WO2021143937A1 - 吡咯类化合物、其制备方法和药物组合物与用途 - Google Patents

吡咯类化合物、其制备方法和药物组合物与用途 Download PDF

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WO2021143937A1
WO2021143937A1 PCT/CN2021/074874 CN2021074874W WO2021143937A1 WO 2021143937 A1 WO2021143937 A1 WO 2021143937A1 CN 2021074874 W CN2021074874 W CN 2021074874W WO 2021143937 A1 WO2021143937 A1 WO 2021143937A1
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pyrrole
phenyl
ureido
carboxylic acid
cyclohexylamino
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French (fr)
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赖宜生
葛书山
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 present invention belongs to the field of new compounds, and specifically relates to a class of pyrrole compounds as indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors, their metabolites, metabolic precursors, prodrugs, solvates, crystals or The pharmaceutically acceptable salts thereof, their preparation methods, pharmaceutical compositions containing these compounds, and the use of these compounds or compositions in the treatment of diseases related to IDO1-mediated immunosuppression.
  • IDO1 indoleamine 2,3-dioxygenase 1
  • IDO1 Indoleamine 2,3-dioxygenase 1
  • cytokines such as IFN- ⁇ , TNF- ⁇ , IL-1 ⁇ and IL-6 can induce IDO1 expression.
  • IDO1 can participate in the regulation of the body's innate immunity and adaptive immunity by catalyzing the oxidative metabolism of tryptophan. IDO1 achieves its regulatory effect on the immune system by catalyzing the local depletion of tryptophan and the accumulation of its metabolites by catalyzing tryptophan. On the one hand, the depletion of tryptophan can induce the T cell division cycle to stop at G1 by activating the GCN2 pathway.
  • helper T cell 17 helper T cell 17
  • tryptophan metabolites such as kynurenine are cytotoxic It can kill T cells and natural killer (NK) cells, and these metabolites can also induce CD4 + T cells to differentiate into regulatory T cells (Treg) by activating aryl hydrocarbon receptors (AhR), and promote dendrites Cells (DC) are transformed into tolerogenic DCs; in addition, tryptophan metabolites can inhibit the function of NK cells by down-regulating the expression of NK cell receptors, which can further inhibit the body's immune response.
  • IDO1 is related to many physiological and pathological processes. Studies have shown that IDO1 plays an important role in host immune defense and maternal-fetal immune tolerance and other physiological stress processes. During this period, the secretion of cytokines such as IFN- ⁇ significantly increases, thereby inducing IDO1 expression, leading to tryptophan depletion and kynurenine, etc. The accumulation of metabolites inhibits the mother's T cell response, induces the mother's immune tolerance, and ensures that the fetus is not rejected by the mother's immune system. The depletion of tryptophan in the host microenvironment makes it unable to provide the necessary tryptophan for the replication of pathogenic microorganisms, leading to the death of pathogenic microorganisms.
  • IDO1-mediated immunosuppression can avoid excessive activation of the body's immune system.
  • IDO1 also exerts an immunosuppressive effect on the survival of transplanted tissue in the new host.
  • IDO1-mediated immune tolerance is closely related to tumor immune escape, viral infection, neurodegenerative diseases, organ transplant rejection, autoimmune diseases, neuropsychiatric diseases and cataracts.
  • the local depletion of tryptophan and the accumulation of its metabolites mediated by over-expression of IDO1 can inhibit the activation of T cells, leading to immune tolerance of the body.
  • IDO1 catalyzed tryptophan metabolites such as kynurenine and quinolinic acid are neurotoxic, and these metabolites are related to neurodegenerative diseases such as memory disorders, Alzheimer's disease (AD), and dementia. , Alzheimer's disease, Parkinson's disease, Parkinson's syndrome and movement disorders are closely related to the occurrence.
  • Neuropsychiatric diseases such as depression, schizophrenia, and anxiety are also related to the overexpression of IDO1 and increased levels of metabolites such as kynurenine.
  • IDO1 causes tryptophan depletion, thereby reducing the amount of tryptophan used to synthesize the neurotransmitter serotonin, leading to serotonin deficiency, plus neurotoxic kynurenine and quinolinic acid, etc.
  • IDO1 overexpression also exists in various autoimmune diseases.
  • the DCs of synovial joint tissues highly express IDO1.
  • the serum tryptophan concentration of the patients decreases, while the kynurenine concentration and the kynurenine/tryptophan ratio are significantly increased.
  • IDO1 Immune suppression induced by IDO1 plays an important role in tumor immune escape. IDO1 is overexpressed in various tumors and cells in their microenvironment such as DC cells and stromal cells, leading to local tumor depletion of tryptophan and accumulation of tryptophan metabolites, thereby inducing tumor immune escape and helping tumor cells to escape the body's immune system. attack.
  • IDO1 inhibitors can reduce the accumulation of tryptophan metabolism and kynurenine and other metabolites, thereby reversing the immunosuppressive effect mediated by IDO1, restoring the proliferation and function of T cells and NK cells, and inhibiting the proliferation of Treg cells, thereby enhancing The immune response of the body, therefore IDO1 inhibitors can be used to treat or prevent the above-mentioned related diseases caused by IDO1 mediated immunosuppression, including cancer, viral infections, neurodegenerative diseases, cataracts, organ transplant rejection, depression and autoimmunity Diseases etc.
  • IDO1 inhibitors can also be combined with other chemotherapeutics, targeted antitumor drugs, immune checkpoint inhibitors, immune checkpoint agonists, antitumor vaccines, antiviral agents, antiviral vaccines, cytokine therapy, and adoptive cellular immunity Treatment and radiotherapy are used in combination to achieve the purpose of synergistic or enhanced therapy.
  • the technical problem to be solved by the present invention is to provide a compound with the structural characteristics of general formula (I), its metabolites, metabolic precursors, prodrugs, solvates, crystals and pharmaceutically acceptable salts thereof, Preparation method, pharmaceutical composition and use.
  • the compound of the present invention has excellent IDO1 inhibitory activity and can be used to treat and/or prevent various related diseases caused by IDO1 mediated immunosuppression.
  • the present invention provides pyrrole compounds, their metabolites, metabolic precursors, prodrugs, solvates, crystals or their pharmaceutically acceptable salts with the structural characteristics of general formula (I):
  • R 1 represents cyano, -CO 2 R 6 or -CONR 7 R 8 ;
  • R 2 represents hydrogen, halogen, cyano, hydroxyl or nitro
  • R 3 and R 4 each independently represent hydrogen, C 1 -C 8 alkyl, C 3 -C 8 cycloalkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 1 -C 8 alkane
  • the amino group or R 3 and R 4 together with the nitrogen atom attached to them form a 5-7 membered heterocyclic ring; wherein the heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; wherein The heterocyclic ring may be optionally substituted by one or more of the following groups: halogen, nitro, cyano, hydroxyl, amino, C 1 -C 8 alkyl, C 1 -C 8 alkoxy or C 3 -C 6 cycloalkyl;
  • R 5 represents an aryl group or an aromatic heterocyclic ring, wherein the aryl group or an aromatic heterocyclic ring may be optionally substituted by one or more R 9;
  • R 6 , R 7 and R 8 each independently represent hydrogen, C 1 -C 8 alkyl, C 3 -C 8 cycloalkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 1- C 8 alkylamino;
  • R 9 represents hydrogen, halogen, cyano, hydroxyl, mercapto, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 1 -C 8 alkylamino or haloalkyl;
  • 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 straight-chain alkylamino, branched alkylamino or cyclic alkylamino;
  • said alkenyl represents straight-chain alkenyl, branched alkenyl or cyclic alkenyl;
  • said alkynyl represents straight-chain alkynyl or branched Alkynyl
  • the aryl group represents phenyl, naphthyl, acenaphthyl or tetrahydronaphthyl; the aromatic heterocycle represents pyrrolyl, pyrazolyl, imidazolyl, furyl, thienyl, oxazolyl, isoxazole Monocyclic heterocycles of pyridyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl; or quinolinyl, quinoxalinyl, indolyl, benzimidazolyl, benzoxanyl Azolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzofuranyl, benzothienyl, 2,3-dihydrobenzo[1,4]dioxanyl Group, or a bicyclic heterocycle of benzo[1,3]d
  • the halogenated alkyl group is a linear or branched saturated hydrocarbon group with 1-8 carbon atoms, or a cyclic saturated hydrocarbon group with 3-8 carbon atoms, or a straight chain with 1-8 carbon atoms. Or a cyclic saturated hydrocarbon group with 3-8 carbon atoms of a branched saturated hydrocarbon group; wherein one or more hydrogen atoms are replaced by one or more halogen atoms.
  • R 1 represents cyano, -CO 2 R 6 or -CONR 7 R 8 ;
  • R 2 represents hydrogen or halogen
  • R 3 and R 4 each independently represent hydrogen, C 1 -C 8 alkyl, C 3 -C 8 cycloalkyl, C 1 -C 8 alkylamino or R 3 and R 4 are formed together with the nitrogen atom to which they are attached 5-7 membered heterocyclic ring; wherein the heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; wherein the heterocyclic ring may optionally be substituted by one or more of the following groups Group substitution: C 1 -C 8 alkyl or C 3 -C 6 cycloalkyl;
  • R 5 represents an aryl group or an aromatic heterocyclic ring, wherein the aryl group or an aromatic heterocyclic ring may be optionally substituted by one or more R 9;
  • R 6 , R 7 and R 8 each independently represent hydrogen, C 1 -C 8 alkyl or C 3 -C 8 cycloalkyl;
  • R 9 represents hydrogen, halogen, cyano, hydroxyl, mercapto, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 1 -C 8 alkylamino or haloalkyl.
  • R 1 represents cyano, -CO 2 R 6 or -CONR 7 R 8 ;
  • R 2 represents hydrogen or halogen
  • R 3 and R 4 each independently represent hydrogen, C 1 -C 4 alkyl, C 3 -C 6 cycloalkyl, C 1 -C 6 alkylamino or R 3 and R 4 are formed together with the nitrogen atom to which they are attached 5-7 membered heterocyclic ring; wherein the heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; wherein the heterocyclic ring may optionally be substituted by one or more methyl groups replace;
  • R 5 represents a benzene ring or an isoxazolyl group, wherein the benzene ring may be optionally substituted by one or more R 9;
  • R 6 , R 7 and R 8 each independently represent hydrogen, C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl;
  • R 9 represents hydrogen, halogen, cyano, C 1 -C 5 alkyl, C 1 -C 5 alkoxy or trifluoromethyl.
  • R 1 stands for COOH
  • R 2 represents hydrogen or halogen
  • R 3 and R 4 each independently represent a C 1 -C 4 alkyl group or a C 3 -C 6 cycloalkyl group;
  • R 5 represents a benzene ring, and the benzene ring may be optionally substituted by one or more R 9;
  • R 9 represents hydrogen, halogen, cyano, C 1 -C 5 alkyl or C 1 -C 5 alkoxy.
  • pyrrole 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 being any one of the following methods:
  • Method 1 Using substituted nitrobenzene as raw material, reacting with amine compound HNR 3 R 4 under the action of alkali to prepare intermediate i, i and pyrrole-2-carboxylate are reacted with Ullmann to prepare intermediate ii, ii Intermediate iii is obtained by reduction, and compound iv is obtained by condensation of iii with substituted benzene isocyanate R 5 NCO, or iii firstly forms active intermediate with 4-nitrophenyl chloroformate, and then reacts with amine compound R 5 NH 2
  • the target compound iv is prepared; iv is hydrolyzed to obtain the target compound v; v is reacted with oxalyl chloride or thionyl chloride to form acid chloride and then reacted with the amine compound HNR 7 R 8 to obtain the target compound vi; vi is dehydrated at high temperature Obtain the target compound vii;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are as defined above;
  • the base is selected from triethylamine, DIPEA, Na 2 CO 3 , K 2 CO 3 or Cs 2 CO 3 ;
  • the reducing agent is selected from zinc powder and ammonium chloride or iron powder and ammonium chloride;
  • Phosphorus oxychloride undergoes high-temperature dehydration to obtain the target compound vii.
  • Method 2 Using 2-fluoro-5-bromonitrobenzene as the raw material, reacting with the amine compound HNR 3 R 4 under the action of a base to obtain intermediate i, and obtaining intermediates viii, viii and pyrrole via NCS chlorination reaction.
  • -2-carboxylic acid ester is reacted by Ullmann to obtain intermediate ix, ix is reduced by reducing agent to obtain intermediate x, x is condensed with substituted phenyl isocyanate R 5 NCO to obtain intermediate xi, and xi is hydrolyzed to obtain target compound xii;
  • R 3 , R 4 , R 5 and R 6 are as defined above.
  • the base described in step 1) is selected from triethylamine, DIPEA, Na 2 CO 3 , K 2 CO 3 or Cs 2 CO 3 .
  • the reducing agent in step 4) is selected from zinc powder and ammonium chloride or iron powder and ammonium chloride.
  • 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 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 use of the compound of general formula (I), its pharmaceutically acceptable salt or the pharmaceutical composition in the preparation of indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor drugs use.
  • IDO1 inhibitor drugs are used to treat IDO1 mediated immune suppression related diseases, and the related diseases include cancer, viral infections, neurodegenerative diseases, cataracts, organ transplant rejection, depression or autoimmune diseases.
  • 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 medicines for the treatment of cancer, viral infections, neurodegenerative diseases, and cataracts , Organ transplant rejection, depression or autoimmune disease.
  • 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 viral infections include but are not limited to: human immunodeficiency virus, hepatitis B virus, hepatitis C virus, influenza virus, polio virus, cytomegalovirus, coxsackie virus, human papilloma virus, Infection caused by one or more of Stan-Barr virus and varicella-zoster virus.
  • the neurodegenerative diseases include, but are not limited to: one or more of memory disorder, Alzheimer's disease, dementia, Alzheimer's disease, Parkinson's disease, and movement disorders.
  • the autoimmune diseases include but are not limited to: rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, scleroderma, nodular vasculitis, multiple sclerosis, nephropathy, myasthenia gravis, mixed One or more of sexual connective tissue disease, psoriasis, liver disease, endocrine-related disease, and autoimmune response due to infection.
  • the present invention also provides that the compound of general formula (I), its pharmaceutically acceptable salt or the pharmaceutical composition can be used in combination with one or more other types of therapeutic agents and/or therapeutic methods for treatment Related diseases mediated by IDO1.
  • 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.
  • the compound of the present invention has high inhibitory activity on IDO1.
  • the results of pharmacological experiments show that these azole compounds can effectively reverse the immunosuppressive effect mediated by IDO1, promote the proliferation of CD8 + T lymphocytes, increase the secretion of granzyme B and interferon- ⁇ , and reduce CD4 + CD25 + Foxp3 + regulation
  • the generation of T cells reduces the expression of PCNA protein.
  • the results of in vivo pharmacodynamic evaluation showed that the compounds of the present invention can significantly inhibit the growth of transplanted tumors in mice with various tumor types, but have no effect on the growth of transplanted tumors in nude mice with immune system defects, indicating that these compounds are activated by the host Immune response and play an anti-tumor effect.
  • Figure 1 shows the effect of the compounds of the present invention on IDO1 protein expression, in which: Figure 1A shows the effects of compounds 2, 14, 26, 27 and 39 on IDO1 protein expression, and Figure 1B shows the gray-scale scanning statistical results of Figure 1A;
  • Figure 2 shows the effect of the compound of the present invention on the growth of B16F1 melanoma transplanted tumors in mice.
  • Figure 2A shows the effect of compound 2 on the volume of B16F1 melanoma transplanted tumors
  • Figure 2B shows the effect of compound 2 on the weight of B16F1 melanoma transplanted tumors;
  • Figure 3 shows the effect of the compound of the present invention on the growth of CT26 colorectal cancer BALB/c mice transplanted tumor
  • Figure 4 shows that the compound of the present invention does not inhibit transplanted tumors in nude mice with immune system deficiency
  • Figure 5 shows the effect of the compound of the invention on the growth of PAN02 pancreatic cancer transplanted tumor in mice
  • Figure 6 shows the effect of the compound of the present invention on IDO1 activity at different temperatures.
  • Figure 6A shows the effect of Epacadostat on IDO1 activity at different temperatures.
  • Figure 6B shows the effect of compound 26 on IDO1 activity at different temperatures.
  • Figure 6C shows the effect of compound 26 on IDO1 activity at different temperatures. The effect of compound 39 on IDO1 activity at different temperatures.
  • Reagents and materials The reagents required for the experiment are all commercially available chemical or analytical products without special instructions.
  • 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.
  • Dissolve 2 (1g, 1.96mmol) in 15mL dry dichloromethane, cool in an ice bath, add 2mL oxalyl chloride, react overnight at room temperature, remove excess oxalyl chloride under reduced pressure, add 15mL dry dichloromethane to dissolve, ice bath After cooling, 3 mL of ammonia was added dropwise, reacted at room temperature for 5 hours, extracted with dichloromethane, and purified by column chromatography to obtain 0.5 g of white solid with a yield of 50.1%.
  • HeLa cell line ATCC, centrifuge: Eppendorf (CHINA), electrothermal constant temperature blast drying oven (DHG-924385-III): Shanghai Xinmiao Medical Equipment Manufacturing Co., Ltd.
  • Acetic acid (glacial acetic acid): Nanjing Chemical Reagent Co., Ltd., Trifluoroacetic acid: Shanghai Lingfeng Chemical Reagent Co., Ltd., electronic balance: Sartorius, p-dimethylaminobenzaldehyde (CAS: 100-10-7): Aladdin, Recombinant Human IFN- ⁇ (Catalog#AF-300-02): PEPROTECH.
  • HeLa cells purchased from ATCC are stored in the minimum basal medium (2mM L-glutamine and Earle adjusted to contain 1.5g/L sodium bicarbonate, 0.1mM non-essential amino acids, 1mM sodium propionate and 10% fetal bovine serum BSS). The HeLa cells were stored in a humidity-controlled incubator with 5% CO 2 at 37°C.
  • HeLa cells were seeded in a 96-well culture plate at a density of 5 ⁇ 10 3 /well and cultured overnight. On the next day, IFN- ⁇ (final concentration 100 ng/mL) and serial dilutions of the compound (total volume 200 ⁇ L of medium) were added to the cells. After 24 hours of incubation, transfer 140 ⁇ L of supernatant/well to a new 96-well plate, add 10 ⁇ L of 6.1 mol/L trichloroacetic acid, and incubate in a constant temperature oven at 50°C for 30 minutes to produce N-formyl dogs. Urine is hydrolyzed to kynurenine.
  • the reaction mixture was then centrifuged at 4000 rpm for 10 min to remove the precipitate. Transfer 100 ⁇ L of supernatant/well to another 96-well plate and mix with an equal volume of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. Use a microplate reader to detect the absorbance at 480nm, and calculate the result using an IC 50 calculator.
  • the experiment has 3 multiple holes.
  • the MTT method was used to detect the survival rate of HeLa cells in each group in order to investigate whether the compound inhibits the activity of IDO1 by inhibiting the proliferation of HeLa cells.
  • BMS-52 is compound No. 52 in WO2015031295A1.
  • the purpose of this experiment is to investigate whether the compound of the present invention inhibits the activity of IDO1 by down-regulating the expression of IDO1 protein.
  • the immunoblotting method was used to detect the effect of the compound on the expression of IDO1 protein.
  • HeLa cells were seeded in a 6-well plate at a density of 2 ⁇ 10 5 per well, and cultured at 37°C and 5% CO 2 for 12 hours. Blank control (only add culture medium), model group (add IFN- ⁇ , corresponding positive drug), drug treatment group (add IFN- ⁇ , corresponding compound), culture at 37°C, 5% CO 2 for 24h, collect cells , Western blot detection of IDO1 expression.
  • T lymphocytes are the core executors of the immune system in the human body and play a central role in tumor immune response.
  • the local tryptophan depletion and kynurenine accumulation caused by IDO1 overexpression can inhibit the proliferation of T lymphocytes and induce their apoptosis.
  • it can also promote the differentiation of initial T lymphocytes into regulatory T lymphocytes and inhibit cytokines such as IFN. -Secretion of ⁇ , IL-2 and TNF- ⁇ .
  • the purpose of this experiment is to test the ability of the compounds of the present invention to reverse IDO1-mediated immunosuppression.
  • B16F1 cell treatment aspirate the medium (high sugar DMEM, 10% FBS), wash 1-2 times with PBS. Add 0.25% trypsin for digestion. Aspirate the trypsin, add medium, pipette down the cells, transfer to a 1.5mL centrifuge tube, centrifuge, aspirate the supernatant, add 1mL DMEM medium to resuspend the cells. Add mitomycin C (final concentration 25 ⁇ g/mL), mix by pipetting, 37°C, water bath for 30min, wash 3 times with RP1640, count the cells, and set aside.
  • Preparation of spleen cells Take C57/BL6 mice, remove the eyeballs and bleed to death, remove the spleen aseptically and place it in a 35mm petri dish containing 2mL of sterile pre-cooled RPMI 1640 medium, and gently push the spleen with a 5mL syringe needle. Cell extrusion. Then add 2 mL of medium and pipette repeatedly with a 5 mL pipette until the suspension is uniform. The cell suspension was filtered with a 70 ⁇ m filter, and centrifuged at 300 g for 5 min (4° C.).
  • CD4 + CD25 + Foxp3 + T lymphocytes are an important type of regulatory T lymphocytes, which play an important negative regulatory role in the human immune system.
  • IDO1 can mediate the transformation of initial T cells into regulatory T lymphocytes, leading to an increase in the proportion of regulatory T lymphocytes in the tumor microenvironment, thereby inducing the formation of an immunosuppressed tumor microenvironment.
  • we selected the mouse melanoma B16F1 cell line with high IDO1 expression treated it with compounds, and took its supernatant to co-culture with mouse spleen cells to simulate the tumor microenvironment. After the cells were collected, flow cytometry was used to detect the effect of the compound on the differentiation of naive T cells into regulatory T cells in the co-culture system.
  • B16F1 cells 8 ⁇ 10 4 cells/well
  • splenic lymphocytes (10 6 cells/well, stimulated with 5 ⁇ g/mL ConA) into a 24-well plate
  • Granzyme B is a serine protease commonly found in cytotoxic T lymphocytes (CTL) and natural killer (NK) cell granules, and is the main effector of CTL and NK cells to exert cytotoxicity.
  • 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 levels of granzyme B, IFN- ⁇ and PCNA in tumor tissues.
  • 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 CT26, B16F1, PAN02 cells in logarithmic growth phase, centrifuge at 180g for 5min (4°C), wash twice with pre-cooled PBS, pipette evenly, and the final cell concentration is 1 ⁇ 10 7 /mL , Ice bath for spare.
  • Tumor cell transplantation CT26, B16F1, and PAN02 cell suspension were respectively inoculated into the right axillary of the mouse subcutaneously, and the number of tumor cells inoculated was 1 ⁇ 10 6 /mouse.
  • the tumor size of the mouse was measured with a vernier caliper every two days, and the mouse weight was weighed once.
  • 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.
  • Model group PBS+2% Tween 20+2% DMSO, ip, qd.
  • positive control group cisplatin, dose: 1 mg/kg, ip, qod.
  • administration group 1-3 Example Compound 2, dosage: 1.25mg/kg, 2.5mg/kg, 5mg/kg, ip, qd.).
  • mice transplanted with CT26 colorectal tumors were divided into 5 groups with 8 mice in each group.
  • Model group PBS+2% Tween 20+2% DMSO, ip, qd.
  • positive control group (5-FU, dose: 25 mg/kg, ip, qod.)
  • administration group (Example Compound 2 , Dose: 5mg/kg, ip, qd.).
  • mice transplanted with CT26 colorectal tumors were divided into 3 groups, each with 8 mice.
  • Model group PBS+2% Tween 20+2% DMSO, ip, qd.
  • positive control group (5-FU, dose: 25 mg/kg, ip, qod.)
  • administration group (Example Compound 2 , Dose: 10mg/kg, ip, qd.).
  • Model group PBS+2% Tween 20+2% DMSO, ig, qd.
  • positive control group 1 gemcitabine, dose: 30 mg/kg, ip, qod.
  • positive control group 2 Epacadostat, dose: 50 mg/kg, ig, bid.
  • administration group Example Compound 9, dose: 15 mg/kg, ig, qd.).
  • the compound of the present invention inhibits the growth of transplanted tumors of B16F1 melanoma mice in a dose-dependent manner
  • the compound of the present invention can significantly inhibit the growth of CT26 colorectal cancer BALB/c mice transplanted tumor
  • Example Compound 2 can significantly inhibit the growth of CT26 colorectal cancer BALB/c mice transplanted tumors, and there is no obvious fibrosis and inflammation in the liver of the mice, and other organs such as the heart, The morphology and structure of the kidney, spleen and lung did not change significantly, indicating that these compounds did not have significant drug-induced damage to the various organs of mice.
  • the body weight of the mice in the 5-FU group began to decrease after the 9th day, while the body weight of the mice in the compound 2 administration group remained stable, indicating that the compound 2 did not affect the body weight of the mice.
  • the compound of the present invention does not inhibit the transplanted tumor of nude mice with defective immune system
  • the compound of the present invention can significantly inhibit the growth of PAN02 pancreatic cancer transplanted tumor in mice
  • the other compounds in the present invention also show significant anti-tumor effects in various tumor types such as CT26, EMT6, B16F1, PAN02, LLC and other mouse xenograft tumor models.
  • compounds 20, 23, 24, 26, 27, 28, 30, 31, 33, 34, 35, 36, 38, 39, 40, and 41 will defecate at low doses (2.5 mg/kg to 15 mg/kg).
  • the results of immunohistochemistry and TUNEL experiments on tumor tissues show that these compounds can also increase CD8 + T cell infiltration, promote the secretion of granzyme B, increase the expression of IFN- ⁇ in tumor tissues, and reduce Foxp3 + Treg.
  • the number of cell populations reduces the expression of PCNA protein.
  • CETSA Cell Thermal Migration Test
  • B16F1 cells were stimulated with IFN- ⁇ (final concentration 100ng/mL) for 24 hours and then divided into 2 groups evenly: control group and compound treatment group. Add compounds 2 and 27 to ensure that the final concentration is 1 ⁇ M. Add an equal volume of DMSO to the control group. After 3 hours, collect the cells, wash twice with PBS, resuspend the cells in 500 ⁇ L PBS, and divide them into 10 parts and place them in imported PCR tubes. .
  • MST Micro thermophoresis
  • the IDO1 inhibitors developed in the early stage mainly inhibit the activity of IDO1 by combining with heme-containing IDO1 (holo-IDO1).
  • Holo-IDO1 inhibitors represented by Epacadostat mainly occupy the catalytic pocket of IDO1 by complexing with heme iron ions, thereby producing inhibitory activity.
  • the purpose of this experiment is to verify the binding mode of the compound of the present invention and IDO1 protein.
  • test substance and IDO1 protein of different concentrations Divide the test substance and IDO1 protein of different concentrations into two groups, mix them and add them to the EP tube, pre-incubate at 25°C and 37°C for 2h, add catalase solution, methylene blue solution, and TritonX-100 solution , VCNa solution, incubate at 37°C for 15 min. Add D-Trp solution, the total reaction system is 250 ⁇ L, continue to incubate for 60min, and perform detection according to 1.2 detection method.

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Abstract

本发明公开了一种具有式(I)结构特征的吡咯类化合物、其代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐、其制备方法、以及它们在制备吲哚胺2,3-双加氧酶1(IDO1)抑制剂药物的用途。实验结果表明，本发明的吡咯类化合物对IDO1的活性具有显著抑制作用，能够有效地促进T淋巴细胞增殖，抑制初始T淋巴细胞分化为调节性T细胞，逆转IDO1介导的免疫抑制作用，可以用于治疗具有IDO1介导的犬尿氨酸代谢途径的病理学特征的相关疾病，包括癌症、病毒感染、神经变性疾病、白内障、器官移植排斥、抑郁症和自身免疫性疾病等。

Description

吡咯类化合物、其制备方法和药物组合物与用途 技术领域
本发明属于新化合物领域，具体涉及一类作为吲哚胺2,3-双加氧酶1(IDO1)抑制剂的吡咯类化合物、其代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐，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IDO1介导的免疫抑制的相关疾病方面的用途。
背景技术
吲哚胺2,3-双加氧酶1(IDO1)是人体肝脏外催化色氨酸的犬尿氨酸代谢途径中的限速酶。IDO1在多种组织(如肺、肾、脑、胎盘、胸腺)以及多种细胞(如巨噬细胞和树突状细胞)中表达，细胞因子如IFN-γ、TNF-α、IL-1β和IL-6可诱导IDO1表达。
IDO1可以通过催化色氨酸氧化代谢参与机体的固有免疫和适应性免疫的调控。IDO1主要是通过催化色氨酸导致色氨酸局部耗竭及其代谢产物蓄积来实现其对免疫***的调控作用：一方面，色氨酸的耗竭可通过激活GCN2通路诱导T细胞***周期停滞于G1期，从而抑制T细胞的增殖，同时还抑制初始CD4 +T细胞分化为辅助性T细胞17(Th17)，进而产生免疫抑制；另一方面，犬尿氨酸等色氨酸代谢产物具有细胞毒性，可以杀灭T细胞和自然杀伤(NK)细胞，而且这些代谢产物还可以通过激活芳香烃受体(AhR)来诱导CD4 +T细胞分化为调节性T细胞(Treg)，并促进树突状细胞(DC)转化成致耐受性DC；此外，色氨酸代谢产物可以通过下调NK细胞受体的表达来抑制NK细胞的功能，这些都可以进一步抑制机体的免疫反应。
IDO1与很多生理病理过程有关。研究表明，IDO1在宿主免疫防御和母胎免疫耐受等生理应激过程中起重要作用，期间细胞因子如IFN-γ分泌显著增加，从而诱导IDO1表达，导致色氨酸耗竭和犬尿氨酸等代谢产物聚积，从而抑制母体的T细胞反应，诱导母体免疫耐受，确保胎儿不被母体的免疫***排斥。而在宿主微环境中的色氨酸耗竭使其不能为病原微生物复制提供所必需的色氨酸，从而导致病原微生物死亡，与此同时IDO1介导的免疫抑制可以避免机体免疫***的过度激活。IDO1对移植组织在新宿主中的存活也发挥免疫抑制作用。这些研究结果说明IDO1是一种免疫调节酶，参与机体的免疫耐受。
众多研究表明，IDO1介导的免疫耐受与肿瘤免疫逃逸、病毒感染、神经变性疾病、器官移植排斥、自身免疫性疾病、神经精神疾病和白内障等疾病的密切相关。在这些疾病中，过度表达的IDO1所介导的色氨酸局部耗竭及其代谢产物的聚积可以抑制T细胞的激活，导致机体的免疫耐受。
此外，IDO1催化的色氨酸代谢产物如犬尿氨酸和喹啉酸等具有神经毒性，并且这些代谢产物与神经变性疾病如记忆障碍症、阿尔茨海默病(AD)、认知障碍症、老年痴呆症、帕金森病、帕金森综合症和运动障碍性疾病的发生密切相关。神经精神疾病如抑郁症、精神***症、焦虑症也与IDO1过度表达和犬尿氨酸等代谢产物水平升高有关。IDO1的过度表达造成色氨酸耗竭，从而减少用于合成神经递质5-羟色胺的色氨酸的量，导致5-羟色胺缺乏，再加上具有神经毒性的犬尿氨酸和喹啉酸等代谢产物的聚积，共同促进神经精神疾病的发生，而且是多种心境障碍的因素。
IDO1过度表达所介导的色氨酸耗竭也存在于各种自身免疫性疾病中。在类风湿关节炎患者滑膜关节组织的DCs高表达IDO1，患者血清中色氨酸浓度降低，而犬尿氨酸浓度和犬尿氨酸/色氨酸比值均明显升高。
IDO1诱导的免疫抑制在肿瘤免疫逃逸中起重要作用。IDO1过度表达于各类肿瘤及其微环境中的细胞如DC细胞和基质细胞，导致肿瘤局部色氨酸耗竭和色氨酸代谢产物聚积，从而诱导肿瘤免疫逃逸，帮助肿瘤细胞逃避机体免疫***的攻击。
IDO1抑制剂可以降低色氨酸代谢和犬尿氨酸等代谢产物的聚积，从而逆转IDO1介导的免疫抑制作用，恢复T细胞和NK细胞的增殖和功能，并且抑制Treg细胞的增殖，从而增强机体的免疫应答，因此IDO1抑制剂可用于治疗或预防由IDO1介导的免疫抑制所引起的上述相关疾病，包括癌症、病毒感染、神经变性疾病、白内障、器官移植排斥、抑郁症和自身免疫性疾病等。此外，IDO1抑制剂还可以和其他化疗剂、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和放射治疗联合使用，达到协同或增强疗法的目的。
发明内容
发明目的：本发明所要解决的技术问题在于提供了一种具有通式(Ⅰ)结构特征的化合物、其代谢产物、代谢前体、前药、溶剂化物、结晶及其药学上可接受的盐、制备方法、药物组合物及用途。本发明的化合物具有优异的IDO1抑制活性，可以用于治疗和/或预防IDO1介导的免疫抑制所引起的各种相关疾病。
技术方案：本发明提供了通式(I)结构特征的吡咯类化合物、其代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐：
Figure PCTCN2021074874-appb-000001
其中：
R 1代表氰基、-CO 2R 6或-CONR 7R 8；
R 2代表氢、卤素、氰基、羟基或硝基；
R 3和R 4各自独立地代表氢、C 1-C 8烷基、C 3-C 8环烷基、C 2-C 8烯基、C 2-C 8炔基、C 1-C 8烷氨基或R 3和R 4与跟它们连接的氮原子一起形成5-7元杂环；其中所述的杂环可任选地包含一个或多个选自O、S或N杂原子；其中所述的杂环上可任选地被一个或多个以下基团取代：卤素、硝基、氰基、羟基、氨基、C 1-C 8烷基、C 1-C 8烷氧基或C 3-C 6环烷基；
R 5代表芳基或芳杂环，其中所述的芳基或芳杂环可任选地被一个或多个R 9取代；
R 6、R 7和R 8各自独立地代表氢、C 1-C 8烷基、C 3-C 8环烷基、C 2-C 8烯基、C 2-C 8炔基、C 1-C 8烷氨基；
R 9代表氢、卤素、氰基、羟基、巯基、C 1-C 8烷基、C 1-C 8烷氧基、C 1-C 8烷氨基或卤代烷基；
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；所述的烯基代表直链烯基、支链烯基或环状烯基；所述的炔基代表直链炔基或支链炔基；
所述的芳基代表苯基、萘基、苊基或四氢萘基；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；
所述的卤代烷基为具有1-8个碳原子的直链或支链饱和烃基，或为具有3-8个碳原子的环状饱和烃基，或为连接具有1-8个碳原子的直链或支链饱和烃基的具有3-8个碳原子的环状饱和烃基；其中一个或多个氢原子被一个或多个卤原子取代。
优选，所述吡咯类化合物及其衍生物结构中：
R 1代表氰基、-CO 2R 6或-CONR 7R 8；
R 2代表氢或卤素；
R 3和R 4各自独立地代表氢、C 1-C 8烷基、C 3-C 8环烷基、C 1-C 8烷氨基或R 3和R 4与跟它们连接的氮原子一起形成5-7元杂环；其中所述的杂环可任选地包含一个或多个选自O、S或N杂原子；其中所述的杂环上可任选地被一个或多个以下基团取代：C 1-C 8烷基或C 3-C 6环烷基；
R 5代表芳基或芳杂环，其中所述的芳基或芳杂环可任选地被一个或多个R 9取代；
R 6、R 7和R 8各自独立地代表氢、C 1-C 8烷基或C 3-C 8环烷基；
R 9代表氢、卤素、氰基、羟基、巯基、C 1-C 8烷基、C 1-C 8烷氧基、C 1-C 8烷氨基或卤代烷基。
优选，所述吡咯类化合物及其衍生物结构中：
R 1代表氰基、-CO 2R 6或-CONR 7R 8；
R 2代表氢或卤素；
R 3和R 4各自独立地代表氢、C 1-C 4烷基、C 3-C 6环烷基、C 1-C 6烷氨基或R 3和R 4与跟它们连接的氮原子一起形成5-7元杂环；其中所述的杂环可任选地包含一个或多个选自O、S或N杂原子；其中所述的杂环上可任选地被一个或多个甲基取代；
R 5代表苯环或异噁唑基，其中所述的苯环可任选地被一个或多个R 9取代；
R 6、R 7和R 8各自独立地代表氢、C 1-C 3烷基或C 3-C 6环烷基；
R 9代表氢、卤素、氰基、C 1-C 5烷基、C 1-C 5烷氧基或三氟甲基。
优选，所述吡咯类化合物及其衍生物结构中：
R 1代表COOH；
R 2代表氢或卤素；
R 3和R 4各自独立地代表C 1-C 4烷基或C 3-C 6环烷基；
R 5代表苯环，所述的苯环可任选地被一个或多个R 9取代；
R 9代表氢、卤素、氰基、C 1-C 5烷基或C 1-C 5烷氧基。
更具体地，所述吡咯类化合物为以下任一化合物：
Figure PCTCN2021074874-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，所述制备方法为以下任一方法：
方法一：以取代硝基苯为原料，在碱作用下与胺类化合物HNR 3R 4反应制得中间体i，i与吡咯-2-羧酸酯经Ullmann反应制得中间体ii，ii经还原制得中间体iii，iii与取代苯异氰酸酯R 5NCO缩合制得化合物iv，或者iii先与氯甲酸-4-硝基苯酯形成活性中间体，然后再与胺类化合物R 5NH 2反应制得目标化合物iv；iv经水解制得目标化合物v；v与草酰氯或氯化亚砜反应制成酰氯后再与胺类化合物HNR 7R 8反应制得目标化合物vi；vi经高温脱水制得目标化合物vii；
Figure PCTCN2021074874-appb-000003
其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7和R 8的定义如前所述；
所述的碱选自三乙胺、DIPEA、Na 2CO 3、K 2CO 3或Cs 2CO 3；所述的还原剂选自锌粉和氯化铵或铁粉和氯化铵；vi在三氯氧磷中经高温脱水制得目标化合物vii。
方法二：以2-氟-5-溴硝基苯为原料，在碱作用下与胺类化合物HNR 3R 4反应制得中间体i，i经NCS氯代反应得到中间体viii，viii与吡咯-2-羧酸酯经Ullmann反应制得中间体ix，ix经还原剂还原制得中间体x，x与取代苯异氰酸酯R 5NCO缩合制得中间体xi，xi经水解制得目标化合物xii；
Figure PCTCN2021074874-appb-000004
其中，R 3、R 4、R 5和R 6的定义如前所述。
其中，步骤1)所述的碱选自三乙胺、DIPEA、Na 2CO 3、K 2CO 3或Cs 2CO 3。
其中，步骤4)所述的还原剂选自锌粉和氯化铵或铁粉和氯化铵。
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。
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。
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。
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)。
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。
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。
本发明所述的组合物在药物组合物中的含量物特殊限制，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％。
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。
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吲哚胺2,3-双加氧酶1(IDO1)抑制剂药物中的用途。所述的IDO1抑制剂药物用于治疗IDO1介导的免疫抑制的相关疾病，所述的相关疾病包括癌症、病毒感染、神经变性疾病、白内障、器官移植排斥、抑郁症或自身免疫性疾病。
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、病毒感染、神经变性疾病、白内障、器官移植排斥、抑郁症或自身免疫性疾病。
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、食管癌中的一种或多种。
所述的病毒感染包括但不限于：由人类免疫缺陷病毒、乙型肝炎病毒、丙型肝炎病毒、流感病毒、脊髓灰质病毒、巨细胞病毒、柯萨奇病毒、人类***状瘤病毒、爱泼斯坦-巴尔病毒、水痘-带状疱疹病毒中的一种或多种引起的感染。
所述的神经变性疾病包括但不限于：记忆障碍症、阿尔茨海默病、认知障碍症、老年痴呆症、帕金森症、运动障碍性疾病中的一种或多种。
所述的自身免疫性疾病包括但不限于：类风湿性关节炎、***性红斑狼疮、皮肌炎、硬皮病、结节性脉管炎、多发性硬化症、肾病、重症肌无力、混合性***病、银屑病、肝病、内分泌相关疾病、由于感染引起的自身免疫反应中的一种或多种。
进一步，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IDO1介导的相关疾病。
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。
有益效果：与现有技术相比，本发明具有以下显著优点：
本发明的化合物对IDO1具有很高的抑制活性。药理实验结果表明，这些吡咯类化合物能够有效逆转IDO1介导的免疫抑制作用，促进CD8 +T淋巴细胞的增殖，提高颗粒酶B和干扰素-γ的分泌，减少CD4 +CD25 +Foxp3 +调节性T细胞的生成，降低PCNA蛋白的表达。体内药效学评价结果表明，本发明 的化合物能够显著抑制各种肿瘤类型的小鼠移植瘤的生长，而对免疫***缺陷的裸鼠移植瘤的生长则无影响，说明这些化合物是通过激活宿主免疫应答而起抗肿瘤作用。
附图说明
图1为本发明化合物对IDO1蛋白表达的影响，其中：图1A为化合物2、14、26、27和39对IDO1蛋白表达的影响，图1B为图1A的灰度扫描统计结果；
图2为本发明化合物对B16F1黑色素瘤小鼠移植瘤生长的影响，其中：图2A为化合物2对B16F1黑色素移植瘤体积的影响，图2B为化合物2对B16F1黑色素移植瘤重量的影响；
图3为本发明化合物对CT26结直肠癌BALB/c小鼠移植瘤生长的影响；
图4为本发明化合物对免疫***缺陷的裸鼠移植瘤不产生抑制；
图5为本发明化合物对PAN02胰腺癌小鼠移植瘤生长的影响；
图6为本发明化合物在不同温度下对IDO1活性的影响，其中：图6A为Epacadostat在不同温度下对IDO1活性的影响，图6B为化合物26在不同温度下对IDO1活性的影响，图6C为化合物39在不同温度下对IDO1活性的影响。
具体实施方式
下面结合实施例对本发明的技术方案作进一步说明。
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。
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。
实施例1：1-(3-(3-(4-甲基苯基)脲基)-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酸甲酯(1)的合成
Figure PCTCN2021074874-appb-000005
(1)2-硝基-4-溴-N,N-(乙基)环己基苯胺(1A)的合成
将2-氟-5-溴硝基苯(3g,13.6mmol)、N-乙基环己胺(2.61g,20.5mmol)和N,N-二异丙基乙胺(3.53g,27.3mmol)加入N,N-二甲基二酰胺(50mL)中，80℃搅拌4小时，乙酸乙酯萃取(100mL×3)，柱层析纯化，得红色固体4.03g，收率90.4％。 1H NMR(300MHz,Chloroform-d)δ(ppm)7.75(t,J＝2.1Hz,1H),7.49(dt,J＝8.8,2.1Hz,1H),7.08(dd,J＝8.8,1.7Hz,1H),3.13(m,2H),3.00-2.85(m,1H),1.75(d,J＝15.0Hz,3H),1.63-1.55(m,1H),1.47-1.01(m,6H),0.95(td,J＝7.1,1.7Hz,3H)；MS(EI)m/z 325.1[M-H] -.
(2)1-(3-硝基-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酸甲酯(1B)的合成
将1A(3g,9.2mmol)、吡咯-2-甲酸甲酯(1.15g,9.2mmol)、碘化亚铜(3.49g,18.4mmol)、碳酸铯(5.97g,18.4mmol)和L-脯氨酸(2.12g,18.4mmol)加入DMF(50mL)中，氮气保护80℃下搅拌12小时，乙酸乙酯萃取(100mL×3)，柱层析纯化，得黄色固体1.52g，收率44.6％。 1H NMR(300MHz,DMSO-d 6)δ(ppm)7.76(d,J＝2.5Hz,1H),7.57-7.37(m,2H),7.30(t,J＝2.2Hz,1H),7.05(dd,J＝3.9,1.88Hz,1H),6.33(dd,J＝3.9,2.7Hz,1H),3.65(s,3H),3.15(q,J＝7.0Hz,2H),2.95(tt,J＝11.7, 2.7Hz,1H),1.84-1.63(m,4H),1.55(d,J＝11.3Hz,1H),1.37(td,J＝14.6,13.7,6.8Hz,2H),1.28-1.01(m,3H),0.91(t,J＝7.0Hz,3H)；MS(EI)m/z 370.5[M-H] -.
(3)1-(3-氨基-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酸甲酯(1C)的合成
将1B(1.52g,4.09mmol)、锌粉(1.33g,20.46mmol)和氯化铵(1.31g,20.46mmol)加入20mL乙醇中，氮气保护下常温反应4小时，乙酸乙酯萃取，减压浓缩，得淡黄色固体1.34g。
(4)1-(3-(3-(4-甲基苯基)脲基)-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酸甲酯(1)的合成
将1C(1.54g,3.92mmol)溶于无水四氢呋喃(20mL)中，加入对甲苯异氰酸酯(0.53g,3.92mmol)，常温反应4小时，减压浓缩，柱层析纯化，得白色固体1.32g，收率63.7％。 1H NMR(300MHz,DMSO-d 6)δ(ppm)9.50(s,1H),8.6(s,1H),8.17(d,J＝2.4Hz,1H),7.36(d,J＝8.1Hz,2H),7.30-7.17(m,2H),7.14-7.00(m,3H),6.88(dd,J＝8.6,2.6Hz,1H),6.30(t,J＝3.2Hz,1H),3.61(s,3H),3.05(q,J＝7.0Hz,2H),2.77(s,1H),2.24(s,3H),1.96(d,J＝14.6Hz,2H),1.71(s,2H),1.55(d,J＝11.9Hz,1H),1.18(s,5H),0.87(t,J＝6.8Hz,3H)；MS(EI)m/z 473.2[M-H] -.
实施例2：1-(3-(3-(4-甲基苯基)脲基)-4-(环己基(乙基)氨基)苯基)-1H-吡咯-2-甲酸(2)的合成
将1(1.32g,2.78mmol)和氢氧化钠(0.56g,13.92mmol)加入20mL乙醇中，65℃下搅拌8小时，减压浓缩，稀盐酸(1M)调pH至3～4，乙酸乙酯萃取，柱层析纯化，得白色固体0.68g，收率53.1％。 1H NMR(300MHz,Chloroform-d)δ(ppm)8.51(s,1H),8.26(d,J＝2.4Hz,1H),7.28(s,1H),7.21(d,J＝2.0Hz,3H),7.16(dt,J＝3.9,2.0Hz,1H),7.12-6.99(m,3H),6.91(dd,J＝8.4,2.4Hz,1H),6.27(dd,J＝3.9,2.6Hz,1H),2.91-2.8(m,2H),2.55(s,1H),2.37(s,3H),1.59(d,J＝9.7Hz,5H),1.07(t,J＝12.9Hz,5H),0.77(t,J＝6.9Hz,3H)；MS(EI)m/z 459.3[M-H] -.
实施例3：1-(3-(3-(4-甲基苯基)脲基)-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酰胺(3)的合成
将2(1g,1.96mmol)溶于15mL无水二氯甲烷中，冰浴冷却，加入2mL草酰氯，室温反应过夜，减压除去过量的草酰氯，加入15mL无水二氯甲烷溶解，冰浴冷却，滴入3mL氨水，室温反应5小时，二氯甲烷萃取，柱层析纯化，得白色固体0.5g，收率50.1％。 1H NMR(300MHz,Chloroform-d)δ(ppm)8.58(d,J＝2.1Hz,1H),7.80(dd,J＝7.4,1.5Hz),7.80(s,1H),7.28(s,1H),7.22(dd,J＝7.6,2.3Hz,3H),7.09(dd,J＝7.5,1.5Hz,1H),6.96(dd,J＝7.5,1.3Hz,2H),6.81(d,J＝7.5Hz,1H),6.76(s,2H),6.64(t,J＝7.5Hz,1H),3.29(q,J＝8.0Hz,2H),2.33(d,J＝1.2Hz,3H),1.79-1.67(m,4H),1.60(t,J＝5.6Hz,2H),1.45-1.29(m,4H),1.18(t,J＝8.0Hz,3H)；MS(EI)m/z 458.3[M-H] -.
实施例4：1-(2-((乙基)环己基氨基)-5-(2-氰基-1H-吡咯基-1)苯基)-3-(4-甲基苯基)脲(4)的合成
将3(0.4g,0.87mmol)溶于10mL二氯甲烷中，加入3mL三氯氧磷，65℃反应8小时。冷却，将反应液倒入冰水中，加入氢氧化钠水溶液(4M)调pH值至9，二氯甲烷萃取，柱层析纯化，得白色固体0.2g，收率52.0％。 1H NMR(300MHz,Chloroform-d)δ(ppm)8.53(s,1H),8.45(d,J＝2.6Hz,1H),7.28(s,3H),7.23-7.12(m,3H),7.10(dd,J＝8.4,2.6Hz,1H),6.99(dd,J＝4.0,1.6Hz,1H),6.33(t,J＝3.3Hz,2H),2.88(q,J＝7.1Hz,2H),2.56(t,J＝10.8Hz,1H),2.39(s,3H),1.27(d,J＝2.9Hz,4H),1.16-0.99(m,3H),0.90(t,J＝8.8Hz,3H),0.80(t,J＝7.0Hz,3H)；MS(EI)m/z 440.3[M-H] -.
采用与实施例1和实施例2相似的操作，制得下列化合物：
Figure PCTCN2021074874-appb-000006
Figure PCTCN2021074874-appb-000007
Figure PCTCN2021074874-appb-000008
Figure PCTCN2021074874-appb-000009
Figure PCTCN2021074874-appb-000010
实施例5：药理活性评价
1.基于HeLa细胞的IDO1抑制活性测试
1.1实验材料和主要仪器
HeLa细胞株：ATCC，离心机：Eppendorf(CHINA)，电热恒温鼓风干燥箱(DHG-924385-Ⅲ)：上海新苗医疗器械制造有限公司，乙酸(冰醋酸)：南京化学试剂股份有限公司，三氟乙酸：上海凌峰化学试剂有限公司，电子天平：Sartorius，对二甲氨基苯甲醛(CAS：100-10-7)：Aladdin，Recombinant  Human IFN-γ(Catalog#AF-300-02)：PEPROTECH。
1.2实验方法
从ATCC购买的HeLa细胞保存在最低基础培养基(2mM L-谷氨酰胺和调成含有1.5g/L碳酸氢钠、0.1mM非必需氨基酸、1mM丙铜酸钠和10％胎牛血清的Earle氏BSS)中。在37℃下将HeLa细胞保存在提供5％CO 2的控湿培养箱中。
按5×10 3/孔的密度将HeLa细胞接种在96孔培养板中，并培养过夜。第二天，将IFN-γ(终浓度100ng/mL)和化合物的系列稀释液(总体积200μL培养基)加给细胞。温育24小时后将140μL上清液/孔移至新的96孔板中，加入10μL 6.1mol/L的三氯乙酸，在恒温烘箱中50℃温育30min以使产生的N-甲酰基犬尿氨酸水解为犬尿氨酸。然后以4000rpm将反应混合物离心10min以去除沉淀物。将100μL上清液/孔移至另一96孔板中，与等体积2％(w/v)对-二甲氨基苯甲醛的乙酸溶液混合。使用酶标仪在480nm处检测吸光值，所得结果利用IC 50计算器计算。实验设3个复孔。
此外，采用MTT法检测各组HeLa细胞的存活率，目的是为了考察化合物是否是通过抑制HeLa细胞的增殖来抑制IDO1的活性。
具体操作：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。
1.3实验结果
实验结果(表1)表明，本发明的化合物对IDO1的活性具有显著的抑制作用。其中，化合物27的活性最强(IC 50:0.010nM)。此外，MTT检测结果表明，各组HeLa细胞的存活率均保持在90％以上，表明这些化合物不是通过抑制HeLa细胞的增殖来抑制IDO1的活性。
表1.本发明化合物对IDO1的抑制活性
Figure PCTCN2021074874-appb-000011
*阳性对照：BMS-52是WO2015031295A1中的第52号化合物。
2.本发明化合物对IDO1蛋白表达的影响
本实验的目的是为了考察本发明化合物是否通过下调IDO1蛋白的表达来抑制IDO1的活性。使用免疫印迹方法检测了化合物对IDO1蛋白表达的影响。
2.1实验方法
将HeLa细胞以2×10 5每孔的密度种于6孔板培养，于37℃，5％CO 2条件下培养12h。空白对照(只加培养基)，模型组(加入IFN-γ、对应阳性药)，药物处理组(加入IFN-γ、对应化合物)，于37℃，5％CO 2条件下培养24h，收集细胞，Western blot检测IDO1表达。
2.2实验结果
实验结果(图1)表明，本发明的化合物不会影响IDO1蛋白的表达，同时灰度扫描结果也显示加药组的IDO1蛋白/Actin蛋白比值与对照组相比没有变化。说明本发明化合物不是通过下调IDO1蛋白的表达来抑制IDO1的活性。
3.本发明化合物对T淋巴细胞增殖和IFN-γ释放的影响
T淋巴细胞是人体内免疫***的核心执行者，在肿瘤免疫应答中起核心作用。IDO1过度表达造成的局部色氨酸耗竭和犬尿氨酸聚积会抑制T淋巴细胞的增殖和诱导其凋亡，同时还能促进初始T淋巴细胞向调节性T淋巴细胞分化，抑制细胞因子如IFN-γ、IL-2和TNF-α等分泌。本实验的目的是检测本发明化合物逆转IDO1介导的免疫抑制的能力。
3.1实验方法
B16F1细胞处理：吸去培养基(高糖DMEM，10％FBS)，PBS洗1-2次。加入0.25％胰酶消化。吸去胰酶，加入培养基，将细胞吹打下来，转移至1.5mL离心管中，离心，吸去上清，加入1mL DMEM培养基重新悬浮细胞。加入丝裂霉素C(终浓度25μg/mL)，吹打混勾，37℃，水浴30min，RP1640洗3次，细胞计数，待用。
脾脏细胞的制备：取C57/BL6小鼠，摘眼球放血处死，无菌取出脾脏放入含有2mL无菌的预冷RPMI 1640培养基的35mm的培养皿中，用5mL注射器针芯轻轻将脾细胞挤出。再加入2mL培养基，用5mL移液管反复吹打直至悬液均匀。将细胞悬液用70μm滤器过滤，300g离心5min(4℃)。弃上清后脾细胞加入10mL Tris-NH 4Cl，吹均，静置2-3min，300g离心5min(4℃)，去除红细胞。弃上清后，用PRMI 1640洗涤两次，待用。
1)将处理过的B16F1细胞2×10 4个/孔(剌激细胞)，脾脏淋巴细胞1×10 6个/孔(反应细胞)，加入96孔板，加入RP1640(10％FBS)，补齐至200μL。
2)分组：给药组(剌激细胞+反应细胞+对应化合物)，空白对照(只加反应细胞)，模型组(剌激细胞+反应细胞)，除空白对照外，其他组均加入ConA(终浓度5μg/mL)，置于37℃、湿度95％、5％CO 2的培养箱中培养，培养48h。
3)加入20μL MTT(终浓度4mg/mL)培养箱中继续培养4h，酶标仪测定570nm波长处吸光度值；计算T淋巴细胞增殖率：
Figure PCTCN2021074874-appb-000012
3.2实验结果
实验结果表明，在混合淋巴细胞体系中，B16F1细胞高表达IDO1，对T淋巴细胞的增殖能够产生抑制作用。当加入化合物2、14、26、27、39(3倍IC 50浓度)培养48h后，利用MTT检测T淋巴细 胞的增殖，这些化合物均能显著增加T淋巴细胞的增殖，增殖率达到139.8％～173.0％，而且这些化合物还能提高细胞因子IFN-γ的释放(提高率：125.8％～134.5％)。这些实验说明本发明化合物能够有效逆转IDO1介导的免疫抑制，从而增强T淋巴细胞的增殖能力，促进IFN-γ的分泌，提高T细胞的免疫功能。
4.本发明化合物对调节性T淋巴细胞的影响
CD4 +CD25 +Foxp3 +T淋巴细胞是一类重要的调节性T淋巴细胞，在人体免疫***中起重要的负性调节作用。研究表明，IDO1能够介导初始T细胞向调节性T淋巴细胞转化，导致肿瘤微环境中调节性T淋巴细胞比例增高，从而诱导形成免疫抑制的肿瘤微环境。为此，我们选择高表达IDO1的小鼠黑色素瘤B16F1细胞株，对其加化合物处理后，取其上清与小鼠脾脏细胞进行共培养，模拟肿瘤微环境。收集细胞后使用流式细胞仪检测在共培养体系中化合物对初始T细胞向调节性T细胞分化的影响。
4.1实验方法
将处理过的B16F1细胞(8×10 4个/孔)，脾脏淋巴细胞(10 6个/孔，使用5μg/mL的ConA刺激)加入24孔板中，加入对应浓度的化合物后置于37℃、湿度95％、5％CO 2的培养箱中培养48h；收集上清液测试ELISA，使用抗CD4、抗CD25、抗Foxp3抗体染色，在流式细胞仪中检测T细胞的分化。
4.2实验结果
实验结果表明，当初始T淋巴细胞与黑色素瘤B16F1细胞株共培养时，调节性T淋巴细胞的数量和仅含初始T淋巴细胞的实验组(3.2％)相比上升了4倍(12.7％)。当化合物2、14、26、27、39(3倍IC 50浓度)加入体系中后能够显著逆转这种效应，其将调节性T淋巴细胞比例分别下调至7.8％、5.3％、6.2％、4.7％和4.5％，说明本发明化合物能够通过抑制IDO1的活性逆转初始T淋巴细胞向调节性T淋巴细胞的分化。
5.本发明化合物体内药效学评价
颗粒酶B是常见于细胞毒性T淋巴细胞(CTL)和天然杀伤(NK)细胞颗粒中的丝氨酸蛋白酶，是CTL和NK细胞发挥细胞毒的主要效应因子。而增殖细胞核抗原(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颗粒酶B、IFN-γ和PCNA水平。
5.1实验方法
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。
肿瘤细胞的处理：分别采集处于对数生长期的CT26、B16F1、PAN02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。
肿瘤细胞的移植：分别将CT26、B16F1、PAN02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。肿瘤的体积按照以下公式计算：V(体积)＝A×B 2/2，其中A是肿瘤长边的长度，B是肿瘤短边的长度。当肿瘤体积均值达到40mm 3左右时，开始给药。
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。
5.1.1 B16F1黑色素瘤小鼠移植瘤模型
移植B16F1黑色素瘤的C57BL/6雌鼠分为5组，每组6只。模型组(PBS+2％的吐温20+2％DMSO，i.p.，qd.)，阳性对照组(顺铂，剂量：1mg/kg，i.p.，qod.)，给药组1-3(实施例化合物2，剂量：1.25mg/kg、2.5mg/kg、5mg/kg，i.p.，qd.)。
5.1.2 CT26结直肠癌BALB/c小鼠移植瘤模型
移植CT26结直肠肿瘤的BALB/c雌鼠分为5组，每组8只。模型组(PBS+2％的吐温20+2％DMSO，i.p.，qd.)，阳性对照组(5-FU，剂量：25mg/kg，i.p.，qod.)，给药组(实施例化合物2，剂量：5mg/kg，i.p.，qd.)。
5.1.3 CT26结直肠癌裸鼠移植瘤模型
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。
移植CT26结直肠肿瘤的BALB/c(nu/nu)裸鼠分为3组，每组8只。模型组(PBS+2％的吐温20+2％DMSO，i.p.，qd.)，阳性对照组(5-FU，剂量：25mg/kg，i.p.，qod.)，给药组(实施例化合物2，剂量：10mg/kg，i.p.，qd.)。
5.1.4 PAN02胰腺癌小鼠移植瘤模型
移植PAN02胰腺癌的C57BL/6雌鼠分为4组，每组6只。模型组(PBS+2％的吐温20+2％DMSO，i.g.，qd.)，阳性对照组1(吉西他滨，剂量：30mg/kg，i.p.，qod.)，阳性对照组2(Epacadostat，剂量：50mg/kg，i.g.，bid.)，给药组(实施例化合物9，剂量：15mg/kg，i.g.,qd.)。
5.2实验结果
5.2.1本发明化合物呈剂量依赖性抑制B16F1黑色素瘤小鼠移植瘤的生长
实验结果(图2)表明，随着给药剂量的升高，化合物2对B16F1黑色素瘤小鼠移植瘤的抑制效果也随之增加，显示剂量依赖性。所有给药组小鼠体重保持在20-23g之间，说明化合物2并不影响小鼠的体重。免疫组化和TUNEL实验表明，化合物2(5mg/kg)可以增加CD8 +T细胞浸润，减少Foxp3 +Treg细胞的数量，促进颗粒酶B和IFN-γ的分泌，并且增加肿瘤细胞的凋亡。
5.2.2本发明化合物能够显著抑制CT26结直肠癌BALB/c小鼠移植瘤的生长
实验结果(图3)表明，实施例化合物2能够显著抑制CT26结直肠癌BALB/c小鼠移植瘤的生长，并且小鼠的肝脏未见明显的纤维化和炎症表现，其余脏器如心脏、肾脏、脾脏和肺的形态结构也没有发生明显改变，说明这些化合物对小鼠各器官没有出现显著的药物性损伤影响。此外，与模型组相比，5-FU组的小鼠体重自第9天后开始下降，而化合物2的给药组小鼠的体重保持稳定，说明化合物2并不影响小鼠的体重。
免疫组化和TUNEL实验结果表明，化合物2可以增加CD8 +T细胞浸润，提高肿瘤组织中IFN-γ的表达，同时能够减少Foxp3 +Treg细胞群的数量，并且还可以显著促进颗粒酶B的分泌，降低PCNA蛋白的表达，进一步增加肿瘤细胞的凋亡。
5.2.3本发明化合物对免疫***缺陷的裸鼠移植瘤不产生抑制
实验结果(图4)表明，细胞毒药物5-FU能够明显抑制CT26结直肠癌裸鼠移植瘤的生长，然而，实施例化合物2却对免疫***缺陷的裸鼠移植瘤不产生抑制作用。说明化合物2是通过免疫***发挥抗肿瘤作用。
5.2.4本发明化合物能够显著抑制PAN02胰腺癌小鼠移植瘤的生长
实验结果(图5)表明，相比于模型组，化合物9在PAN02胰腺癌小鼠移植瘤模型中能够显著抑制肿瘤的生长。免疫组化和TUNEL实验表明，化合物9能有效逆转IDO1介导的免疫抑制现象。
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和LLC等小鼠移植瘤模型中同样表现出显著的抗肿瘤作用。例如，化合物20、23、24、26、27、28、30、31、33、34、35、36、38、39、40和41等在低剂量下(2.5mg/kg～15mg/kg)便能显著抑制小鼠移植瘤的生长。此外，对肿瘤组织的免疫组化和TUNEL实验结果表明，这些化合物同样能够增加CD8 +T细胞浸润，促进颗粒酶B的分泌，提高肿瘤组织中IFN-γ的表达，同时还能够减少Foxp3 +Treg细胞群的数量，降低PCNA蛋白的表达。这些实验表明本发明化合物能够有效逆转IDO1介导的免疫抑制作用。
6.本发明化合物与IDO1蛋白的相互作用
6.1实验方法
1)细胞热迁移实验(CETSA)：其原理是随着温度的升高，蛋白质的三级结构会受到影响进而逐渐降解，但当小分子与蛋白结合后，则可以增加蛋白质的热稳定性，减缓降解趋势。
具体操作：B16F1细胞加入IFN-γ(终浓度100ng/mL)刺激24h之后平均分成2组：对照组和化合物处理组。加入化合物2和27，确保终浓度为1μM，对照组加入等体积的DMSO，3h后收集细胞，用PBS洗两遍，500μL PBS重悬细胞，将其平均分为10份置于进口PCR管中。将PCR仪器设置10个温度(43、46、49、52、55、58、61、64、67、70℃)，将对照组与实验组的每个样品按照对应温度进行加热：每组加热3min后室温放置3min，然后置于冰上。最后，将样品放置在-80℃过夜，第二天取出，置室温融化后利用液氮反复冻融3次。将处理好的样本转移到1.5mL EP管中，12000g，20min离心，取上清40μL加入6×loading混匀，然后进行免疫印迹分析。
2)微量热泳动(MST)：该技术是基于分子在温度梯度中的定向运动从而引起分子性质的变化，如分子大小、电荷和水化层及构象等。MST是一种新型的测量分子间相互作用的技术，可以测量不同的结合模式，包括二聚化、协同作用和竞争作用。该技术适应性强，可用于不同的环境、不同的生物分子和不同的溶液，可以在复杂的生物溶液甚至细胞溶解液中完成而无需样品纯化。
具体操作：将IDO蛋白进行荧光标记，进行正式操作前先测量一定稀释倍数下蛋白的荧光值，以观察是否存在吸附等问题。随后，将待测化合物2的27设置16个不同浓度梯度，并在进口PCR管中用蛋白Buffer配好，每管体积为10μL。向PCR管中加入稀释后的蛋白，总体积20μL。用毛细管吸取10μL上机检测并分析数据。
6.2实验结果
CETSA实验结果表明，对照组(仅含DMSO)的IDO1蛋白在58℃就已经完全降解，而与对照组相比，本发明化合物2和27在61℃高温下依然能维持IDO1蛋白的稳定性，使其不易被降解。说明化合物2和27能够进入B16F1细胞，并与IDO1蛋白结合。
MST实验结果显示，本发明化合物2和27与IDO1蛋白的解离常数K D分别为3.9×10 -8M和1.3×10 -10M，说明化合物2和27与IDO1蛋白之间的亲和力很强，并且能够对IDO1有极强的选择结合力。
7.本发明化合物与IDO1蛋白的结合方式
早期研发的IDO1抑制剂主要是通过与含血红素的IDO1(holo-IDO1)结合来抑制IDO1的活性。以Epacadostat为代表的holo-IDO1抑制剂主要通过与血红素铁离子络合而占据IDO1催化口袋，从而产 生抑制活性。本实验的目的是为了验证本发明化合物与IDO1蛋白的结合方式。
7.1实验方法
1)不同温度下酶学实验：研究表明，IDO1蛋白的稳定性会随着温度的变化而变化。在25℃时，血红素与IDO1蛋白的结合比较牢固，此时当其与apo-IDO1抑制剂共孵育时，抑制剂无法将血红素挤出IDO1蛋白的结合口袋。而当温度升高至37℃时，血红素与IDO1蛋白结合的稳定性会降低，此时血红素就易被apo-IDO1抑制剂挤出IDO1的结合口袋。然而，holo-IDO1抑制剂如Epacadostat无论在什么温度下都能通过与铁离子络合稳定血红素与IDO1蛋白的结合。因此可以利用不同温度下酶学实验检测抑制剂与IDO1的结合方式(Ortizmeoz RF,et al.bioRxiv,2018)。
具体操作：将不同浓度的受试物和IDO1蛋白分为两组，混匀加入EP管中，分别在25℃和37℃预孵育2h，加入过氧化氢酶溶液、亚甲基蓝溶液、TritonX-100溶液、VCNa溶液，在37℃下孵育15min。加入D-Trp溶液，反应总体系为250μL，继续孵育60min，按照1.2检测方法进行检测。
2)X射线衍射实验：为了培养本发明化合物与IDO1蛋白结合的复合物晶体，在50mM Tris buffer,pH 7.4的条件下，用42℃的水浴孵育化合物39和hIDO1，持续2h。15000rpm离心5min后，将上清用96孔的sitting-drop trays进行坐滴，再加上结晶溶液后进行晶体生长，获得39/hIDO1复合物晶体后在上海光源BL19U线站采集X射线衍射的数据。
7.2实验结果
酶学实验结果表明，无论是在25℃还是37℃下，Epacadostat都能与含有血红素的IDO1结合，因此在两种不同温度下对IDO1都具有较强的抑制活性(图6A)。然而，在25℃下，实施例化合物26和39对IDO1的抑制作用很弱，只有在37℃时才显示出极强的抑制活性(图6B和图6C)。表明本发明化合物主要是通过与血红素竞争性结合IDO1蛋白而抑制其活性。
进一步地，通过晶体培养获得了实施例化合物39与IDO1蛋白复合物晶体，经X射线衍射结果显示，实施例化合物39能够与apo-IDO1蛋白结合，这进一步证实了本发明化合物是一类apo-IDO1抑制剂。

Claims (10)

  1. 一种通式(I)所示的吡咯类化合物、其代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐：
    Figure PCTCN2021074874-appb-100001
    其中：
    R 1代表氰基、-CO 2R 6或-CONR 7R 8；
    R 2代表氢、卤素、氰基、羟基或硝基；
    R 3和R 4各自独立地代表氢、C 1-C 8烷基、C 3-C 8环烷基、C 2-C 8烯基、C 2-C 8炔基、C 1-C 8烷氨基或R 3和R 4与跟它们连接的氮原子一起形成5-7元杂环；其中所述的杂环可任选地包含一个或多个选自O、S或N杂原子；其中所述的杂环上可任选地被一个或多个以下基团取代：卤素、硝基、氰基、羟基、氨基、C 1-C 8烷基、C 1-C 8烷氧基或C 3-C 6环烷基；
    R 5代表芳基或芳杂环，其中所述的芳基或芳杂环可任选地被一个或多个R 9取代；
    R 6、R 7和R 8各自独立地代表氢、C 1-C 8烷基、C 3-C 8环烷基、C 2-C 8烯基、C 2-C 8炔基、C 1-C 8烷氨基；
    R 9代表氢、卤素、氰基、羟基、巯基、C 1-C 8烷基、C 1-C 8烷氧基、C 1-C 8烷氨基或卤代烷基。
  2. 根据权利要求1所述的吡咯类化合物、其代谢产物、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：
    R 1代表氰基、-CO 2R 6或-CONR 7R 8；
    R 2代表氢或卤素；
    R 3和R 4各自独立地代表氢、C 1-C 8烷基、C 3-C 8环烷基、C 1-C 8烷氨基或R 3和R 4与跟它们连接的氮原子一起形成5-7元杂环；其中所述的杂环可任选地包含一个或多个选自O、S或N杂原子；其中所述的杂环上可任选地被一个或多个以下基团取代：C 1-C 8烷基或C 3-C 6环烷基；
    R 5代表芳基或芳杂环，其中所述的芳基或芳杂环可任选地被一个或多个R 9取代；
    R 6、R 7和R 8各自独立地代表氢、C 1-C 8烷基或C 3-C 8环烷基；
    R 9代表氢、卤素、氰基、羟基、巯基、C 1-C 8烷基、C 1-C 8烷氧基、C 1-C 8烷氨基或卤代烷基。
  3. 根据权利要求1所述的吡咯类化合物、其代谢产物、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：
    R 1代表氰基、-CO 2R 6或-CONR 7R 8；
    R 2代表氢或卤素；
    R 3和R 4各自独立地代表氢、C 1-C 4烷基、C 3-C 6环烷基、C 1-C 6烷氨基或R 3和R 4与跟它们连接的氮原子一起形成5-7元杂环；其中所述的杂环可任选地包含一个或多个选自O、S或N杂原子；其中所述的杂环上可任选地被一个或多个甲基取代；
    R 5代表苯环或异噁唑基，其中所述的苯环可任选地被一个或多个R 9取代；
    R 6、R 7和R 8各自独立地代表氢、C 1-C 3烷基或C 3-C 6环烷基；
    R 9代表氢、卤素、氰基、C 1-C 5烷基、C 1-C 5烷氧基或三氟甲基。
  4. 根据权利要求1所述的吡咯类化合物、其代谢产物、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：
    R 1代表COOH；
    R 2代表氢或卤素；
    R 3和R 4各自独立地代表C 1-C 4烷基或C 3-C 6环烷基；
    R 5代表苯环，所述的苯环可任选地被一个或多个R 9取代；
    R 9代表氢、卤素、氰基、C 1-C 5烷基或C 1-C 5烷氧基。
  5. 根据权利要求1所述的吡咯类化合物、其代谢产物、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，所述吡咯类化合物选自以下任一化合物：
    1-(3-(3-(4-甲基苯基)脲基)-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酸甲酯(1)，
    1-(3-(3-(4-甲基苯基)脲基)-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酸(2)，
    1-(3-(3-(4-甲基苯基)脲基)-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酰胺(3)，
    1-(2-((乙基)环己基氨基)-5-(2-氰基-1H-吡咯基-1)苯基)-3-(4-甲基苯基)脲(4)，
    1-(3-(3-(4-氯苯基)脲基)-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酸(5)，
    1-(3-(3-(3-三氟甲基苯基)脲基)-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酸(6)，
    1-(3-(3-(4-氯-3-三氟甲基苯基)脲基)-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酸(7)，
    1-(3-(3-(3,4-二氯苯基)脲基)-4-((乙基)环己基氨基)苯基)-1H-吡咯-2-甲酸(8)，
    1-(3-(3-(4-甲基苯基)脲基)-4-(二异丁氨基)苯基)-1H-吡咯-2-甲酸(9)，
    1-(3-(3-(4-氟苯基)脲基)-4-(二异丁氨基)苯基)-1H-吡咯-2-甲酸(10)，
    1-(3-(3-(4-氯苯基)脲基)-4-(二异丁氨基)苯基)-1H-吡咯-2-甲酸(11)，
    1-(3-(3-(4-三氟甲基苯基)脲基)-4-(二异丁氨基)苯基)-1H-吡咯-2-甲酸(12)，
    1-(3-(3-(2,4-二氟苯基)脲基)-4-(二异丁氨基)苯基)-1H-吡咯-2-甲酸(13)，
    1-(3-(3-(2-氟-4-氯苯基)脲基)-4-(二异丁氨基)苯基)-1H-吡咯-2-甲酸(14)，
    1-(3-(3-(3-三氟甲基苯基)脲基)-4-(二异丁氨基)苯基)-1H-吡咯-2-甲酸(15)，
    1-(3-(3-(4-氯-3-三氟甲基苯基)脲基)-4-(二异丁氨基)苯基)-1H-吡咯-2-甲酸(16)，
    1-(3-(3-(3,4-二氯苯基)脲基)-4-(二异丁氨基)苯基)-1H-吡咯-2-甲酸(17)，
    1-(3-(3-(3-甲基异噁唑-5-基)脲基)-4-(二异丁氨基)苯基)-1H-吡咯-2-甲酸(18)，
    1-(3-氯-4-(二异丁氨基)-5-(3-(4-甲基苯基)脲基)苯基)-1H-吡咯-2-甲酸(19)，
    1-(3-氯-4-(二异丁氨基)-5-(3-(4-甲氧基苯基)脲基)苯基)-1H-吡咯-2-甲酸(20)，
    1-(3-氯-4-(二异丁氨基)-5-(3-(4-氟苯基)脲基)苯基)-1H-吡咯-2-甲酸(21)，
    1-(3-氯-4-(二异丁氨基)-5-(3-(4-氯苯基)脲基)苯基)-1H-吡咯-2-甲酸(22)，
    1-(3-氯-4-(二异丁氨基)-5-(3-(4-溴苯基)脲基)苯基)-1H-吡咯-2-甲酸(23)，
    1-(3-氯-4-(二异丁氨基)-5-(3-(4-氰基苯基)脲基)苯基)-1H-吡咯-2-甲酸(24)，
    1-(3-氯-4-(二异丁氨基)-5-(3-(2-氯苯基)脲基)苯基)-1H-吡咯-2-甲酸(25)，
    1-(3-氯-4-(二异丁氨基)-5-(3-(2,4-二氟苯基)脲基)苯基)-1H-吡咯-2-甲酸(26)，
    1-(3-氯-4-(二异丁氨基)-5-(3-(2-氟-4-氯苯基)脲基)苯基)-1H-吡咯-2-甲酸(27)，
    1-(3-氯-4-(二异丁氨基)-5-(3-(2-氟-4-氰基苯基)脲基)苯基)-1H-吡咯-2-甲酸(28)，
    1-(3-氯-4-(二异丁氨基)-5-(3-(3-氟-4-氯苯基)脲基)苯基)-1H-吡咯-2-甲酸(29)，
    1-(3-(3-(4-甲基苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(30)，
    1-(3-(3-(4-甲氧基苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(31)，
    1-(3-(3-(4-氟苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(32)，
    1-(3-(3-(4-氯苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(33)，
    1-(3-(3-(4-溴苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(34)，
    1-(3-(3-(4-氰基苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(35)，
    1-(3-(3-(2-氟苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(36)，
    1-(3-(3-(2-氯苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(37)，
    1-(3-(3-(2,4-二氟苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(38)，
    1-(3-(3-(2-氟-4-氯苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(39)，
    1-(3-(3-(2-氟-4-溴苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(40)，
    1-(3-(3-(2-氟-4-氰基苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(41)，
    1-(3-(3-(2,4-二氯苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(42)，
    1-(3-(3-(3-氟-4-氯苯基)脲基)-4-((异丁基)环己基氨基)苯基)-1H-吡咯-2-甲酸(43)，
    1-(3-(3-(3-三氟甲基苯基)脲基)-4-((甲基)环己基氨基)苯基)-1H-吡咯-2-甲酸(44)，
    1-(3-(3-(4-氯-3-三氟甲基苯基)脲基)-4-((甲基)环己基氨基)苯基)-1H-吡咯-2-甲酸(45)，
    1-(3-(3-(3,4-二氯苯基)脲基)-4-((甲基)环己基氨基)苯基)-1H-吡咯-2-甲酸(46)。
  6. 一种权利要求1所述的吡咯类化合物的制备方法，其特征在于，所述制备方法为以下任一方法：
    方法一：以取代硝基苯为原料，在碱作用下与胺类化合物HNR 3R 4反应制得中间体i，i与吡咯-2-羧酸酯经Ullmann反应制得中间体ii，ii经还原制得中间体iii，iii与取代苯异氰酸酯R 5NCO缩合制得化合物iv，或者iii先与氯甲酸-4-硝基苯酯形成活性中间体，然后再与胺类化合物R 5NH 2反应制得目标化合物iv；iv经水解制得目标化合物v；v与草酰氯或氯化亚砜反应制成酰氯后再与胺类化合物HNR 7R 8反应制得目标化合物vi；vi经高温脱水制得目标化合物vii；
    Figure PCTCN2021074874-appb-100002
    其中，R 2、R 3、R 4、R 5、R 6、R 7和R 8的定义如权利要求1所述；
    方法二：以2-氟-5-溴硝基苯为原料，在碱作用下与胺类化合物HNR 3R 4反应制得中间体i，i经NCS氯代反应得到中间体viii，viii与吡咯-2-羧酸酯经Ullmann反应制得中间体ix，ix经还原剂还原制得中间体x，x与取代苯异氰酸酯R 5NCO缩合制得中间体xi，xi经水解制得目标化合物xii；
    Figure PCTCN2021074874-appb-100003
    其中，R 3、R 4、R 5和R 6的定义如权利要求1所述。
  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中任一项所述的吡咯类化合物、其代谢产物、代谢前体、前药、溶剂化物、结晶或其药学上可接受的盐。
  8. 一种权利要求1-5中任一项所述的吡咯类化合物、其代谢产物、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吲哚胺2,3-双加氧酶1抑制剂药物中的应用。
  9. 一种权利要求1-5中任一项所述的吡咯类化合物、其代谢产物、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吲哚胺2,3-双加氧酶1介导的免疫抑制相关疾病药物中的用途。
  10. 根据权利要求8或9所述的用途，其特征在于，所述药物用于治疗癌症、病毒感染、神经变性疾病、白内障、器官移植排斥、抑郁症或自身免疫性疾病。
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