-3.40 (m, 4H), 3.38 (s, 1H), 3.02 (dd, J = 9.6, 4.7 Hz, 1H) ),2.91-2.84(m,1H),2.31(dd,J=15.7,8.9Hz,1H),2.21(s,3H),2.17-2.07(m,1H),1.67(ddd,J=36.0,13.7 ,7.5Hz,5H),1.07-1.00(m,1H),0.73(t,J=7.4Hz,3H).
化合物20～32的制备参照实施例3。Refer to Example 3 for the preparation of compounds 20 to 32.
Figure PCTCN2021074875-appb-000007
Figure PCTCN2021074875-appb-000007
Figure PCTCN2021074875-appb-000008
Figure PCTCN2021074875-appb-000008
Figure PCTCN2021074875-appb-000009
Figure PCTCN2021074875-appb-000009
实施例4：(2-((3-氰基苄基)氧基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)-L-苏氨酸(33)的合成Example 4: (2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)-5- Synthesis of nitrobenzyl)-L-threonine (33)
Figure PCTCN2021074875-appb-000010
Figure PCTCN2021074875-appb-000010
(1)2,4-羟基-3-硝基苯甲醛(33A)的合成(1) Synthesis of 2,4-hydroxy-3-nitrobenzaldehyde (33A)
将2,4-羟基苯甲醛(10.0g,72.4mmol)溶于乙酸(20mL)，冰浴冷却，滴入浓硝酸(20mL)，室温反应2小时，倒入200mL饱和食盐水，抽滤，干燥，得淡黄色固体11.6g，收率88％。MS(EI)m/z182[M-H] -； 1H NMR(300MHz,DMSO-d 6)δ11.83(s,2H),10.08(d,J＝4.5Hz,1H),8.29(d,J＝4.6Hz,1H),6.62(d,J＝4.3Hz,1H). Dissolve 2,4-hydroxybenzaldehyde (10.0g, 72.4mmol) in acetic acid (20mL), cool in an ice bath, drop in concentrated nitric acid (20mL), react at room temperature for 2 hours, pour into 200mL saturated brine, filter with suction, and dry , 11.6 g of light yellow solid was obtained, and the yield was 88%. MS (EI) m/z182[MH] - ; 1 H NMR (300MHz, DMSO-d 6 ) δ 11.83 (s, 2H), 10.08 (d, J = 4.5 Hz, 1H), 8.29 (d, J = 4.6Hz, 1H), 6.62 (d, J = 4.3Hz, 1H).
(2)2-羟基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苯甲醛(33B)的合成(2) Synthesis of 2-hydroxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)-5-nitrobenzaldehyde (33B)
将2,4-羟基-3-硝基苯甲醛(1.3g,7.2mmol)、NaHCO 3(1.5g,18.1mmol)和3-(溴甲基)-2-甲基-1,1'-联苯(2.1g,7.9mmol)溶于20mL乙腈，回流反应12h，旋除溶剂，加15mL水，乙酸乙酯萃取，无水硫酸镁干燥，柱层析纯化[石油醚:乙酸乙酯＝4:1(V:V)]，得白色固体0.8g，收率31％。MS(EI)m/z 362[M-H] -； 1H NMR(300MHz,DMSO-d 6)δ12.15-11.78(m,1H),10.02(d,J＝12.9Hz,1H),8.21(s,1H),7.42(d,J＝7.1Hz,1H),7.32(d,J＝6.9Hz,2H),7.26(d,J＝6.6Hz,1H),7.19(t,J＝7.0Hz,3H),7.10(d,J＝7.5Hz,1H),6.82(d,J＝7.8Hz,1H),5.27(s,2H),2.08(s,3H). Combine 2,4-hydroxy-3-nitrobenzaldehyde (1.3g, 7.2mmol), NaHCO 3 (1.5g, 18.1mmol) and 3-(bromomethyl)-2-methyl-1,1'-linked Benzene (2.1g, 7.9mmol) was dissolved in 20mL acetonitrile, and the reaction was refluxed for 12h. The solvent was removed by rotating. 15mL water was added, extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and purified by column chromatography [Petroleum ether: ethyl acetate = 4: 1(V:V)], 0.8 g of white solid was obtained, and the yield was 31%. MS(EI)m/z 362[MH] - ; 1 H NMR(300MHz,DMSO-d 6 )δ12.15-11.78(m,1H),10.02(d,J=12.9Hz,1H), 8.21(s ,1H),7.42(d,J=7.1Hz,1H),7.32(d,J=6.9Hz,2H),7.26(d,J=6.6Hz,1H),7.19(t,J=7.0Hz,3H ), 7.10 (d, J = 7.5 Hz, 1H), 6.82 (d, J = 7.8 Hz, 1H), 5.27 (s, 2H), 2.08 (s, 3H).
(3)3-((2-甲酰基-5-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-4-硝基苯氧基)甲基)苄腈(33C)的合成(3) 3-((2-Formyl-5-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)-4-nitrophenoxy)methyl ) Synthesis of benzonitrile (33C)
将33B(0.8g,2.2mmol)、间氰基苄溴(0.5g,2.4mmol)和K 2CO 3(0.6g,4.4mmol)溶于10mL DMF中，室温反应1小时，加50mL饱和食盐水，抽滤，干燥，得白色固体0.7g，收率67％。MS(EI) m/z 477[M-H] -； 1H NMR(300MHz,DMSO-d 6)δ10.21(s,1H),8.36(s,1H),8.00(s,1H),7.89(s,1H),7.87(d,J＝1.7Hz,1H),7.68(t,J＝7.7Hz,1H),7.57(d,J＝6.4Hz,1H),7.47(s,1H),7.45(s,1H),7.42-7.38(m,1H),7.35(dd,J＝6.0,2.3Hz,3H),7.32(d,J＝7.5Hz,1H),7.28-7.23(m,1H),5.59(s,2H),5.51(s,2H),2.26(s,3H). Dissolve 33B (0.8g, 2.2mmol), m-cyanobenzyl bromide (0.5g, 2.4mmol) and K 2 CO 3 (0.6g, 4.4mmol) in 10mL DMF, react at room temperature for 1 hour, add 50mL saturated brine , Suction filtration, drying, to obtain 0.7 g of white solid, with a yield of 67%. MS(EI) m/z 477[MH] - ; 1 H NMR(300MHz,DMSO-d 6 )δ10.21(s,1H),8.36(s,1H),8.00(s,1H),7.89(s ,1H),7.87(d,J=1.7Hz,1H),7.68(t,J=7.7Hz,1H),7.57(d,J=6.4Hz,1H),7.47(s,1H),7.45(s ,1H),7.42-7.38(m,1H),7.35(dd,J=6.0,2.3Hz,3H),7.32(d,J=7.5Hz,1H),7.28-7.23(m,1H),5.59( s, 2H), 5.51 (s, 2H), 2.26 (s, 3H).
(4)(3-硝基-4-((2-甲基-[1,1’-联苯]-3-基)甲氧基)苄基)-L-苏氨酸甲酯(33D)的合成(4) (3-Nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-threonine methyl ester (33D) Synthesis
将33C(0.22g,0.46mmol)溶于5mL DMF中，依次加入L-苏氨酸甲酯盐酸盐(0.14g,0.92mmol)、TEA(0.12g,1.38mmol)、冰醋酸(0.14g,2.3mmol)和氰基硼氢化钠(0.14g,2.3mmol)，室温反应24h，加10mL水，乙酸乙酯萃取，减压浓缩，直接投入下一步反应。Dissolve 33C (0.22g, 0.46mmol) in 5mL DMF, add L-threonine methyl ester hydrochloride (0.14g, 0.92mmol), TEA (0.12g, 1.38mmol), glacial acetic acid (0.14g, 2.3mmol) and sodium cyanoborohydride (0.14g, 2.3mmol), react at room temperature for 24h, add 10mL water, extract with ethyl acetate, concentrate under reduced pressure, and directly put into the next reaction.
(5)(2-((3-氰基苄基)氧基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)-L-苏氨酸(33)的合成(5)(2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)-5-nitro Synthesis of benzyl)-L-threonine (33)
将33D溶于5mL甲醇中，加入LiOH(50mg)，反应5h，旋除溶剂，加入10mL饱和氯化钠溶液，冰浴下滴加6M盐酸将反应液pH值调至2，抽滤，干燥，得白色固体65mg，收率54％。MS(EI)m/z 566[M-H] -； 1H NMR(300MHz,DMSO-d 6)δ8.12(s,1H),8.02(s,1H),7.86(d,J＝18.7Hz,2H),7.63(s,2H),7.46(s,3H),7.40(s,1H),7.30(d,J＝6.5Hz,3H),7.19(d,J＝16.3Hz,2H),5.42(s,2H),5.37(s,2H),3.92(s,2H),3.63(s,2H),3.15(s,2H),2.21(s,3H). Dissolve 33D in 5mL methanol, add LiOH (50mg), react for 5h, spin off the solvent, add 10mL saturated sodium chloride solution, add 6M hydrochloric acid dropwise under ice bath to adjust the pH value of the reaction solution to 2, suction filter, dry, 65 mg of white solid was obtained, and the yield was 54%. MS(EI)m/z 566[MH] - ; 1 H NMR(300MHz,DMSO-d 6 )δ8.12(s,1H),8.02(s,1H),7.86(d,J=18.7Hz,2H ), 7.63 (s, 2H), 7.46 (s, 3H), 7.40 (s, 1H), 7.30 (d, J = 6.5 Hz, 3H), 7.19 (d, J = 16.3 Hz, 2H), 5.42 (s , 2H), 5.37 (s, 2H), 3.92 (s, 2H), 3.63 (s, 2H), 3.15 (s, 2H), 2.21 (s, 3H).
化合物34～49的制备参照实施例4。Refer to Example 4 for the preparation of compounds 34 to 49.
Figure PCTCN2021074875-appb-000011
Figure PCTCN2021074875-appb-000011
Figure PCTCN2021074875-appb-000012
Figure PCTCN2021074875-appb-000012
Figure PCTCN2021074875-appb-000013
Figure PCTCN2021074875-appb-000013
实施例5：药理活性评价Example 5: Evaluation of Pharmacological Activity
1.本发明化合物对PD-1/PD-L1相互作用的抑制活性1. The inhibitory activity of the compound of the present invention on the interaction of PD-1/PD-L1
1.1主要实验材料1.1 Main experimental materials
购买的试剂盒(Cisbio公司PD-1/PD-L1binding assay kit)中含有PD-1、PD-L1、Anti-tag1-Eu、Anti-tag2-XL665、Dilute Buffer和Detection Buffer等实验所需的试剂；阳性药BMS-1018；
Figure PCTCN2021074875-appb-000014
M1000多功能酶标仪；Echo520超声波微量液体转移***(Labcyte)。 The purchased kit (PD-1/PD-L1 binding assay kit from Cisbio) contains the reagents needed for experiments such as PD-1, PD-L1, Anti-tag1-Eu, Anti-tag2-XL665, Dilute Buffer and Detection Buffer ; Positive drug BMS-1018;
Figure PCTCN2021074875-appb-000014
M1000 Multifunctional Microplate Reader; Echo520 Ultrasonic Micro Liquid Transfer System (Labcyte).
1.2实验方法1.2 Experimental method
1)将化合物用100％DMSO配置成所设置的浓度，然后将化合物的DMSO溶液加入到稀释缓冲液中，混合均匀后加入2μL到384孔白色酶标板中；1) Prepare the compound with 100% DMSO to the set concentration, then add the DMSO solution of the compound to the dilution buffer, mix well and add 2 μL to the 384-well white microtiter plate;
2)将PD-1蛋白和PD-L1蛋白用稀释缓冲液稀释，分别加入4μL到上述384孔板中，常温条件下孵育15分钟；2) Dilute PD-1 protein and PD-L1 protein with dilution buffer, add 4 μL to the above 384-well plate respectively, and incubate for 15 minutes at room temperature;
3)将10μL的Anti-tag1-Eu和Anti-tag2-XL665混合液加入到检测缓冲溶液中，混合均匀后加入到上述384孔板中，然后在室温条件下孵育1到24个小时；3) Add 10 μL of Anti-tag1-Eu and Anti-tag2-XL665 mixture to the detection buffer solution, mix well and add it to the above 384-well plate, and then incubate at room temperature for 1 to 24 hours;
4)用
Figure PCTCN2021074875-appb-000015
M1000多功能酶标仪检测665nm和620nm处的荧光信号，
Figure PCTCN2021074875-appb-000016
根据荧光比值计算化合物对蛋白结合的抑制率。 4) Use
Figure PCTCN2021074875-appb-000015
M1000 multifunctional microplate reader detects the fluorescence signal at 665nm and 620nm,
Figure PCTCN2021074875-appb-000016
Calculate the inhibition rate of the compound to protein binding based on the fluorescence ratio.
1.3实验结果1.3 Experimental results
实施例化合物对PD-1/PD-L1蛋白-蛋白相互作用的抑制活性如表1所示。结果表明，本发明化合物能显著抑制PD-1/PD-L1的相互作用。The inhibitory activity of the compounds of the examples on the PD-1/PD-L1 protein-protein interaction is shown in Table 1. The results show that the compound of the present invention can significantly inhibit the interaction of PD-1/PD-L1.
表1本发明化合物对PD-1/PD-L1相互作用的抑制活性Table 1 Inhibitory activity of the compounds of the present invention on the interaction of PD-1/PD-L1
Figure PCTCN2021074875-appb-000017
Figure PCTCN2021074875-appb-000017
Figure PCTCN2021074875-appb-000018
Figure PCTCN2021074875-appb-000018
*对照组：BMS-1018是专利WO 2015160641 A2中的第1018号化合物。*Control group: BMS-1018 is the No. 1018 compound in patent WO 2015160641 A2.
2.本发明化合物对细胞因子INF-γ释放的影响2. The effect of the compound of the present invention on the release of cytokine INF-γ
细胞因子是一类具有效应及调节双重作用的独特蛋白，在淋巴细胞应答中有重要的免疫调节作用。活化的人外周血单个核细胞(PBMC)可以释放IFN-γ、IL-2和TNF-α等细胞因子，而当PBMC膜上表达的PD-1与其配体PD-L1结合后将会抑制细胞因子的释放。本实验的目的是检测本发明化合物逆转PD-1/PD-L1抑制PBMC分泌INF-γ的能力。Cytokines are a class of unique proteins with dual effects and regulation. They play an important role in immune regulation in the response of lymphocytes. Activated human peripheral blood mononuclear cells (PBMC) can release cytokines such as IFN-γ, IL-2 and TNF-α, and when PD-1 expressed on the PBMC membrane binds to its ligand PD-L1, it will inhibit the cell Factor release. The purpose of this experiment is to test the ability of the compound of the present invention to reverse PD-1/PD-L1's inhibition of PBMC secretion of INF-γ.
2.1实验方法2.1 Experimental method
使用人淋巴细胞分离液提取人外周血单个核细胞，接种到24孔板，加入终浓度1μg/mL的anti-CD3/anti-CD28，终浓度为2μg/mL配体蛋白和不同剂量的化合物，48h后离心取上清100μL，使用达科为的INF-γ酶联免疫吸附试剂盒检测上清中INF-γ的表达量。Use human lymphocyte separation solution to extract human peripheral blood mononuclear cells, inoculate it into a 24-well plate, add anti-CD3/anti-CD28 at a final concentration of 1μg/mL, a final concentration of 2μg/mL ligand protein and different doses of compounds, After 48h, 100 μL of the supernatant was collected by centrifugation, and the INF-γ expression level in the supernatant was detected using Daktronics' INF-γ enzyme-linked immunosorbent kit.
2.2实验结果2.2 Experimental results
实验结果(图1)表明，与模型组相比，当加入anti-CD3/anti-CD28时能明显促进INF-γ的释放，而加入PD-L1后则显著降低INF-γ的水平，表明PD-1/PD-L1明显抑制INF-γ的释放。当加入不同浓度的实施例化合物33后便能够显著提高INF-γ的水平，并且呈现剂量依赖性。此外，本发明中的其他化合物如3、8、9、13、27、28、30、32、33、36、37、39、40、41、43、46、47、48和49等在10nM和100nM浓度下均可显著提高INF-γ的水平，这说明本发明化合物能够阻断PD-1/PD-L1对PBMC的抑制作用，从而促进INF-γ的分泌。The experimental results (Figure 1) show that, compared with the model group, the addition of anti-CD3/anti-CD28 can significantly promote the release of INF-γ, while the addition of PD-L1 significantly reduces the level of INF-γ, indicating PD -1/PD-L1 obviously inhibits the release of INF-γ. When adding different concentrations of Example Compound 33, the level of INF-γ can be significantly increased, and it is dose-dependent. In addition, other compounds in the present invention, such as 3, 8, 9, 13, 27, 28, 30, 32, 33, 36, 37, 39, 40, 41, 43, 46, 47, 48 and 49 etc. are at 10 nM and The level of INF-γ can be significantly increased at a concentration of 100 nM, which indicates that the compound of the present invention can block the inhibitory effect of PD-1/PD-L1 on PBMC, thereby promoting the secretion of INF-γ.
3.本发明化合物对细胞的毒性实验3. Toxicity test of the compound of the present invention on cells
采用MTT法检测本发明化合物对Lewis肺癌细胞活性的影响，目的是为了考察本发明化合物是否存在细胞毒性。The MTT method is used to detect the effect of the compound of the present invention on the activity of Lewis lung cancer cells, and the purpose is to investigate whether the compound of the present invention has cytotoxicity.
3.1实验方法3.1 Experimental method
于96孔板中每孔加入20μL 4mg/mL MTT溶液，放入细胞培养箱孵育4小时，将96孔板进行离心，小心吸去孔内液体，每孔加入200μL二甲基亚砜，放置在摇床上300r振荡10min，使紫色结晶物质充分溶解。最后在酶标仪570nm处检测吸光值。根据吸光度用Bliss法计算抑制率。Add 20μL 4mg/mL MTT solution to each well of a 96-well plate, place it in the cell incubator and incubate for 4 hours, centrifuge the 96-well plate, carefully aspirate the liquid in the well, add 200μL dimethyl sulfoxide to each well, and place it in the Shake at 300r for 10min on a shaker to fully dissolve the purple crystalline substance. Finally, the absorbance value was detected at 570nm of the microplate reader. According to the absorbance, the inhibition rate was calculated by the Bliss method.
Figure PCTCN2021074875-appb-000019
Figure PCTCN2021074875-appb-000019
3.2实验结果3.2 Experimental results
实验结果(图2)表明，与模型组相比，实施例化合物33在各种检测浓度下对Lewis肺癌细胞的活力均没有明显的影响，说明本发明化合物33无明显的细胞毒性。The experimental results (Figure 2) show that, compared with the model group, Example Compound 33 has no obvious effect on the viability of Lewis lung cancer cells at various concentrations, indicating that Compound 33 of the present invention has no obvious cytotoxicity.
4.本发明化合物的体内药效学评价4. In vivo pharmacodynamic evaluation of the compound of the present invention
增殖细胞核抗原(PCNA)则是真核细胞DNA合成所必需的一种核蛋白，检测PCNA可以客观评价肿瘤细胞的增殖状态。为此，在开展体内药效学评价过程中，利用免疫组化和TUNEL分析检测肿瘤组织中的T淋巴细胞的浸润以及IFN-γ和PCNA水平。Proliferating cell nuclear antigen (PCNA) is a nuclear protein necessary for DNA synthesis in eukaryotic cells. The detection of PCNA can objectively evaluate the proliferation status of tumor cells. To this end, in the process of in vivo pharmacodynamic evaluation, immunohistochemistry and TUNEL analysis were used to detect the infiltration of T lymphocytes in tumor tissues and the levels of IFN-γ and PCNA.
4.1实验方法4.1 Experimental method
小鼠的培养：选择7～8周的雌鼠，在SPF级动物饲养室饲养一周，每只小鼠体重大约在18～20g。Cultivation of mice: select female mice aged 7-8 weeks and raise them in an SPF-class animal breeding room for one week. Each mouse weighs about 18-20g.
肿瘤细胞的处理：采集处于对数生长期的肿瘤细胞，180g离心5min(4℃)，使用预冷的PBS洗 2次，吹打均匀，终细胞浓度为1×10 7/mL，冰浴备用。 Treatment of tumor cells: Collect tumor cells in logarithmic growth phase, centrifuge at 180g for 5 min (4°C), wash twice with pre-cooled PBS, pipette evenly, and make a final cell concentration of 1×10 7 /mL, ice bath for later use.
肿瘤细胞的移植：将肿瘤细胞悬浮液接种至小鼠右侧腋窝皮下，接种的肿瘤细胞数为1×10 6/只。每两天使用游标卡尺测量小鼠肿瘤大小一次，称小鼠体重一次。当肿瘤体积均值达到40mm 3左右时，开始给药。 Tumor cell transplantation: The tumor cell suspension was inoculated subcutaneously into the right axilla of the mouse, and the number of tumor cells inoculated was 1×10 6 per mouse. The tumor size of the mouse was measured with a vernier caliper every two days, and the mouse weight was weighed once. When the average tumor volume reaches about 40mm 3 , the drug is started.
当肿瘤体积达到一定大小后，结束动物实验。称量小鼠体重，对其进行眼球取血，并对小鼠实施安乐死，剥取肿瘤组织，对肿瘤组织进行称重并拍照。同时，将部分组织置于10％中性固定液中，送样进行石蜡包埋组织、制作石蜡组织切片，并开展H&E染色、TUNEL和免疫组化分析。实验操作参考检测试剂盒说明书。When the tumor volume reached a certain size, the animal experiment was ended. Weigh the weight of the mice, take blood from their eyeballs, and euthanize the mice, strip the tumor tissues, weigh the tumor tissues and take pictures. At the same time, some tissues were placed in 10% neutral fixative, samples were sent for paraffin-embedded tissues, paraffin tissue sections were made, and H&E staining, TUNEL and immunohistochemical analysis were performed. Refer to the test kit instructions for experimental operations.
(1)Lewis肺癌小鼠移植瘤模型(1) Lewis lung cancer mouse xenograft tumor model
移植Lewis肺癌的BALB/c雌鼠分为4组，每组6只。模型组(溶剂：PBS+2％的吐温20+2％DMSO，灌胃给药，每天一次)，阳性对照组(BMS-1018，灌胃给药，每天一次，剂量：5mg/kg)，药物处理组1(化合物33，灌胃给药，每天一次，剂量：2mg/kg)，药物处理组2(化合物33，灌胃给药，每天一次，剂量：5mg/kg)。The BALB/c female mice transplanted with Lewis lung cancer were divided into 4 groups with 6 mice in each group. Model group (solvent: PBS+2% Tween 20+2% DMSO, intragastric administration, once a day), positive control group (BMS-1018, intragastric administration, once a day, dose: 5mg/kg), Drug treatment group 1 (compound 33, intragastric administration, once a day, dose: 2 mg/kg), drug treatment group 2 (compound 33, intragastric administration, once a day, dose: 5 mg/kg).
(2)B16F1黑色素瘤小鼠移植瘤模型(2) B16F1 melanoma transplanted tumor model in mice
移植B16F1黑色素瘤的C57BL/6雌鼠分为4组，每组6只。模型组(溶剂：PBS+2％的吐温20+2％DMSO，灌胃给药，每天一次)，阳性对照组(BMS-1018，灌胃给药，每天一次，剂量：5mg/kg)，药物处理组1(化合物33，灌胃给药，每天一次，剂量：2mg/kg)，药物处理组2(化合物33，灌胃给药，每天一次，剂量：5mg/kg)。C57BL/6 female mice transplanted with B16F1 melanoma were divided into 4 groups with 6 mice in each group. Model group (solvent: PBS+2% Tween 20+2% DMSO, intragastric administration, once a day), positive control group (BMS-1018, intragastric administration, once a day, dose: 5mg/kg), Drug treatment group 1 (compound 33, intragastric administration, once a day, dose: 2 mg/kg), drug treatment group 2 (compound 33, intragastric administration, once a day, dose: 5 mg/kg).
(3)CT26结直肠癌BALB/c小鼠移植瘤模型(3) CT26 colorectal cancer BALB/c mouse transplanted tumor model
移植CT26结直肠肿瘤的BALB/c雌鼠分为5组，每组6只。模型组(溶剂：PBS+2％的吐温20+2％DMSO，灌胃给药，每天一次)，阳性对照组-1(5-FU，剂量：25mg/kg，腹腔注射，每两天一次)，阳性对照组-2(BMS-1018，灌胃给药，每天一次，剂量：5mg/kg)，药物处理组(化合物33，灌胃给药，每天一次，剂量：5mg/kg)，联合给药组(5-FU，剂量：25mg/kg，腹腔注射，每两天一次；化合物33，灌胃给药，每天一次，剂量：5mg/kg)。The BALB/c female mice transplanted with CT26 colorectal tumors were divided into 5 groups with 6 mice in each group. Model group (solvent: PBS+2% Tween 20+2% DMSO, intragastric administration, once a day), positive control group-1 (5-FU, dose: 25mg/kg, intraperitoneal injection, once every two days ), positive control group-2 (BMS-1018, intragastric administration, once a day, dose: 5mg/kg), drug treatment group (compound 33, intragastric administration, once a day, dose: 5mg/kg), combined Administration group (5-FU, dose: 25 mg/kg, intraperitoneal injection, once every two days; compound 33, intragastric administration, once a day, dose: 5 mg/kg).
(4)CT26结直肠癌裸鼠移植瘤模型(4) CT26 colorectal cancer transplanted tumor model in nude mice
为了验证本发明化合物是否是通过免疫***发挥抗肿瘤作用，在免疫***缺陷的小鼠上构建了CT26结肠癌移植瘤模型。In order to verify whether the compound of the present invention exerts an anti-tumor effect through the immune system, a CT26 colon cancer xenograft model was constructed on mice with immune system deficiency.
移植CT26结直肠肿瘤的BALB/c(nu/nu)裸鼠分为4组，每组6只。模型组(溶剂：PBS+2％的吐温20+2％DMSO，灌胃给药，每天一次)，阳性对照组-1(5-FU，剂量：25mg/kg，腹腔注射，每两天一次)，阳性对照组-2(BMS-1018，灌胃给药，每天一次，剂量：5mg/kg)，药物处理组(化合物33，灌胃给药，每天一次，剂量：5mg/kg)。BALB/c(nu/nu) nude mice transplanted with CT26 colorectal tumors were divided into 4 groups with 6 mice in each group. Model group (solvent: PBS+2% Tween 20+2% DMSO, intragastric administration, once a day), positive control group-1 (5-FU, dose: 25mg/kg, intraperitoneal injection, once every two days ), positive control group-2 (BMS-1018, intragastric administration, once a day, dose: 5mg/kg), drug treatment group (compound 33, intragastric administration, once a day, dose: 5mg/kg).
(5)PAN02胰腺癌小鼠移植瘤模型(5) PAN02 pancreatic cancer mouse transplanted tumor model
移植PAN02胰腺癌的C57BL/6雌鼠分为3组，每组8只。模型组(溶剂：PBS+2％的吐温20+2％DMSO，灌胃给药，每天一次)，阳性对照组(BMS-1018，灌胃给药，每天一次，剂量：5mg/kg)，药物处理组(化合物33，灌胃给药，每天一次，剂量：5mg/kg)。C57BL/6 female mice transplanted with PAN02 pancreatic cancer were divided into 3 groups, each with 8 mice. Model group (solvent: PBS+2% Tween 20+2% DMSO, intragastric administration, once a day), positive control group (BMS-1018, intragastric administration, once a day, dose: 5mg/kg), Drug treatment group (Compound 33, intragastric administration, once a day, dose: 5mg/kg).
4.2实验结果4.2 Experimental results
(1)Lewis肺癌小鼠移植瘤模型(1) Lewis lung cancer mouse xenograft tumor model
实验结果(图3)表明，与模型组相比，实施例化合物33在2mg/kg和5mg/kg给药剂量下都可以显著抑制Lewis肺癌小鼠移植瘤的生长，呈现剂量依赖性，并且不影响小鼠的体重。此外，在相同给药剂量下(5mg/kg)，实施化合物33对移植瘤的抑制活性(抑瘤率为47.8％)明显优于对照组BMS-1018(抑瘤率为19.6％)。The experimental results (Figure 3) show that, compared with the model group, Example Compound 33 can significantly inhibit the growth of Lewis lung cancer transplanted tumors in mice at 2 mg/kg and 5 mg/kg, showing a dose-dependent, and no Affect the weight of mice. In addition, under the same dosage (5mg/kg), the inhibitory activity of compound 33 on transplanted tumors (tumor inhibition rate 47.8%) was significantly better than that of the control group BMS-1018 (tumor inhibition rate 19.6%).
免疫组化和TUNEL实验结果表明，与Vehicle组相比，实施例化合物33在2mg/kg和5mg/kg剂量下都能够显著促进肿瘤组织中T淋巴细胞的浸润，并且提高IFN-γ的水平，降低PCNA蛋白的表达，而且在5mg/kg剂量下，化合物33逆转PD-1/PD-L1介导的免疫抑制作用强于BMS-1018对照组。The results of immunohistochemistry and TUNEL experiments show that, compared with the Vehicle group, Example Compound 33 can significantly promote the infiltration of T lymphocytes in tumor tissues at the doses of 2 mg/kg and 5 mg/kg, and increase the level of IFN-γ, Reduce the expression of PCNA protein, and at a dose of 5 mg/kg, compound 33 reverses PD-1/PD-L1 mediated immunosuppression stronger than the BMS-1018 control group.
(2)B16F1黑色素瘤小鼠移植瘤模型(2) B16F1 melanoma transplanted tumor model in mice
实验结果表明，与模型组相比，实施例化合物33在2mg/kg和5mg/kg剂量下呈现剂量依赖性抑制B16F1黑色素瘤小鼠移植瘤的生长，对小鼠的体重无影响，而且在相同剂量下(5mg/kg)，化合物33对移植瘤的抑制活性优于BMS-1018对照组。免疫组化和TUNEL实验结果表明，实施例化合物33给药组也能够显著促进肿瘤组织中T淋巴细胞的浸润，提高IFN-γ的水平，降低PCNA蛋白的表达，并且在相同剂量下效果强于BMS-1018。The experimental results showed that, compared with the model group, Example Compound 33 showed a dose-dependent inhibition of the growth of B16F1 melanoma transplanted tumors in mice at the doses of 2 mg/kg and 5 mg/kg, and had no effect on the body weight of the mice. At the dose (5 mg/kg), the inhibitory activity of compound 33 on transplanted tumors was better than that of the BMS-1018 control group. The results of immunohistochemistry and TUNEL experiments show that the compound 33 administration group of Example can also significantly promote the infiltration of T lymphocytes in tumor tissues, increase the level of IFN-γ, reduce the expression of PCNA protein, and the effect is stronger than that at the same dose. BMS-1018.
(3)CT26结直肠癌BALB/c小鼠移植瘤模型(3) CT26 colorectal cancer BALB/c mouse transplanted tumor model
实验结果表明，与模型组相比，实施例化合物33能够抑制CT26结直肠癌BALB/c小鼠移植瘤的生长，而且与5-FU联合用药后能提高后者的抑瘤率。The experimental results show that, compared with the model group, Example Compound 33 can inhibit the growth of CT26 colorectal cancer BALB/c mice transplanted tumors, and the combination with 5-FU can increase the tumor inhibition rate of the latter.
(4)CT26结直肠癌裸鼠移植瘤模型(4) CT26 colorectal cancer transplanted tumor model in nude mice
实验结果表明，与模型组相比，化合物33给药组的肿瘤体积和肿瘤质量均无变化，表明实施例化合物对免疫***缺陷的裸鼠移植瘤不产生抑制作用。相反，5-FU却能够明显抑制裸鼠移植瘤的生长，结合上例实验表明，本发明化合物是通过免疫***发挥抗肿瘤作用。The experimental results showed that, compared with the model group, the tumor volume and tumor mass of the compound 33 administration group did not change, indicating that the compounds of the examples have no inhibitory effect on transplanted tumors in nude mice with immune system deficiency. On the contrary, 5-FU can obviously inhibit the growth of transplanted tumors in nude mice. Combined with the experiment of the above example, it is shown that the compound of the present invention exerts an anti-tumor effect through the immune system.
(5)PAN02胰腺癌小鼠移植瘤模型(5) PAN02 pancreatic cancer mouse transplanted tumor model
实验结果表明，相比于模型组，实施例化合物33在5mg/kg剂量下能有效抑制PAN02胰腺癌小鼠移植瘤的生长。免疫组化和TUNEL实验表明，化合物33能够阻断PD-1/PD-L1介导的免疫抑制作用。Experimental results show that, compared with the model group, Example Compound 33 can effectively inhibit the growth of PAN02 pancreatic cancer transplanted tumors in mice at a dose of 5 mg/kg. Immunohistochemistry and TUNEL experiments show that compound 33 can block the immunosuppressive effect mediated by PD-1/PD-L1.
5.本发明化合物对肿瘤微环境T淋巴细胞浸润的影响5. The effect of the compound of the present invention on the infiltration of T lymphocytes in the tumor microenvironment
T淋巴细胞是人体免疫***的核心执行者，在肿瘤免疫应答中起重要作用。肿瘤浸润淋巴细胞(TIL)是指那些离开血流进入到肿瘤中的白细胞。当肿瘤微环境中存在大量的肿瘤浸润淋巴细胞时，表明机体启动了对抗肿瘤的免疫反应。PD-1/PD-L1信号通路的激活会抑制抗肿瘤免疫微环境，导致淋巴细胞的浸润减少。本实验的目的分析本发明化合物对肿瘤微环境中T淋巴细胞浸润的影响。T lymphocytes are the core executors of the human immune system and play an important role in tumor immune response. Tumor infiltrating lymphocytes (TIL) are those white blood cells that leave the bloodstream and enter the tumor. When there are a large number of tumor-infiltrating lymphocytes in the tumor microenvironment, it indicates that the body has initiated an immune response against tumors. The activation of the PD-1/PD-L1 signaling pathway will inhibit the anti-tumor immune microenvironment, resulting in a decrease in lymphocyte infiltration. The purpose of this experiment is to analyze the effect of the compound of the present invention on the infiltration of T lymphocytes in the tumor microenvironment.
5.1实验方法5.1 Experimental method
取部分实验4中剥取的肿瘤组织，剪碎转入15mL离心管中，加入胶原酶IV(0.5mg/mL)、DNA酶I(0.5mg/mL)，37℃消化30min，过滤掉剩余组织碎片，离心重悬细胞后使用不同通道的CD45、CD3、CD4、CD8流式抗体避光染色30min，流式细胞仪检测。Take part of the tumor tissue stripped in Experiment 4, cut into pieces and transfer to a 15mL centrifuge tube, add collagenase IV (0.5mg/mL), DNase I (0.5mg/mL), digest at 37°C for 30 minutes, and filter out the remaining tissue After centrifugation to resuspend the cells, the fragments were stained with CD45, CD3, CD4, and CD8 flow cytometry antibodies in different channels for 30 minutes in the dark, and then detected by flow cytometry.
5.2实验结果5.2 Experimental results
实验结果(图4)表明，与模型组相比，实施例化合物33在2mg/kg和5mg/kg剂量下能够显著促进CD45 +白细胞、CD45 +CD3 +T淋巴细胞、CD8 +CD45 +CD3 +细胞毒性T细胞的浸润，并且呈现剂量依赖性，而对CD4 +CD45 +CD3 +调节性T淋巴细胞的影响很弱。此外，在相同剂量(5mg/kg)下，实施例化合物33促进淋巴细胞浸润的能力强于BMS-1018，特别是对CD45 +CD3 +CD8 +细胞毒性T淋巴 细胞的浸润增加更为突出。这些实验说明本发明的化合物能够有效地逆转PD-1/PD-L1介导的免疫抑制作用，重塑抗肿瘤免疫微环境。 The experimental results (Figure 4) show that, compared with the model group, Example Compound 33 can significantly promote CD45 + white blood cells, CD45 + CD3 + T lymphocytes, CD8 + CD45 + CD3 + cells at the doses of 2 mg/kg and 5 mg/kg. The infiltration of toxic T cells is dose-dependent, while the effect on CD4 + CD45 + CD3 + regulatory T lymphocytes is weak. In addition, under the same dose (5 mg/kg), the ability of Example Compound 33 to promote lymphocyte infiltration is stronger than that of BMS-1018, especially the increase in CD45 + CD3 + CD8 + cytotoxic T lymphocyte infiltration. These experiments show that the compound of the present invention can effectively reverse the immunosuppressive effect mediated by PD-1/PD-L1 and reshape the anti-tumor immune microenvironment.
需要指出的是，本发明中的其他化合物在多种肿瘤类型如CT26、EMT6、B16F1、PAN02、LLC等小鼠移植瘤模型中表现出显著的抗肿瘤作用。例如，实施例化合物3、8、28、30、33、34、36、39、41、43、47和48等在低剂量下(2mg/kg～15mg/kg)便可显著抑制小鼠移植瘤的生长，而且这些化合物能够促进淋巴细胞对肿瘤微环境的浸润，提高肿瘤组织中IFN-γ的分泌，降低PCNA蛋白的表达。这些实验说明本发明化合物能够阻断PD-1/PD-L1介导的免疫抑制作用，激活免疫应答起抗肿瘤作用。It should be pointed out that other compounds in the present invention show significant anti-tumor effects in various tumor types such as CT26, EMT6, B16F1, PAN02, LLC and other mouse xenograft tumor models. For example, the example compounds 3, 8, 28, 30, 33, 34, 36, 39, 41, 43, 47, and 48 can significantly inhibit transplanted tumors in mice at low doses (2 mg/kg to 15 mg/kg) These compounds can promote the infiltration of lymphocytes into the tumor microenvironment, increase the secretion of IFN-γ in tumor tissues, and reduce the expression of PCNA protein. These experiments show that the compound of the present invention can block the PD-1/PD-L1 mediated immunosuppressive effect, activate the immune response and play an anti-tumor effect.

Claims (10)

  1. 一种通式(I)所示的硝基苯醚类化合物、其立体异构体、代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐：A nitrophenyl ether compound represented by general formula (I), its stereoisomers, metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof:
    Figure PCTCN2021074875-appb-100001
    Figure PCTCN2021074875-appb-100001
    其中：in:
    R 1选自：甲基或溴； R 1 is selected from: methyl or bromine;
    R 2选自：氢、C 1-C 4烷氧基或-O(CH 2) nAr；其中，n选自0-4的整数；Ar选自芳基或芳杂环；所述的芳杂环可任选地包含一个或多个选自O、S或N杂原子；所述的芳基或芳杂环可任选地被一个或多个W基团取代；W选自：氢、卤素、氰基、羟基、巯基、羧基、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氨基或C 1-C 6卤代烷基； R 2 is selected from: hydrogen, C 1 -C 4 alkoxy or -O(CH 2 ) n Ar; wherein, n is selected from an integer of 0-4; Ar is selected from an aryl group or an aromatic heterocyclic ring; The heterocycle may optionally contain one or more heteroatoms selected from O, S or N; the aryl or aromatic heterocycle may optionally be substituted by one or more W groups; W is selected from: hydrogen, Halogen, cyano, hydroxyl, mercapto, carboxyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino or C 1 -C 6 haloalkyl;
    R 3和R 4各自独立地选自：氢、C 1-C 8烷基、C 1-C 8烷氧基、C 3-C 8环烷基或R 3和R 4跟它们连接的氮原子一起形成5-7元杂环；所述的烷基、烷氧基或环烷基可任选地被一个或多个X基团取代；X选自：氢、卤素、羟基、巯基、甲巯基、羧基、氨基、呋喃基、二甲胺基、羟基取代的异丙胺基、二乙胺基、四氢吡咯基、吗啉基、N-甲基哌嗪基或-NHCOR 5；其中R 5选自C 1-C 8烷基；所述的杂环可被羧基或-CONR 6R 7取代； R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen atom to which R 3 and R 4 are connected Together to form a 5-7 membered heterocyclic ring; the alkyl, alkoxy or cycloalkyl group may be optionally substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, mercapto, and methylmercapto , Carboxyl, amino, furanyl, dimethylamino, hydroxy-substituted isopropylamino, diethylamino, tetrahydropyrrolyl, morpholinyl, N-methylpiperazinyl or -NHCOR 5 ; where R 5 is selected From C 1 -C 8 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
    R 6和R 7各自独立地选自：氢、C 1-C 8烷基、C 1-C 8烷氧基、C 3-C 8环烷基或R 6和R 7跟它们连接的氮原子一起形成5-7元杂环；所述的烷基、烷氧基、环烷基或杂环可任选地被一个或多个Y基团取代；Y选自：氢、卤素、羟基、巯基、羧基、氨基或乙酰胺基。 R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl, or the nitrogen atom to which R 6 and R 7 are connected Together to form a 5-7 membered heterocyclic ring; the alkyl, alkoxy, cycloalkyl or heterocyclic ring may be optionally substituted by one or more Y groups; Y is selected from: hydrogen, halogen, hydroxyl, mercapto , Carboxyl, amino or acetamido.
  2. 根据权利要求1所述的硝基苯醚类化合物、其立体异构体、代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐，其特征在于：The nitrophenyl ether compound, its stereoisomers, metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof according to claim 1, characterized in that:
    R 1选自：甲基或溴； R 1 is selected from: methyl or bromine;
    R 2选自：氢或-OCH 2Ar；其中，Ar选自芳基或芳杂环；所述的芳杂环可任选地包含一个或多个选自O、S或N杂原子；所述的芳基或芳杂环可任选地被一个或多个W基团取代；W选自：氢、卤素、氰基、羟基、巯基、羧基、C 1-C 4烷基或C 1-C 4烷氧基； R 2 is selected from: hydrogen or -OCH 2 Ar; wherein, Ar is selected from an aryl group or an aromatic heterocyclic ring; the aromatic heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; The aryl or aromatic heterocycle may be optionally substituted by one or more W groups; W is selected from: hydrogen, halogen, cyano, hydroxyl, mercapto, carboxy, C 1 -C 4 alkyl or C 1- C 4 alkoxy;
    R 3和R 4各自独立地选自：氢、C 1-C 8烷基、C 1-C 8烷氧基、C 3-C 8环烷基或R 3和R 4跟它们连接的氮原子一起形成5-7元杂环；所述的烷基、烷氧基或环烷基可任选地被一个或多个X基团取代；X选自：氢、卤素、羟基、巯基、甲巯基、羧基、氨基、呋喃基、二甲胺基、羟基取代的异丙胺基、二乙胺基、四氢吡咯基、吗啉基、N-甲基哌嗪基或-NHCOR 5；其中R 5选自C 1-C 8烷基；所述的杂环可被羧基或-CONR 6R 7取代； R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen atom to which R 3 and R 4 are connected Together to form a 5-7 membered heterocyclic ring; the alkyl, alkoxy or cycloalkyl group may be optionally substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, mercapto, and methylmercapto , Carboxyl, amino, furanyl, dimethylamino, hydroxy-substituted isopropylamino, diethylamino, tetrahydropyrrolyl, morpholinyl, N-methylpiperazinyl or -NHCOR 5 ; where R 5 is selected From C 1 -C 8 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
    R 6和R 7各自独立地选自：氢、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 8环烷基或R 6和R 7跟它们连接的氮原子一起形成5-7元杂环；所述的烷基、烷氧基、环烷基或杂环可任选地被一个或多个Y基团取代；其中的Y选自：氢、卤素、羟基、巯基、羧基、氨基或乙酰胺基。 R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen atom to which R 6 and R 7 are connected Together to form a 5-7 membered heterocyclic ring; the alkyl, alkoxy, cycloalkyl or heterocyclic ring may be optionally substituted by one or more Y groups; wherein Y is selected from: hydrogen, halogen, hydroxy , Sulfhydryl, carboxyl, amino or acetamide group.
  3. 根据权利要求1所述的硝基苯醚类化合物、其立体异构体、代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐，其特征在于：The nitrophenyl ether compound, its stereoisomers, metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof according to claim 1, characterized in that:
    R 1选自：甲基或溴； R 1 is selected from: methyl or bromine;
    R 2选自：氢或-OCH 2Ar；其中，Ar选自芳基或芳杂环；所述的芳杂环可任选地包含一个或多个N 原子；所述的芳基或芳杂环可任选地被一个或多个W基团取代；W选自：氢或氰基； R 2 is selected from: hydrogen or -OCH 2 Ar; wherein, Ar is selected from aryl or aromatic heterocycle; said aromatic heterocycle may optionally contain one or more N atoms; said aryl or aromatic heterocycle The ring may be optionally substituted with one or more W groups; W is selected from: hydrogen or cyano;
    R 3和R 4各自独立地选自：氢、C 1-C 8烷基、C 1-C 8烷氧基、C 3-C 8环烷基或R 3和R 4跟它们连接的氮原子一起形成5-6元杂环；所述的烷基或烷氧基可任选地被一个或多个X基团取代；X选自：氢、卤素、羟基、羧基、甲巯基、氨基、呋喃基、二甲胺基、羟基取代的异丙胺基或-NHCOR 5；其中R 5选自C 1-C 4烷基；所述的杂环可被羧基或-CONR 6R 7取代； R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 3 -C 8 cycloalkyl or the nitrogen atom to which R 3 and R 4 are connected Together to form a 5-6 membered heterocycle; the alkyl group or alkoxy group may be optionally substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, carboxyl, methylmercapto, amino, and furan , Dimethylamino, hydroxy-substituted isopropylamino or -NHCOR 5 ; wherein R 5 is selected from C 1 -C 4 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
    其中R 6和R 7各自独立地选自：氢、C 1-C 6烷基、C 1-C 6烷氧基或R 6和R 7跟它们连接的氮原子一起形成5-6元杂环；其中所述的烷基、烷氧基或杂环可任选地被一个或多个Y基团取代；其中的Y选自：氢、卤素、羟基、羧基、氨基或乙酰胺基。 Wherein R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or R 6 and R 7 together with the nitrogen atom to which they are connected to form a 5-6 membered heterocyclic ring ; Wherein the alkyl group, alkoxy group or heterocyclic ring may be optionally substituted by one or more Y groups; wherein Y is selected from: hydrogen, halogen, hydroxyl, carboxyl, amino or acetamido.
  4. 根据权利要求1所述的硝基苯醚类化合物、其立体异构体、代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐，其特征在于：The nitrophenyl ether compound, its stereoisomers, metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof according to claim 1, characterized in that:
    R 1选自：甲基或溴； R 1 is selected from: methyl or bromine;
    R 2选自：氢或-OCH 2Ar；其中，Ar选自芳基或5-6元芳杂环；所述的芳杂环可任选地包含一个或多个N原子；所述的芳基或芳杂环可任选地被一个或多个W基团取代；W选自：氢或氰基； R 2 is selected from: hydrogen or -OCH 2 Ar; wherein, Ar is selected from an aryl group or a 5-6 membered aromatic heterocyclic ring; the aromatic heterocyclic ring may optionally contain one or more N atoms; the aromatic The group or aromatic heterocyclic ring may be optionally substituted by one or more W groups; W is selected from: hydrogen or cyano;
    R 3和R 4各自独立地选自：氢、C 1-C 8烷基或R 3和R 4跟它们连接的氮原子一起形成5-6元杂环；所述的烷基可任选地被一个或多个X基团取代；X选自：氢、卤素、羟基、羧基、甲基巯基、氨基、呋喃基、二甲胺基、羟基取代的二甲胺基或-NHCOR 5；其中R 5选自C 1-C 4烷基；所述的杂环可被羧基或-CONR 6R 7取代； R 3 and R 4 are each independently selected from: hydrogen, C 1 -C 8 alkyl or R 3 and R 4 together with the nitrogen atom to which they are attached form a 5-6 membered heterocyclic ring; the alkyl group may optionally Substituted by one or more X groups; X is selected from: hydrogen, halogen, hydroxyl, carboxyl, methylmercapto, amino, furanyl, dimethylamino, hydroxy-substituted dimethylamino or -NHCOR 5 ; wherein R 5 is selected from C 1 -C 4 alkyl; the heterocyclic ring may be substituted by carboxy or -CONR 6 R 7;
    其中R 6和R 7各自独立地选自：氢、C 1-C 6烷基或R 6和R 7跟它们连接的氮原子一起形成5-6元杂环；所述的烷基或杂环可任选地被一个或多个Y基团取代；其中的Y选自：氢、卤素、羟基、羧基、氨基或乙酰胺基。 Wherein R 6 and R 7 are each independently selected from: hydrogen, C 1 -C 6 alkyl or R 6 and R 7 together with the nitrogen atom to which they are connected to form a 5-6 membered heterocyclic ring; the alkyl or heterocyclic ring It can be optionally substituted by one or more Y groups; where Y is selected from: hydrogen, halogen, hydroxyl, carboxyl, amino, or acetamido.
  5. 根据权利要求1所述的硝基苯醚类化合物、其立体异构体、代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐，其特征在于，所述硝基苯醚类化合物选自以下任一化合物：The nitrophenyl ether compound, its stereoisomers, metabolites, metabolic precursors, prodrugs, solvates, crystals, or pharmaceutically acceptable salts thereof according to claim 1, wherein the nitrophenyl ether The phenyl ether compound is selected from any of the following compounds:
    (3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)-L-丝氨酸(1)，(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-serine (1),
    (3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)-L-苏氨酸(2)，(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-threonine (2),
    (3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)-L-脯氨酸(3)，(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-proline (3),
    (S)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)哌啶-2-羧酸(4)，(S)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxylic acid ( 4),
    2-甲基-1-((3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)氨基)丙烷-2-醇(5)，2-methyl-1-((3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propane-2- Alcohol (5),
    2-甲基-2-((3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)氨基)丙烷-1,3-二醇(6)，2-methyl-2-((3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propane-1, 3-diol (6),
    2,2'-((3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)氮杂二基)双(乙烷-1-醇)(7)，2,2'-((3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)azadiyl)bis(ethyl Alkan-1-ol) (7),
    2-((3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)氨基)丙烷-1,3-二醇(8)，2-((3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propane-1,3-diol( 8),
    2-((3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)氨基)乙-1-醇(9)，2-((3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)ethan-1-ol (9),
    N-(2-((3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)氨基)乙基)乙酰胺(10)，N-(2-((3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)ethyl)acetamide( 10),
    N-甲基-N-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)甘氨酸(11)，N-methyl-N-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)glycine (11),
    3-((3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)氨基)丙酸(12)，3-((3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propionic acid (12),
    (3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)-L-蛋氨酸(13)，(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-methionine (13),
    1-(呋喃-3-基)-N-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)甲胺(14)，1-(Furan-3-yl)-N-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)methylamine (14),
    N 1,N 1-二甲基-N 2-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)乙烷-1,2-二胺(15)， N 1 ,N 1 -Dimethyl-N 2 -(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)ethyl Alkyl-1,2-diamine (15),
    3-氯-N-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)丙-1-胺(16)，3-chloro-N-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)propan-1-amine (16) ,
    2-((3-((3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)氨基)丙基)氨基)丙烷-1,3-二醇(17)，2-((3-((3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)amino)propyl)amino) Propane-1,3-diol (17),
    (3-硝基-4-((2-溴-[1,1'-联苯]-3-基)甲氧基)苄基)-L-苏氨酸(18)，(3-nitro-4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-threonine (18),
    (S)-N-(1-羟基-2-(羟甲基)丁-2-基)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)吡咯烷-2-羧酰胺(19)，(S)-N-(1-hydroxy-2-(hydroxymethyl)butan-2-yl)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl ]-3-yl)methoxy)benzyl)pyrrolidine-2-carboxamide (19),
    (S)-N-(2-羟基-2-甲基丙基)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)吡咯烷-2-羧酰胺(20)，(S)-N-(2-Hydroxy-2-methylpropyl)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl) (Methoxy)benzyl)pyrrolidine-2-carboxamide (20),
    (3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)-L-脯氨酰-L-丝氨酸(21)，(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-prolyl-L-serine (21),
    (S)-N-(2-羟乙基)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)吡咯烷-2-羧酰胺(22)，(S)-N-(2-Hydroxyethyl)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl Yl)pyrrolidine-2-carboxamide (22),
    (S)-N-(1,3-二羟基-2-甲基丙烷-2-基)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)吡咯烷-2-羧酰胺(23)，(S)-N-(1,3-Dihydroxy-2-methylpropan-2-yl)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl ]-3-yl)methoxy)benzyl)pyrrolidine-2-carboxamide (23),
    (S)-N-((S)-1-羟基丙烷-2-基)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)吡咯烷-2-羧酰胺(24)，(S)-N-((S)-1-Hydroxypropan-2-yl)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3- Yl)methoxy)benzyl)pyrrolidine-2-carboxamide (24),
    (3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)-L-脯氨酰基-L-苏氨酸(25)，(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-L-prolyl-L-threonine (25 ),
    (S)-N-(2-乙酰氨基乙基)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)吡咯烷-2-羧酰胺(26)，(S)-N-(2-Acetylaminoethyl)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy) Benzyl)pyrrolidine-2-carboxamide (26),
    (2S)-N-(2,3-二羟丙基)-N-甲基-1-(3-硝基-4-((2-甲基-[1,1’-联苯]-3-基)甲氧基)苄基)吡咯烷-2-羧酰胺(27)，(2S)-N-(2,3-Dihydroxypropyl)-N-methyl-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3 -Yl)methoxy)benzyl)pyrrolidine-2-carboxamide (27),
    (S)-N-(2-羟基-2-甲基丙基)-1-(3-硝基-4-((2-甲基-[1,1’-联苯]-3-基)甲氧基)苄基)哌啶-2-羧酰胺(28)，(S)-N-(2-Hydroxy-2-methylpropyl)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl) (Methoxy)benzyl)piperidine-2-carboxamide (28),
    ((S)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)哌啶-2-羰基)-L-苏氨酸(29)，((S)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carbonyl) -L-threonine (29),
    ((S)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)哌啶-2-羰基)丙氨酸(30)，((S)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carbonyl) Alanine (30),
    ((S)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)哌啶-2-羰基)-L-脯氨酸(31)，((S)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carbonyl) -L-Proline (31),
    ((S)-1-(3-硝基-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)苄基)哌啶-2-羰基)-L-丝氨酸(32)，((S)-1-(3-nitro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carbonyl) -L-serine (32),
    (2-((3-氰基苄基)氧基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)-L-苏氨酸(33)，(2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)-5-nitrobenzyl )-L-threonine (33),
    (2-((3-氰基苄基)氧基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)-L-丝氨酸(34)，(2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)-5-nitrobenzyl )-L-serine (34),
    (2-((3-氰基苄基)氧基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)-L-脯氨酸(35)，(2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)-5-nitrobenzyl )-L-Proline(35),
    (S)-1-(2-(3-氰基苄基)氧基)-4-(((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)哌啶-2-羧酸(36)，(S)-1-(2-(3-cyanobenzyl)oxy)-4-(((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)- 5-nitrobenzyl)piperidine-2-carboxylic acid (36),
    N-(2-((2-((3-氰基苄基)氧基)-4-(((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)氨基)乙基)乙酰胺(37)，N-(2-((2-((3-cyanobenzyl)oxy)-4-(((2-methyl-[1,1'-biphenyl]-3-yl)methoxy) -5-nitrobenzyl)amino)ethyl)acetamide (37),
    3-((2-((1,3-二羟基丙烷-2-基)氨基)甲基)-5-(((2-甲基-[1,1'-联苯]-3-基)甲氧基)-4-硝基苯氧基)甲基)苄腈(38)，3-((2-((1,3-Dihydroxypropan-2-yl)amino)methyl)-5-(((2-methyl-[1,1'-biphenyl]-3-yl) (Methoxy)-4-nitrophenoxy)methyl)benzonitrile (38),
    3-((2-((2-羟乙基)氨基)基)-5-(((2-甲基-[1,1'-联苯]-3-基)甲氧基)-4-硝基苯氧基)甲基)苄腈(39)，3-((2-((2-hydroxyethyl)amino)yl)-5-(((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)-4- Nitrophenoxy)methyl)benzonitrile (39),
    N-(2-((3-氰基苄基)氧基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)-N-甲基甘氨酸(40)，N-(2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)-5-nitro Benzyl)-N-methylglycine (40),
    (S)-1-(2-((3-氰基苄基)氧基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)-N-(2-羟基-2-甲基丙基)哌啶-2-羧酰胺(41)，(S)-1-(2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)- 5-nitrobenzyl)-N-(2-hydroxy-2-methylpropyl)piperidine-2-carboxamide (41),
    ((S)-1-(2-((3-氰基苄基)氧基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)哌啶-2-羰基)-L-脯氨酸(42)；((S)-1-(2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy) -5-nitrobenzyl)piperidine-2-carbonyl)-L-proline (42);
    ((S)-1-(2-(3-氰基苄基)氧基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)哌啶-2-羰基)-L-丝氨酸(43)，((S)-1-(2-(3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)- 5-nitrobenzyl)piperidine-2-carbonyl)-L-serine (43),
    (4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基-2-(吡啶-3-基甲氧基)苄基)-L-丝氨酸(44)，(4-((2-Methyl-[1,1'-biphenyl]-3-yl)methoxy)-5-nitro-2-(pyridin-3-ylmethoxy)benzyl)- L-serine (44),
    (4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基-2-(吡啶-3-基甲氧基)苄基)-L-苏氨酸(45)，(4-((2-Methyl-[1,1'-biphenyl]-3-yl)methoxy)-5-nitro-2-(pyridin-3-ylmethoxy)benzyl)- L-threonine (45),
    (S)-1-(4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基-2-(吡啶-3-基甲氧基)苄基)哌啶-2-羧酸(46)，(S)-1-(4-((2-Methyl-[1,1'-biphenyl]-3-yl)methoxy)-5-nitro-2-(pyridin-3-ylmethoxy (Yl)benzyl)piperidine-2-carboxylic acid (46),
    (2-((2-氰基吡啶-4-基)甲基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)-L-丝氨酸(47)，(2-((2-Cyanopyridin-4-yl)methyl)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)-5-nitro Benzyl)-L-serine (47),
    (2-((2-氰基吡啶-4-基)甲基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)-L-苏氨酸(48)，(2-((2-Cyanopyridin-4-yl)methyl)-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)-5-nitro Benzyl)-L-threonine (48),
    (S)-1-(2-(((2-氰基吡啶-4-基)甲氧基)-4-((2-甲基-[1,1'-联苯]-3-基)甲氧基)-5-硝基苄基)哌啶-2-羧酸(49)。(S)-1-(2-(((2-cyanopyridin-4-yl)methoxy)-4-((2-methyl-[1,1'-biphenyl]-3-yl) Methoxy)-5-nitrobenzyl)piperidine-2-carboxylic acid (49).
  6. 一种权利要求1所述的硝基苯醚类化合物的制备方法，其特征在于，所述制备方法如下：A method for preparing a nitrophenyl ether compound according to claim 1, wherein the preparation method is as follows:
    以a为原料，与取代苯甲醛b或c分别得到中间体i和ii；i与胺类化合物HNR 3R 4经还原胺化制得通式(I)化合物，或再进一步经缩合反应和水解反应中的一种或几种组合的方法制得通式(I)化合物；ii与溴代物经醚化反应制得中间体iii，iii与胺类化合物HNR 3R 4经还原胺化制得通式(I)化合物，或再进一步经缩合反应和水解反应中的一种或几种组合的方法制得通式(I)化合物； Using a as a raw material, and substituted benzaldehyde b or c to obtain intermediates i and ii, respectively; i and amine compound HNR 3 R 4 are reductively amination to obtain a compound of general formula (I), or further subjected to condensation reaction and hydrolysis One or a combination of methods in the reaction to obtain the compound of general formula (I); ii and bromide are etherified to obtain intermediate iii, iii and amine compound HNR 3 R 4 are reductively amination to obtain the intermediate The compound of formula (I), or the compound of general formula (I) can be prepared by one or more methods of condensation reaction and hydrolysis reaction;
    Figure PCTCN2021074875-appb-100002
    Figure PCTCN2021074875-appb-100002
    其中，R 1、R 2、R 3和R 4的定义如权利要求1所述。 Wherein, R 1 , R 2 , R 3 and R 4 are as defined in claim 1.
  7. 一种药物组合物，其特征在于，所述药物组合物包括在治疗上有效量的活性组分和药学上可接受的辅料；所述的活性组分包括如权利要求1-5中任一项所述的硝基苯醚类化合物、其立体异构体、代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐。A pharmaceutical composition, characterized in that, the pharmaceutical composition includes a therapeutically effective amount of active components and pharmaceutically acceptable auxiliary materials; the active components include any one of claims 1-5 The nitrophenyl ether compound, its stereoisomers, metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof.
  8. 一种权利要求1-5任意一项所述的硝基苯醚类化合物、其立体异构体、代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐在制备PD-1/PD-L1抑制剂药物中的应用。A nitrophenyl ether compound, its stereoisomers, metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof according to any one of claims 1 to 5 are prepared Application of PD-1/PD-L1 inhibitor drugs.
  9. 一种权利要求1-5中任一项所述的硝基苯醚类化合物、其立体异构体、代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐在制备治疗PD-1/PD-L1介导的免疫抑制相关疾病的药物中的用途。A nitrophenyl ether compound according to any one of claims 1-5, its stereoisomers, metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof Use in the preparation of a medicine for treating PD-1/PD-L1 mediated immune suppression related diseases.
  10. 根据权利要求9所述的应用，其特征在于，所述PD-1/PD-L1介导的免疫抑制的相关疾病包括癌症。The application according to claim 9, wherein the PD-1/PD-L1 mediated immune suppression related diseases include cancer.
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