CN106905256A - Benzo five-membered heterocyclic IDO1 inhibitor, its preparation method and application - Google Patents

Abstract

The invention belongs to the field of medicines, and in particular relates to a benzo five-membered heterocyclic compound or a pharmaceutically acceptable salt thereof with the structural characteristics of formula (I), a preparation method thereof, and their use as indoleamine 2,3-bis Use of an oxygenase 1 (IDO1) inhibitor. The experimental results show that the compound of the present invention has a significant inhibitory effect on the activity of IDO1, can effectively promote the proliferation of T cells, inhibit the differentiation of initial T cells into regulatory T cells, reverse the immune suppression mediated by IDO1, and can be used for the treatment of IDO1. Diseases associated with pathological features of kynurenine metabolic pathways, including cancer, viral infection, neurodegenerative diseases, cataracts, organ transplant rejection, depression and autoimmune diseases, etc.

Description

Benzo five-membered heterocyclic IDO1 inhibitor, its preparation method and application

technical field

The invention belongs to the field of medicines, and specifically relates to a benzo five-membered heterocyclic compound or a pharmaceutically acceptable salt thereof, a preparation method thereof, and their function as indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors use of the agent.

technical background

Tryptophan is an essential amino acid for human cells to maintain proliferation and survival, and can be used for the biosynthesis of protein, niacin and 5-hydroxytryptamine. Tryptophan is generally ingested from food, more than 95% of tryptophan is metabolized along the kynurenic acid pathway, and the remaining tryptophan is converted into 5-hydroxytryptophan and 5-hydroxytryptophan in the nervous system and intestine, or in the pineal gland Synthetic melatonin. Indoleamine 2,3-dioxygenase 1 (indoleamine 2,3-dioxygenase, IDO1) is the rate-limiting enzyme that catalyzes the metabolism of tryptophan along the kynurenine pathway outside the human liver. IDO1 is expressed in various tissues (eg, lung, kidney, brain, placenta, thymus) and in various cells (eg, macrophages and dendritic cells). IDO1 reduces the concentration of tryptophan in the microenvironment of the body by oxidatively cleaving tryptophan, and produces a series of metabolisms such as kynurenine, 3-hydroxykynurenine, 2-amino-3-hydroxybenzoic acid, and quinolinic acid. product. Cytokines such as IFN-γ, TNF-α, IL-1β and IL-6 can induce up-regulated expression of IDO1 (Bernhardt R. Chem Rev, 1996, 96(1):2841-2888).

A large amount of evidence shows that IDO1 can not only catalyze the oxidative metabolism of tryptophan, but also play an important role in regulating the body's innate immunity and adaptive immunity (Munn DH, et al.Trends Immunol, 2013, 34(3): 137- 143). IDO1 regulates the immune system mainly by catalyzing the metabolism of tryptophan leading to the depletion of tryptophan and the accumulation of its metabolites: on the one hand, depletion of tryptophan can induce the T cell division cycle to stay in the G1 phase by activating the GCN2 pathway , thereby inhibiting the proliferation of T cells, and at the same time inhibiting the differentiation of initial CD4 ⁺ T cells into helper T cells 17 (Th17), thereby producing immunosuppression (Munn DH, et al.Immunity, 2005, 22(5): 633-642 ). On the other hand, tryptophan metabolites such as kynurenine are cytotoxic and can kill T cells and natural killer (NK) cells (Frumento G, et al. JExp Med, 2002, 196(4): 459-468 ; Munn DH, et al.J Clin Invest, 2004, 114 (2): 280-290), and these metabolites can also induce CD4 ⁺ T cells to differentiate into regulatory T ( Treg) cells, and promote the conversion of dendritic cells (DC) into tolerogenic DC (Mezrich JD, et al.J Immunol, 2010, 185(6): 3190-3198; Mezrich JD, et al.J Immunol, 2008, 181 (8): 5396-5404); 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 (Della Chiesa M, et al al. Blood, 2006, 108(13): 4118-4125).

IDO1 is related to many physiological and pathological processes. Studies have shown that IDO1 plays an important immune regulatory role in physiological processes such as host immune defense and maternal-fetal immune tolerance. Under normal conditions, the expression level of IDO1 is low, but in physiological stresses such as placental development and inflammatory response, the secretion of cytokines such as IFN-γ increases significantly, thereby inducing the upregulation of IDO1 expression, leading to tryptophan depletion and kynuria Accumulation of metabolites such as aminoacid acid, thereby inhibiting the mother's T cell response, inducing maternal-fetal immune tolerance, and ensuring 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 what is necessary for the replication of pathogenic microorganisms At the same time, IDO1-mediated immunosuppression can avoid excessive activation of the body's immune system (Mellor AL, et al. Nat Rev Immunol, 2008, 8(1): 74-80; Terness P, et al. Am J Reprod Immunol, 2007, 58(3): 238-254; Divanovic S, et al. J Infect Dis, 2012, 205(1): 152-161). When the IDO1 inhibitor is administered to pregnant mice, it will cause T cell-mediated embryo rejection, resulting in miscarriage of the mice, indicating that IDO1 can prevent the fetus from being rejected by the mother (Munn DH, et al.Science, 1998, 281 (5380 ): 1191-1193). IDO1 also exerts an immunosuppressive effect on the survival of transplanted tissues in new hosts (Radu CA, et al. Plast Reconstr Surg, 2007, 119(7):2023-2028). These findings indicate that IDO is an immunoregulatory enzyme involved in the body's immune tolerance.

Many studies have shown that immune tolerance mediated by IDO1 is closely related to tumor immune escape, viral infection, neurodegenerative diseases, organ transplant rejection, autoimmune diseases, neuropsychiatric diseases and cataracts (Munn DH, et al. Trends Immunol , 2013, 34(3): 137-143; Nguyen NT, et al.Front Immunol, 2014, 5: 551; Myint AM, et al.J Affect Disord, 2007, 98(1-2): 143-151; Mailankot M, et al. LabInvest, 2009, 89(5): 498-512). In these diseases, the depletion of tryptophan and the accumulation of its metabolites mediated by overexpressed IDO1 can inhibit the activation of T cells and lead to the body's immune tolerance.

In the mouse model of virus infection, administration of IDO1 inhibitors can significantly promote the proliferation of CD8 ⁺ T cells, restore the immune response of T cells, and inhibit the mononuclear macrophages of the virus-infected host. During influenza virus infection, the immunosuppressive effect mediated by overexpression of IDO1 easily leads to secondary infection in the lungs (van der Sluijs KF, et al. J InfectDis, 2006, 193(2): 214-222). During HIV infection, IDO1 will be up-regulated to promote the proliferation of Treg cells and inhibit the proliferation of Th17 cells, resulting in an imbalance in the ratio of Tregs/Th17 cells, leading to immunosuppression in patients (Favre D, et al.Sci Transl Med, 2010, 2 (32):32ra36). In addition, IDO1-mediated depletion of tryptophan and increased concentration of its metabolites are also associated with parasitic infection (Knubel CP, et al. FASEB J, 2010, 24(8): 2689-2701).

Studies have shown that tryptophan metabolites catalyzed by IDO1, such as kynurenine and quinolinic acid, are neurotoxic, and these metabolites are associated with neurodegenerative diseases such as memory impairment, Alzheimer's disease (AD), cognitive impairment Syndrome, Alzheimer's disease, Parkinson's disease, Parkinson's syndrome and movement disorders are closely related (Malpass K.Nat RevNeurol, 2011, 7(8): 417; Maddison DC, et al.Semin Cell Dev Biol, 2015, 40: 134-141). The expression of IDO1 and the concentration of quinolinic acid in the brain of AD patients were higher than those of normal people, and the highest content was in microglia and astrocytes around senile plaques. In addition, the concentration of tryptophan in the blood of AD patients is lower than that of normal people, while the concentration of kynurenine is higher than that of normal people, and the ratio of the two is closely related to the degree of cognitive impairment of patients (Guillemin GJ, et al.Neuropathol Appl Neurobiol, 2005, 31(4): 395-404; Widner B, et al. Adv Exp Med Biol, 1999, 467: 133-138). Neuropsychiatric disorders such as depression, schizophrenia, and anxiety are also associated with IDO1 overexpression and elevated levels of metabolites such as kynurenine. Overexpression of IDO1 causes tryptophan depletion, thereby reducing the amount of tryptophan used to synthesize the neurotransmitter serotonin, resulting in serotonin deficiency, coupled with neurotoxic kynurenine and quinolinic acid, etc. The accumulation of metabolites jointly promotes the occurrence of neuropsychiatric diseases, and is a factor of various mood disorders (Myint AM. FEBS J, 2012, 279(8): 1375-1385). Therefore, inhibition of IDO1 is an important therapeutic strategy for patients with neurodegenerative and neuropsychiatric disorders.

Excessive metabolism of tryptophan mediated by high expression of IDO1 also exists in various autoimmune diseases (NguyenNT, et al. Front Immunol, 2014, 5:551). In synovial joint DCs of patients with rheumatoid arthritis, IDO1 is highly expressed, and the concentration of tryptophan in the serum of patients is decreased, while the concentration of kynurenine is increased (Zhu L, et al. J Immunol, 2006, 177 (11): 8226-8233; Ozkan Y. et al. Clin Rheumatol, 2012, 31(1):29-34). IDO1 overexpression and enhanced tryptophan metabolism were also observed in patients with systemic lupus erythematosus. The concentration of tryptophan in the patient's serum decreased, and the concentration of the metabolite kynurenine and the ratio of kynurenine to tryptophan all increased significantly (Widner B, et al. Immunobiology, 2000, 201(5): 621-630). Therefore, inhibition of IDO1 is also an important therapeutic strategy for patients with autoimmune diseases.

A large number of studies have shown that IDO1-induced immunosuppression plays an important role in tumor immune escape. IDO1 is overexpressed in various tumor cells and cells in their microenvironment, such as DC and stromal cells, leading to local tryptophan depletion and accumulation of tryptophan metabolites in the tumor, thereby inducing tumor immune escape and helping tumor cells evade the body's immune system. Attack (Munn DH, et al. Trends Immunol, 2016, 37(3):193-207). Uyttenhove group used immunohistochemical labeling method in melanoma, lung cancer, breast cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, lymphatic cancer, prostate cancer, kidney cancer, brain cancer, head and neck cancer, ovarian cancer, cervical cancer, uterus The expression of IDO1 was detected in 24 kinds of human tumor cells including endometrial cancer, mesothelioma, thyroid cancer, esophageal cancer and liver cancer (Uyttenhove C, et al. Nat Med, 2003, 9(10): 1269-1274). It was further confirmed in ovarian cancer, melanoma, lung cancer, leukemia and other tumor tissues, and it was found that the expression of IDO1 in tumor tissues was closely related to the malignancy of the tumor, and affected the prognosis of tumor patients (Théate I, et al. Cancer Immunol Res, 2015, 3(2): 161-172; Curti A, et al. Blood, 2007, 109(7): 2871-2877; de Jong RA, et al. Int J Gynecol Cancer, 2011, 21(7 ): 1320-1327; OkamotoA, et al. Clin Cancer Res, 2005, 11(16): 6030-6039; Ino K, et al. Br J Cancer, 2006, 95(11): 1555-1561; Speeckaert R, et al. Eur. J. Cancer, 2012, 48(13): 2004-2011). IDO1 inhibitors can activate T cells, overcome tumor immune escape mediated by IDO1, and can also improve the therapy of other tumor therapeutic agents (Koblish HK, et al. Mol Cancer Ther, 2010, 9(2): 489-498; Wainwright DA, et al. Clin Cancer Res, 2014, 20(20):5290-5301).

A number of preclinical and clinical studies have shown that IDO1 inhibitors can reduce the accumulation of metabolites such as tryptophan metabolism and kynurenine, thereby reversing IDO1-mediated immunosuppression, restoring the proliferation and function of T cells and NK cells, and inhibiting The proliferation of Treg cells, thereby enhancing the body's immune response, so IDO1 inhibitors can be used to treat the above-mentioned related diseases caused by IDO1-mediated immunosuppression, including cancer, viral infection, neurodegenerative diseases, cataracts, organ transplant rejection, depression syndrome and autoimmune diseases. In addition, IDO1 inhibitors can also be used in combination with other chemotherapeutic agents, targeted anticancer drugs, immune checkpoint therapeutics, antitumor vaccines, antiviral agents, antiviral vaccines, cytokine therapy, adoptive cellular immunotherapy and radiation therapy , play the role of synergistic or enhanced therapy (Vacchelli E, et al. Oncoimmunology, 2014, 3(10): e957994; Jochems C, et al. . Blood, 2010, 115(17): 3520-3530; Zamarin D, et al. Pharmacol Ther, 2015, 150: 23-32).

Small molecule inhibitors of IDO1 are currently being developed to treat or prevent the above-mentioned IDO1-related diseases. For example, oxadiazole IDO1 inhibitors are reported in CN102164902B, involving the treatment of cancer, viral infection, neurodegenerative disease, trauma, age-related cataract, organ transplant rejection or Patients with autoimmune diseases.

Based on the fact that IDO1 is closely related to the pathogenesis of various diseases such as cancer, viral infection, neurodegenerative disease, cataract, organ transplant rejection, autoimmune disease and depression, IDO1 inhibitors can be used to inhibit the activity of IDO1, thereby reducing color Amino acid metabolism and the accumulation of metabolites such as kynurenine, restore the body's immune function, and then achieve the purpose of treating the above diseases. The compound described in the present invention has IDO1 inhibitory activity, and can be used to treat related diseases caused by IDO1-mediated immunosuppression, including cancer, viral infection, neurodegenerative disease, cataract, organ transplant rejection, autoimmune disease and depression .

Contents of the invention

The technical problem to be solved by the present invention is to provide a benzo five-membered heterocyclic compound or a pharmaceutically acceptable salt thereof, its preparation method, pharmaceutical composition and application. The compound of the present invention has good IDO1 inhibitory activity, and can be used for treating and/or preventing various related diseases caused by IDO1-mediated immunosuppression.

The present invention provides a benzo five-membered heterocyclic compound represented by general formula (I) or a pharmaceutically acceptable salt thereof:

in:

α and β represent a single bond or a double bond; when α is a single bond, β is a double bond; when α is a double bond, β is a single bond;

A stands for C, O, S, NH or N;

B represents O, S or NH ₂ ;

R ₁ represents hydrogen, halogen, nitro, hydroxyl, cyano, amino or C ₁ -C ₁₀ alkyl;

R ₂ represents hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₁₀ alkyl C(O)R ₃ , -C(O)NR ₄ R ₅ , -C(O)R ₃ , C ₆ -C ₁₀ Aryl, (C ₁ -C ₅ )alkyl(C ₆ -C ₁₀ )aryl, C ₁ -C ₁₀ aromatic heterocyclic group or -(C ₁ -C ₅ )alkyl(C ₁ -C ₁₀ )aryl Heterocyclic group; wherein said aromatic heterocyclic group may optionally contain one or more other heteroatoms selected from O, S or N; said alkyl, aryl or aromatic heterocyclic group may optionally are monosubstituted to pentasubstituted by the following identical or different substituents selected from the group consisting of: hydrogen, halogen, hydroxyl, methoxy or R ₃ ;

R ₃ , R ₄ , and R ₅ can be the same or different, representing hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, -(C ₁ -C ₅ ) alkane Base (C ₆ -C ₁₀ ) aryl, C ₁ -C ₁₀ aromatic heterocyclic group or (C ₁ -C ₅ ) alkyl (C ₁ -C ₁₀ ) aromatic heterocyclic group; wherein said aromatic heterocyclic group The group may optionally contain one or more other heteroatoms selected from O, S or N; the alkyl, aryl or aromatic heterocyclic group may optionally be monosubstituted by the following identical or different substituents Substitution to five substitutions, the substituents are selected from: hydrogen, halogen, hydroxyl or methoxy.

Further, the benzo five-membered heterocyclic compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof is characterized in that:

α and β represent a single bond or a double bond; when α is a single bond, β is a double bond; when α is a double bond, β is a single bond;

A represents O, NH or N;

B represents O or NH ₂ ;

R represents hydrogen, halogen, nitro, hydroxyl or amino _;

R ₂ represents hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₁₀ alkyl C(O)R ₃ , -C(O)NR ₄ R ₅ , -C(O)R ₃ , C ₆ -C ₁₀ Aryl, -(C ₁ -C ₅ )alkyl(C ₆ -C ₁₀ )aryl, C ₁ -C ₁₀ aromatic heterocyclic group or -(C ₁ -C ₅ )alkyl(C ₁ -C ₁₀ ) Aromatic heterocyclic group; wherein said aromatic heterocyclic group may optionally contain one or more other heteroatoms selected from O, S or N; said alkyl, aryl or aromatic heterocyclic group may optionally is optionally monosubstituted to pentasubstituted by the following identical or different substituents selected from the group consisting of: hydrogen, halogen, hydroxyl, methoxy or R ₃ ;

R ₃ , R ₄ , R ₅ can be the same or different, representing hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, (C ₁ -C ₅ ) alkyl (C ₆ -C ₁₀ ) aryl, C ₁ -C ₁₀ aromatic heterocyclic group or (C ₁ -C ₅ ) alkyl (C ₁ -C ₁₀ ) aromatic heterocyclic group; wherein said aromatic heterocyclic group Can optionally contain one or more other heteroatoms selected from O, S or N; said alkyl, aryl or aromatic heterocyclic group can be optionally monosubstituted by the same or different substituents below to five substitutions, the substituents are selected from: hydrogen, halogen, hydroxyl or methoxy.

Further, the benzo five-membered heterocyclic compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof is characterized in that:

α and β represent a single bond or a double bond; when α is a single bond, β is a double bond; when α is a double bond, β is a single bond;

A represents O, NH or N;

B represents O or NH ₂ ;

R represents hydrogen, chlorine, fluorine, nitro or amino _;

R is selected from _:

Specifically, the benzo five-membered heterocyclic compound represented by the general formula (I) is preferably selected from the following compounds:

The compound codes involved in the following pharmacological experiments are equal to the compounds corresponding to the codes here.

The present invention also provides the preparation method of the compound shown in general formula (I), it is characterized in that:

a) When A is O and B is O, the preparation method of the compound shown in general formula (I) is: the condensation reaction between different substituted o-aminophenols and CDI produces intermediate 1, 1 and different substituted α-chloro Substituted acetophenone was reacted to obtain 2a-f, 2a-f was reduced by sodium borohydride to obtain 3a-f; 1 was reacted with different substituted aralkyl chlorides to obtain 4a-j, 4i was reduced to obtain 5; 1 and Different acyl chlorides were reacted to prepare 6a-c; 1 was reacted with different amine compounds to obtain 7a-c; the synthetic route was as follows:

Wherein, R ₁ and R ₂ are as defined above;

b) when A is NH, and B is O, the preparation method of the compound shown in general formula (I) is: 2-benzimidazolone reacts with α-chloro-4'-fluoroacetophenone to prepare 8,8 9 was prepared by sodium borohydride reduction; its synthetic route is as follows:

Wherein, R ₁ and R ₂ are as defined above;

c) When A is N and B is NH ₂ , the preparation method of the compound shown in general formula (I) is: 1H-2-aminobenzimidazole is protected by Boc to obtain 10, and 10 reacts with acid chloride to obtain 11,11 The Boc substituent is removed to obtain 12; its synthetic route is as follows:

Wherein, R ₁ and R ₂ are as defined above.

The pharmaceutically acceptable salts of the compounds of general formula (I) can be synthesized by general chemical methods.

In general, salts can be prepared by reacting free bases or acids with equivalent or excess amounts of acids (inorganic or organic) or bases (inorganic or organic) in a suitable solvent or solvent composition.

The present invention also provides a pharmaceutical composition, which is composed of a therapeutically effective amount of an active component and a pharmaceutically acceptable adjuvant; the active component includes a compound of general formula (I) and its pharmaceutically acceptable One or more of salt. In the pharmaceutical composition, the auxiliary materials include pharmaceutically acceptable carriers, diluents and/or excipients.

According to the purpose of treatment, the pharmaceutical composition can be made into various types of administration unit dosage forms, such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories and injections (solutions and suspensions), etc., Tablets, capsules, liquids, suspensions, and injections (solutions and suspensions) are preferred.

For shaping pharmaceutical compositions in the form of tablets, pills or suppositories, any excipient known and widely used in the art may be used.

In order to prepare the pharmaceutical composition in the form of injection, the solution or suspension can be sterilized (preferably adding an appropriate amount of sodium chloride, glucose or glycerin) to make an injection with isotonic pressure with blood. When preparing injections, any commonly used carrier in the art can also be used. For example: water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, fatty acid esters of polyethylene sorbitan, and the like. In addition, general dissolving agents, buffering agents and the like may be added.

The content of the composition of the present invention in the pharmaceutical composition is not particularly limited, and can be selected within a wide range, usually 5-95% by mass, preferably 30-85% by mass.

The administration method of the pharmaceutical composition of the present invention is not particularly limited. Preparations in various dosage forms can be selected and administered according to the patient's age, sex and other conditions and symptoms.

The present invention further provides the application 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. The IDO1 inhibitor is used for treating patients with diseases related to IDO1-mediated immunosuppression, and the related diseases include cancer, viral infection, neurodegenerative disease, cataract, organ transplant rejection, depression and autoimmune disease.

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, and the medicines are used to treat cancer, viral infection and neurodegenerative diseases in patients , cataracts, organ transplant rejection, depression, or autoimmune disease.

The cancers include, but are not limited to: malignant melanoma, lung cancer, breast cancer, gastric 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, mesothelioma, thyroid cancer, liver cancer and esophageal cancer.

The virus infection includes but not limited to: caused by human immunodeficiency virus, hepatitis B virus, hepatitis C virus, influenza virus, poliovirus, cytomegalovirus, Coxsackie virus, human papilloma virus, HIV Infection caused by one or more of Predstein-Barr virus and Varicella-Zoster virus.

The neurodegenerative diseases include, but are not limited to: one or more of memory impairment, Alzheimer's disease, cognitive impairment, senile dementia, Parkinson's disease, Parkinson's syndrome and movement disorders .

The autoimmune diseases include, but are not limited to: rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, scleroderma, nodular vasculitis, multiple sclerosis, kidney disease, 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 treatment methods for the treatment of IDO1 mediated related diseases.

The other types of therapeutic agents and/or treatment methods include, but are not limited to: chemotherapeutic agents, targeted anti-tumor drugs, inhibitors of immune checkpoint costimulatory molecules, agonists of immune checkpoint costimulatory molecules, anti-tumor vaccines, Antiviral agents, antiviral vaccines, cytokine therapy, adoptive cellular immunotherapy or radiation therapy.

The chemotherapeutic agents include, but are not limited to: alkylating agents, tubulin inhibitors, topoase inhibitors, platinum drugs, anti-metabolite drugs or hormone anti-tumor drugs.

The targeted anti-tumor drugs include, but are not limited to: protein kinase inhibitors, proteasome inhibitors, isocitrate dehydrogenase inhibitors, epigenetic-based anti-tumor drugs or cell cycle signaling pathway inhibitors.

The immune checkpoint inhibitors include but are not limited to: CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, TIM-3 inhibitors, VISTA inhibitors, LAG3 inhibitors , TIGIT inhibitors, A2AR inhibitors, or VTCN1 inhibitors.

The immune checkpoint agonists include but not limited to: STING agonists, 4-1BB agonists, OX40 agonists, RORγ agonists or ICOS agonists.

detailed description

In order to further clarify the present invention, a series of examples are given below, these examples are completely illustrative, they are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

Example 1

Synthesis of 2-Benzoxazolinone (1a)

Dissolve o-aminophenol (1.00g, 9.16mmol) and CDI (1.78g, 10.99mmol) in 30mL of anhydrous DMF, under nitrogen protection, react at 80°C for 5h, cool down to room temperature, add water (10mL) to quench the reaction, ethyl acetate ester (3×7 mL), washed the organic layer with saturated ammonium chloride and saturated brine respectively, dried over anhydrous magnesium sulfate, and separated by column chromatography (petroleum ether:ethyl acetate=7:1) to obtain a light red solid 1.10 g, yield 89%, mp 137-141°C. ¹ H NMR (300MHz, Chloroform-d) δ9.81(s, 1H), 8.00-6.85(m, 4H).

Synthesis of 3-(4-fluoroacetophenonyl)-2-benzoxazolinone (2a)

Mix 2-benzoxazolinone (0.10g, 0.74mmol) and potassium carbonate (0.31g, 2.22mmol) with 10mL DMF, react at 80°C for 1h, then lower the temperature to 60°C, and add α-chloro -4'-fluoroacetophenone (0.38g, 2.22mmol), reacted for 3h, cooled to room temperature, added saturated ammonium chloride solution (10mL) to quench the reaction, extracted with ethyl acetate (3×7mL), washed with saturated brine , dried over anhydrous magnesium sulfate, and separated by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain 0.17 g of a light yellow solid, yield 83%, mp 148-150°C. ESI-MS: 294.1[M+Na] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ8.08 (ddd, J=9.0, 5.2, 2.1Hz, 2H), 7.39-6.96 (m, 5H), 6.85 (dq, J=4.8, 3.3, 2.1Hz, 1H), 5.22 (d, J=2.0Hz, 2H).IR(KBr): 3074, 1763, 1694, 1601, 1487, 1232cm ^-1 .

Example 2

Synthesis of 3-(4-chloroacetophenonyl)-2-benzoxazolinone (2b)

Referring to the synthetic method of the target compound 2a, it was prepared from 2-benzoxazolinone and α-chloro-4'-chloroacetophenone as raw materials, with a yield of 85%, and mp 193-195°C. ESI-MS: 288.6[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ8.14-7.86(m, 2H), 7.53(d, J=8.0Hz, 2H), 7.40-7.05(m , 3H), 6.86(s, 1H), 5.23(d, J=3.1Hz, 2H).IR(KBr): 3065, 1763, 1691, 1587, 1487, 1246cm ^-1 .

Example 3

Synthesis of 3-(3-chloroacetophenonyl)-2-benzoxazolinone (2c)

Referring to the synthesis method of the target compound 2a, it was prepared using 2-benzoxazolinone and α-chloro-3'-chloroacetophenone as raw materials. The yield was 85%, and the mp was 150-152°C. ESI-MS: 288.6[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ8.34-7.84(m, 2H), 7.83-7.43(m, 2H), 7.42-7.07(m, 3H) , 7.02-6.76(m, 1H), 5.23(qd, J=8.4, 4.9, 4.3Hz, 2H).IR(KBr): 3071, 1722, 1683, 1601, 1512, 1229cm ^-1 .

Example 4

Synthesis of 5-chloro-2-benzoxazolinone (1b)

Referring to the synthesis method of 1a, it was prepared from 4-chloro-2-aminophenol, with a yield of 87%, and mp 189-193°C. ¹ H NMR (300MHz, Chloroform-d) δ8.87(s, 1H), 7.28(d, J=1.8Hz, 1H), 7.13(dp, J=8.0, 2.1Hz, 2H).

Synthesis of 5-chloro-3-(4-fluoroacetophenonyl)-2-benzoxazolinone (2d)

Referring to the synthesis method of the target compound 2a, it was prepared from 1b and α-chloro-4'-fluoroacetophenone as raw materials, with a yield of 89%, and mp 153-155°C. ESI-MS: 306.7[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ8.08 (d, J=7.4Hz, 2H), 7.20 (dd, J=30.4, 9.8Hz, 4H), 6.85(s, 1H), 5.20(s, 2H). IR(KBr): 3076, 1771, 1690, 1599, 1487, 1232cm ^-1 .

Example 5

Synthesis of 5-chloro-3-(4-chloroacetophenonyl)-2-benzoxazolinone (2e)

Referring to the synthesis method of the target compound 2a, it was prepared from 1b and α-chloro-4'-chloroacetophenone as raw materials, with a yield of 87%, and mp 167-169°C. ESI-MS: 323.1[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ8.05-7.93(m, 2H), 7.60-7.50(m, 2H), 7.24-7.12(m, 2H) , 6.85(d, J=2.0Hz, 1H), 5.21(s, 2H).IR(KBr): 3067, 1772, 1719, 1589, 1485, 1246cm ^-1 .

Example 6

Synthesis of 5-chloro-3-(3-chloroacetophenonyl)-2-benzoxazolinone (2f)

Referring to the synthesis method of the target compound 2a, it was prepared from 1b and α-chloro-3'-chloroacetophenone as raw materials, with a yield of 87%, and mp 134-136°C. ESI-MS: 323.1[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.97(d, J=27.1Hz, 2H), 7.68(s, 1H), 7.54(s, 1H), 7.17(d, J=10.9Hz, 2H), 6.84(s, 1H), 5.22(s, 2H).IR(KBr): 3067, 1778, 1703, 1614, 1487, 1223cm ^-1 .

Example 7

Synthesis of 3-(2-(4-fluorophenyl)-2-hydroxy)ethyl-2-benzoxazolinone (3a)

The target compound 2a (0.10g, 0.37mmol) was dissolved in 10mL of anhydrous tetrahydrofuran, sodium borohydride (0.04g, 1.11mmol) was added under ice-cooling, reacted for 1h under ice-cooling, quenched by adding 10mL of saturated ammonium chloride, ethyl acetate Ester was extracted (3×7 mL), dried over anhydrous magnesium sulfate, and purified by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain 0.10 g of white solid, yield 99%, mp 111-113°C. ESI-MS: 274.3[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.89(s, 1H), 7.53-7.37(m, 2H), 7.26-6.86(m, 5H), 5.79 (t, 1H), 4.41(t, 1H), 4.10(t, 1H).IR(KBr): 3645, 3071, 1722, 1601, 1512, 1229, 1103cm ^-1 .

Example 8

Synthesis of 3-(2-(4-chlorophenyl)-2-hydroxy)ethyl-2-benzoxazolinone (3b)

Referring to the synthesis method of the target compound 3a, it was prepared using 2b as a raw material, with a yield of 99% and a mp of 140-142°C. ESI-MS: 290.7[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.36(q, J=7.5, 6.4Hz, 4H), 7.15(dq, J=14.1, 6.9Hz, 3H ), 6.99(d, J=7.0Hz, 1H), 5.14(s, 1H), 3.95(d, J=10.8Hz, 2H), 3.06(s, 1H).IR(KBr): 3645, 3063, 1748 , 1593, 1485, 1240, 1090cm ^-1 .

Example 9

Synthesis of 3-(2-(3-chlorophenyl)-2-hydroxy)ethyl-2-benzoxazolinone (3c)

Referring to the synthesis method of the target compound 3a, it was prepared from 2c as a raw material, with a yield of 99% and a mp of 113-115°C. ESI-MS: 290.7[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.52-7.42(m, 1H), 7.28(d, J=2.2Hz, 3H), 7.20-7.05(m , 3H), 7.01(dd, J=7.9, 1.7Hz, 1H), 5.11(dd, J=8.2, 4.0Hz, 1H), 4.13-3.83(m, 3H), 3.54(s, 1H).IR( KBr): 3646, 3063, 1759, 1599, 1485, 1240, 1103cm ^-1 .

Example 10

Synthesis of 5-chloro-3-(2-(4-fluorophenyl)-2-hydroxy)ethyl-2-benzoxazolinone (3d)

Referring to the synthesis method of the target compound 3a, it was obtained from 2d with a yield of 99%, mp 142-144°C. ESI-MS: 308.7[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.42(dd, J=8.4, 5.3Hz, 2H), 7.18-6.87(m, 5H), 5.14(dd , J=8.4, 3.7Hz, 1H), 4.17-3.79(m, 2H), 2.94(s, 1H).IR(KBr): 3646, 3089, 1771, 1607, 1485, 1223, 1096cm ^-1 .

Example 11

Synthesis of 5-chloro-3-(2-(4-chlorophenyl)-2-hydroxy)ethyl-2-benzoxazolinone (3e)

Referring to the synthesis method of the target compound 3a, it was obtained from 2e with a yield of 99% and a mp of 127-129°C. ESI-MS: 325.1[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.42-7.32(m, 4H), 7.09(d, J=1.1Hz, 1H), 7.08(s, 1H ), 7.03 (dd, J=1.8, 0.8Hz, 1H), 5.12 (dd, J=8.3, 3.6Hz, 1H), 3.99 (dd, J=14.6, 3.6Hz, 1H), 3.87 (dd, J= 14.5, 8.3Hz, 1H), 2.97(s, 1H).IR(KBr): 3626, 3063, 1767, 1614, 1485, 1252, 1092cm ^-1 .

Example 12

Synthesis of 5-chloro-3-(2-(3-chlorophenyl)-2-hydroxy)ethyl-2-benzoxazolinone (3f)

Referring to the synthesis method of the target compound 3a, it was prepared from 2f with a yield of 99%, and mp 157-159°C. ESI-MS: 325.1[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.45 (dq, J=1.6, 0.9Hz, 1H), 7.43-7.37 (m, 2H), 7.37-7.31 (m, 1H), 7.16-7.05(m, 2H), 6.95(d, J=8.5Hz, 1H), 5.74(t, J=8.3Hz, 1H), 4.38(t, J=8.9Hz, 1H) , 4.01 (dd, J=9.1, 7.9Hz, 1H).IR(KBr): 3647, 3078, 1728, 1599, 1506, 1261, 1099cm ^-1 .

Example 13

Synthesis of 3-phenethyl-2-benzoxazolinone (4a)

Mix 2-benzoxazolinone (0.10g, 0.74mmol) and potassium carbonate (0.31g, 2.22mmol) with 10mL DMF, react at 80°C for 1h, then lower the temperature to 60°C, and add β-bromo Phenylethane (0.41g, 2.22mmol), react for 6h, cool down to room temperature, add 10mL saturated ammonium chloride solution to quench the reaction, extract with ethyl acetate (3×7mL), wash the organic layer with saturated brine, and dry over anhydrous magnesium sulfate , column chromatography (petroleum ether: ethyl acetate = 10:1), to obtain a white solid 0.15g, yield 86%, mp116-118 ° C. ESI-MS: 240.3[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.29(d, J=7.7Hz, 3H), 7.20(s, 3H), 7.10(t, J=5.9 Hz, 2H), 6.79(d, J=7.1Hz, 1H), 4.06(t, J=7.4Hz, 2H), 3.08(t, J=7.2Hz, 2H).IR(KBr): 3061, 1767, 1614, 1489, 1258cm ^-1 .

Example 14

Synthesis of 3-(3-fluorobenzyl)-2-benzoxazolinone (4b)

Referring to the synthesis method of the target compound 4a, it was obtained from 1a and 3-fluorochlorobenzyl, the yield was 88%, and the mp was 119-121°C. ESI-MS: 244.2[M+H] ⁺ ; ¹ H NMR (300MHz, DMSO-d ₆ ) δ7.48-7.31(m, 2H), 7.30-6.86(m, 6H), 5.12-4.86(m, 2H ).IR(KBr): 3042, 1757, 1589, 1487, 1252cm ^-1 .

Example 15

Synthesis of 3-(4-chlorobenzyl)-2-benzoxazolinone (4c)

According to the synthetic method of the target compound 4a, it was obtained from 1a and 4-chlorobenzyl chloride, the yield was 87%, and the mp was 145-147°C. ESI-MS: 260.7[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.57-7.25(m, 4H), 7.23(s, 1H), 7.18-7.01(m, 2H), 6.95 -6.74(m, 1H), 5.26-4.79(m, 2H).IR(KBr): 3005, 1771, 1597, 1485, 1275cm ^-1 .

Example 16

Synthesis of 5-chloro-3-phenethyl-2-benzoxazolinone (4d)

Referring to the synthesis method of the target compound 4a, it was prepared from 1b and β-bromophenylethane, the yield was 88%, and the mp was 102-104°C. ESI-MS: 274.7[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.36-7.24(m, 3H), 7.24-7.15(m, 2H), 7.14-6.99(m, 2H) , 6.67(d, J=2.0Hz, 1H), 4.03(t, J=7.3Hz, 2H), 3.06(t, J=7.3Hz, 2H).IR(KBr): 3057, 1759, 1610, 1485, 1246cm ^-1 .

Example 17

Synthesis of 5-chloro-3-(3-fluorobenzyl)-2-benzoxazolinone (4e)

Referring to the synthesis method of the target compound 4a, it was obtained from 1b and 3-fluorochlorobenzyl, the yield was 92%, and the mp was 134-136°C. ESI-MS: 278.7[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.36(q, J=11.9, 9.7Hz, 1H), 7.22-6.92(m, 5H), 6.81(d , J=14.5Hz, 1H), 4.96 (d, J=9.4Hz, 2H).IR(KBr): 3065, 1772, 1589, 1506, 1256cm ^-1 .

Example 18

Synthesis of 5-chloro-3-(4-chlorobenzyl)-2-benzoxazolinone (4f)

According to the synthetic method of the target compound 4a, it was prepared from 1b and 4-chlorobenzyl, the yield was 91%, and the mp was 122-124°C. ESI-MS: 295.1[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.32(q, J=8.1Hz, 4H), 7.23-6.97(m, 3H), 6.90-6.73(m , 1H), 4.95(s, 2H).IR(KBr): 3067, 1767, 1601, 1483, 1252cm-1.

Example 19

Synthesis of 5-fluoro-2-benzoxazolinone (1c)

Referring to the synthesis method of 1a, it is prepared from 4-fluoro-2-aminophenol, with a yield of 90%, mp 174-176°C. ¹ H NMR (300MHz, Chloroform-d) δ9.60(s, 1H), 7.13(dd, J=8.7, 4.2Hz, 1H), 6.93-6.68(m, 2H).

Synthesis of 5-fluoro-3-(3-fluorobenzyl)-2-benzoxazolinone (4g)

Referring to the synthesis method of the target compound 4a, it was obtained from 1c and 3-fluorochlorobenzyl, the yield was 88%, and the mp was 156-158°C. ¹ HNMR (300MHz, Chloroform-d) δ (ppm) 7.37 (td, J=8.3, 5.6Hz, 1H), 7.23-7.11 (m, 2H), 7.06 (dd, J=10.2, 4.0Hz, 2H), 6.83(td, J=9.2, 2.6Hz, 1H), 6.59(dd, J=7.7, 2.6Hz, 1H), 5.00(s, 2H).MS(EI) m/z 262.1[M+H]+. IR(KBr): 3063, 1765, 1591, 1487, 1250cm ^-1 .

Example 20

Synthesis of 5-fluoro-3-(4-chlorobenzyl)-2-benzoxazolinone (4h)

Referring to the synthesis method of the target compound 4a, it was prepared from 1c and 4-chlorobenzyl, the yield was 82%, and the mp was 103-105°C. ¹ HNMR (300MHz, Chloroform-d) δ (ppm) 7.46-7.21 (m, 4H), 7.13 (dd, J=8.8, 4.2Hz, 1H), 6.79 (td, J=9.2, 2.6Hz, 1H), 6.57(dd, J=7.6, 2.6Hz, 1H), 4.94(s, 2H).MS(EI) m/z 278.2[M+H] ⁺ .IR(KBr): 3061, 1778, 1614, 1489, 1250cm ^-1 .

Example 21

Synthesis of 5-nitro-3-(3-fluorobenzyl)-2-benzoxazolinone (4i)

Referring to the synthesis method of the target compound 4a, it was prepared from 5-nitro-2-benzoxazolinone and 3-fluorochlorobenzyl, with a yield of 81%, mp 116-118°C. ¹ H NMR (300MHz, Chloroform-d) δ (ppm) 8.42 (d, J = 1.8Hz, 1H), 8.10 (dd, J = 7.5, 2.0Hz, 1H), 7.40 (d, J = 7.5Hz, 1H ), 7.35-7.25 (m, 2H), 7.19 (dt, J=8.7, 1.8Hz, 1H), 6.97 (ddt, J=8.9, 6.8, 2.3Hz, 1H), 5.56 (d, J=1.3Hz, 2H).MS(EI) m/z 289.1[M+H] ⁺ .IR(KBr): 3065, 1792, 1614, 1506, 1261cm ^-1 .

Example 22

Synthesis of 5-nitro-3-(4-chlorobenzyl)-2-benzoxazolinone (4j)

Referring to the synthesis method of the target compound 4a, it was prepared from 5-nitro-2-benzoxazolinone and 4-chlorobenzyl chloride, with a yield of 77%, and mp 165-167°C. ¹ H NMR (300MHz, Chloroform-d) δ (ppm) 8.14 (dd, J=8.8, 2.3Hz, 1H), 7.76 (d, J=2.3Hz, 1H), 7.48-7.29 (m, 5H), 5.05 (s, 2H).MS(EI) m/z 303.1[MH] ⁺ .IR(KBr): 3080, 1784, 1595, 1522, 1260cm ^-1 .

Example 23

Synthesis of 5-amino-3-(3-fluorobenzyl)-2-benzoxazolinone (5)

4i (0.10 g, 0.35 mmol) was dissolved in 10 mL of industrial ethanol, and ammonium chloride (0.19 g, 3.47 mmol) was dissolved in 2 mL of water and added to the reaction system. Zinc powder (0.23g, 3.47mmol) was slowly added under ice-bath cooling, the ice-bath reaction was returned to room temperature for 2h, the ethanol was removed, ethyl acetate was extracted, dried over anhydrous magnesium sulfate, and column chromatography separated (petroleum ether: ethyl acetate Ester = 5:1), to obtain 0.70 g of light yellow solid, yield 78%, mp 179-181°C. ¹ H NMR (300MHz, Chloroform-d) δ (ppm) 7.29 (d, J = 21.3Hz, 2H), 7.10 (d, J = 7.7Hz, 1H), 7.03-6.96 (m, 2H), 6.37 (dd , J=8.5, 2.4Hz, 1H), 6.15(d, J=2.2Hz, 1H), 4.92(s, 2H), 3.59(s, 2H). MS(EI) m/z 257.1[MH] ⁺ . IR(KBr): 3356, 3048, 1746, 1591, 1489, 1254cm ^-1 .

Example 24

Synthesis of 3-(2-(4-fluorophenyl)acetyl)-2-benzoxazolinone (6a)

Dissolve 1a (0.10g, 0.74mmol) in 10mL of anhydrous tetrahydrofuran, add triethylamine (0.22g, 2.22mmol) to bind the acid, add dropwise anhydrous tetrahydrofuran solution of p-fluorophenylacetyl chloride (0.15g, 0.89 mmol), after dropping, the temperature of the reaction solution was raised to room temperature, and then heated to 70° C., reacted for 6 h, added 10 mL of water to quench the reaction, and extracted with ethyl acetate (3×7 mL). Purification by column chromatography (petroleum ether: ethyl acetate = 10:1) gave 0.17 g of white solid, yield 86%, mp 180-182°C. ESI-MS: 272.2[M+H] ⁺ ; ¹ H NMR (300MHz, DMSO-d ₆ ) δ7.98-7.89(m, 1H), 7.50-7.41(m, 1H), 7.41-7.28(m, 4H ), 7.24-7.14(m, 2H), 4.41(s, 2H).IR(KBr): 3067, 1796, 1721, 1601, 1514, 1248cm ^-1 .

Example 25

Synthesis of 3-benzoyl-2-benzoxazolinone (6b)

Referring to the synthesis method of the target compound 6a, it was prepared from 1a and benzoyl chloride as raw materials, with a yield of 89%, mp 185-187°C. ESI-MS: 240.2[M+H] ⁺ ; ¹ H NMR (300MHz, DMSO-d ₆ ) δ7.95-7.85(m, 2H), 7.84-7.75(m, 1H), 7.67(t, J=7.4 Hz, 1H), 7.53(t, J=7.6Hz, 2H), 7.49-7.42(m, 1H), 7.40-7.26(m, 2H).IR(KBr): 3069, 1805, 1697, 1599, 1481, 1250cm ^-1 .

Example 26

Synthesis of 5-chloro-3-(2-(4-fluorophenyl)acetyl)-2-benzoxazolinone (6c)

Referring to the synthesis method of the target compound 6a, it was prepared using 1b and p-fluorophenylacetyl chloride as raw materials, with a yield of 87%, mp 193-195°C. ESI-MS: 306.7[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ7.99(d, J=8.2Hz, 2H), 7.56(d, J=8.0Hz, 2H), 7.35- 6.99(m, 2H), 6.85(s, 1H), 3.51(s, 2H).IR(KBr): 3063, 1755, 1715, 1611, 1485, 1246cm ^-1 .

Example 27

Synthesis of 3-(4-methoxybenzyl)carbamoyl-2-benzoxazolinone (7a)

Solid phosgene (0.22g, 0.74mmol) was dissolved with 5mL of anhydrous dichloromethane, and 2-benzoxazolone (0.10g, 0.74mmol) and triethylamine (0.30g, 2.96mmol) were added dropwise under ice-cooling ) in anhydrous dichloromethane solution (5mL), nitrogen protection immediately after dropping, after stirring at room temperature for 1h, p-methoxybenzylamine (0.15g, 1.11mmol) and triethylamine (0.11g, 1.11mmol) in anhydrous dichloromethane solution (5mL), react at room temperature for 3h, add 10mL saturated ammonium chloride to quench, dichloromethane extract (3×7mL), dry over anhydrous magnesium sulfate, column chromatography (petroleum ether : ethyl acetate=7:1), 0.18 g of white solid was obtained, yield 83%, mp 82-84°C. ESI-MS: 299.4[M+H] ⁺ ; ¹ H NMR (300MHz, DMSO-d ₆ ) δ8.56(s, 1H), 7.89(d, 1H), 7.43(s, 1H), 7.41-7.25( m, 4H), 6.90(d, 2H), 4.45(d, 2H), 3.73(s, 3H). IR(KBr): 3266, 3009, 2951, 2870, 1761, 1722, 1612, 1514, 1275, 1260 , 1034cm ^-1 .

Example 28

Synthesis of 5-chloro-3-(3-methoxybenzyl)carbamoyl-2-benzoxazolinone (7b)

Referring to the synthesis method of the target compound 7a, it was prepared from 1b, solid phosgene, and o-methoxybenzylamine as raw materials, with a yield of 90% and a mp of 120-122°C. ESI-MS: 333.7[M+H] ⁺ ; ¹ H NMR (300MHz, Chloroform-d) δ8.37(s, 1H), 8.15(d, 1H), 7.33-7.16(m, 3H), 6.97-6.84 (m, 3H), 4, 60 (d, 2H), 3.83 (s, 3H). IR (KBr): 3354, 3048, 1771, 1724, 1601, 1477, 1265, 1252, 1042cm ^-1 .

Example 29

Synthesis of 5-chloro-3-(4-methoxyphenyl)carbamoyl-2-benzoxazolinone (7c)

Referring to the synthesis method of the target compound 7a, it was prepared from 1b, solid phosgene, and p-methoxyaniline as raw materials, with a yield of 86% and a mp of 162-164°C. ESI-MS: 319.7[M+H] ⁺ ; ¹ H NMR (300MHz, DMSO-d ₆ ) δ9.85(s, 1H), 7.89(d, J=2.3Hz, 1H), 7.53(dd, J= IR (KBr): 3291, 3036, 2957, 2866, 1784, 1734, 1609, 1477, 1246, 1182, 1034cm ^-1 .

Example 30

Synthesis of 1-(2-(4-fluorophenyl)-2carbonylethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (8)

Referring to the synthesis method of the target compound 2a, it was prepared from 2-benzimidazolone and α-chloro-4′-fluoroacetophenone with a yield of 78%, mp 280-282°C. ¹ H NMR (300MHz, Chloroform -d) δ (ppm) 8.22-8.03 (m, 2H), 7.43-7.31 (dd, J = 7.9, 1.5Hz, 1H), 7.26-7.14 (m, 3H), 7.12-7.06 (td, J = 7.6 , 1.5Hz, 1H), 7.05-6.97(td, J=7.6, 1.5Hz, 1H), 5.61(s, 2H).MS(EI) m/z 273.3[M+H] ^+. IR(KBr): 3323, 3018, 1687, 1520, 1439cm ^-1 .

Synthesis of 1-(2-(4-fluorophenyl)-2-hydroxyethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (9)

Referring to the synthesis method of the target compound 3a, using 8 as a raw material, the yield was 73%, mp 276-278°C. ¹ H NMR (300MHz, Chloroform-d) δ (ppm) 7.49-7.43 (m, 2H), 7.39-7.35 (m, 1H), 7.13-7.07 (m, 3H), 7.06-7.01 (td, J=7.6, 1.5Hz, 1H), 7.00-6.96 (dd, J=7.8, 1.5Hz, 1H), 5.25-5.17 (d, J=6.4Hz, 1H), 5.14-5.07(m, 1H), 4.38-4.27(d, J=4.9Hz, 2H).MS(EI) m/z 273.3[M+H] ⁺ .IR (KBr): 3570, 3343, 3028, 1670, 1521, 1449cm ^-1 .

Example 31

Synthesis of tert-butyl(1H-benzo[d]imidazol-2-yl)carbamate (10)

Add Boc ₂ O (3.61g, 16.52mmol) and TEA (3.04g, 30.04mmol) to a solution of 1H-2-aminobenzimidazole (2.00g, 15.02mmol) in THF (30mL), then stir at 60°C for 3h , cooled to room temperature, and the solvent was removed. Washing with petroleum ether gave the crude product 10 (3 g, 85.62%) as a white solid, which was directly used in the next reaction.

Synthesis of tert-butyl(1-(2-(4-fluorophenyl)acetyl)-1H-benzo[d]imidazol-2-yl)carbamate (11)

Referring to the synthesis method of the target compound 6a, it was prepared from 10, with a yield of 53%, mp 310-312 ° C. ¹ H NMR (300 MHz, Chloroform-d) δ (ppm) 8.53 (s, 1H), 7.92-7.80 ( m, 1H), 7.76-7.64(dd, J=7.7, 1.4Hz, 1H), 7.41-7.21(m, 4H), 7.11-6.99(m, 2H), 4.04-3.88(t, J=1.0Hz, 2H), 1.46(s, 9H). MS(EI) m/z 370.4[M+H] ⁺ .IR(KBr): 3353, 3038, 1680, 1531, 1423, 1370cm ^-1 .

Synthesis of 1-(2-amino-1H-benzo[d]imidazol-1-yl)-2-(4-fluorophenyl)ethane (12)

Slowly add HCl solution (4M HCl, 6.8mL) dropwise to a solution of 11 (0.5g, 1.35mmol) in dioxane (5mL) in an ice bath, stir at room temperature for 2h, filter the white solid, wash with dioxane to obtain Hydrochloride, after dissociation, 12 was obtained (0.26g, 71.33%), mp 237-239°C. ¹ H NMR (300MHz, Chloroform-d) δ (ppm) 7.84-7.69 (m, 2H), 7.36-7.22 ( m, 4H), 7.14-7.02(m, 2H), 6.15-6.08(d, J=5.9Hz, 1H), 6.06-6.00(d, J=5.9Hz, 1H), 4.03-3.93(t, J= 1.0Hz, 2H). MS(EI) m/z 270.2[M+H] ⁺ .IR(KBr): 3356, 3148, 3048, 1646, 1591, 1419cm ^-1 .

Example 32

1. IDO1 inhibitory activity test based on Hela cells

1.1 Experimental materials and main instruments

1.2 Experimental method

Hela cells purchased from ATCC were stored in minimal basal medium (2mM L-glutamine and Earle's supplemented with 1.5g/L sodium bicarbonate, 0.1mM non-essential amino acids, 1mM sodium propupuprate and 10% fetal calf serum). 's BSS). Store Hela cells at 37 °C in a humidified incubator supplied with 5% _CO2 .

Hela cells were seeded in a 96-well culture plate at a density of 5×10 ³ /well and cultured overnight. The next day, IFN-γ (final concentration 100 ng/mL) and compound serial dilutions (total volume 200 μL medium) were added to the cells. After 24 hours of incubation, transfer 140 μL of supernatant/well to a new 96-well plate, add 10 μL of 6.1 mol/L trichloroacetic acid, and incubate in a constant temperature oven at 50°C for 30 minutes to make the produced N-formyl canine Uridine is hydrolyzed to kynurenine. The reaction mixture was then centrifuged at 4000 rpm for 10 min to remove the precipitate. 100 μL of supernatant/well was transferred to another 96-well plate, and mixed with an equal volume of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid solution. The absorbance was detected at 480 nm using a microplate reader, and the obtained results were calculated using an IC ₅₀ calculator. Three replicate wells were set up in the experiment.

1.3 Experimental results

The experimental results are shown in Table 1. The results show that the compound of the present invention has significant inhibitory effect on the activity of IDO1. Among them, compound 4f has the strongest activity (IC ₅₀ : 6.08 μM).

Table 1 The inhibitory activity of compounds of the present invention to IDO1

2. Proliferation experiment of T lymphocytes

2.1 Experimental method

B16F1 cell treatment: Aspirate the medium (high glucose DMEM, 10% FBS), wash with PBS 1-2 times. Add 0.25% trypsin for digestion. Aspirate the trypsin, add the medium, blow the cells down, 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 μg/ml), pipette to mix, 37°C, water bath for 30 minutes, wash 3 times with RP1640, count cells, and set aside.

Preparation of spleen cells: Take C57/BL6 mice, remove their eyeballs, and kill them by bloodletting. Aseptically remove the spleen and put it into a 35mm culture dish containing 2 mL of sterile pre-cooled RPMI 1640 medium. Cell extrusion. Then add 2 mL of culture medium, and pipette repeatedly with a 5 mL pipette until the suspension is uniform. The cell suspension was filtered with a 70 μm filter, and centrifuged at 300 g for 5 min (4° C.). After discarding the supernatant, add 10mL Tris-NH ₄ Cl to the splenocytes, blow evenly, let stand for 2-3min, and centrifuge at 300g for 5min (4°C) to remove red blood cells. After discarding the supernatant, wash twice with PRMI 1640 and set aside.

1) Add ² ×10 cells/well of treated B16F1 cells (stimulator cells) and 1× ¹⁰ cells/well of spleen lymphocytes (response cells) to a 96-well plate, add RP1640 (10% FBS), and replenish Make up to 200μL.

2) Grouping: drug administration group (stimulatory cells+responsive cells+corresponding compound), blank control (only reactive cells were added), model group (stimulatory cells+reactive cells), except for the blank control, other groups were added with ConA ( final concentration of 5 μg/ml), placed in an incubator at 37°C, 95% humidity, and 5% CO ₂ for 48 hours.

3) Add 20 μL of MTT (final concentration 4 mg/ml) into the incubator to continue culturing for 4 hours, measure the absorbance value at 570 nm wavelength with a microplate reader; calculate the proliferation rate of T lymphocytes:

T lymphocyte proliferation rate (%)=[administration hole (T lymphocyte+B16F1 cell+IDO1 inhibitor) OD value-control hole (T lymphocyte+B16F1 cell) OD value]/control hole (T lymphocyte+B16F1 Cell) OD value × 100%

2.2 Experimental results

In the mixed lymphocyte reaction system, B16F1 cells highly express IDO1, which can inhibit the proliferation of T lymphocytes. After adding compounds 4e, 4f and 6c (3 times _IC50 concentration) and culturing for 48h, the proliferation of T lymphocytes was detected by MTT, and the proliferation rates were 29.96%, 52.25% and 27.22%, indicating that compounds 4e, 4f and 6c could significantly increase Proliferation of T lymphocytes.

3. Effects on regulatory T lymphocytes

3.1 Experimental method

Add the treated B16F1 cells ( ⁸ ×104 wells) and spleen lymphocytes (106 wells, stimulated with 5 μg/mL ConA) into a 24-well plate, add the corresponding concentration of the compound and place it at 37°C with a humidity of 95°C. %, 5% CO ₂ incubator for 48 hours; collect the supernatant to test ELISA, use anti-CD4, anti-CD25, anti-FOCP3 antibody staining, and detect the differentiation of T cells in flow cytometry.

3.2 Experimental results

When primitive T lymphocytes were co-cultured with melanoma cell line B16F1, the number of regulatory T lymphocytes increased by 2 times (11.7%) compared with the experimental group containing only primitive T lymphocytes (5.1%). When compounds 4e, 4f and 6c (3 times IC ₅₀ concentration) were added to the system, this effect could be significantly reversed (respectively decreased to 6.0%, 8.7% and 7.1%), indicating that compounds 4e, 4f and 6c can inhibit IDO1 Reverses the transformation of naïve T lymphocytes to regulatory T lymphocytes.

4. Effect on IDO1 expression

4.1 Experimental method

Hela cells were cultured 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 (add medium only), model group (add IFN-γ, corresponding positive drug), drug treatment group (add IFN-γ, corresponding compound), culture at 37°C, 5% CO ₂ for 24h, and collect cells , Western blot detection of IDO1 expression.

4.2 Experimental results

The experimental results showed that compounds 4e, 4f and 6c did not affect the expression of IDO1 in Hela cells, indicating that compounds 4e, 4f and 6c reversed IDO1-mediated immunosuppression by inhibiting the activity of IDO1.

Claims (10)

1. A benzo five-membered heterocyclic compound represented by general formula (I) or a pharmaceutically acceptable salt thereof: in: α and β represent a single bond or a double bond; when α is a single bond, β is a double bond; when α is a double bond, β is a single bond; A stands for C, O, S, NH or N; B represents O, S or NH ₂ ; R ₁ represents hydrogen, halogen, nitro, hydroxyl, cyano, amino or C ₁ -C ₁₀ alkyl; R ₂ represents hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₁₀ alkyl C(O)R ₃ , -C(O)NR ₄ R ₅ , -C(O)R ₃ , C ₆ -C ₁₀ Aryl, (C ₁ -C ₅ )alkyl(C ₆ -C ₁₀ )aryl, C ₁ -C ₁₀ aromatic heterocyclic group or -(C ₁ -C ₅ )alkyl(C ₁ -C ₁₀ )aryl Heterocyclic group; wherein said aromatic heterocyclic group may optionally contain one or more other heteroatoms selected from O, S or N; said alkyl, aryl or aromatic heterocyclic group may optionally are monosubstituted to pentasubstituted by the following identical or different substituents selected from the group consisting of: hydrogen, halogen, hydroxyl, methoxy or R ₃ ; R ₃ , R ₄ , and R ₅ can be the same or different, representing hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, -(C ₁ -C ₅ ) alkane Base (C ₆ -C ₁₀ ) aryl, C ₁ -C ₁₀ aromatic heterocyclic group or (C ₁ -C ₅ ) alkyl (C ₁ -C ₁₀ ) aromatic heterocyclic group; wherein said aromatic heterocyclic group The group may optionally contain one or more other heteroatoms selected from O, S or N; the alkyl, aryl or aromatic heterocyclic group may optionally be monosubstituted by the same or different substituents described below Substitution to five substitutions, the substituents are selected from: hydrogen, halogen, hydroxyl or methoxy. 2. The benzo five-membered heterocyclic compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: α and β represent a single bond or a double bond; when α is a single bond, β is a double bond; when α is a double bond, β is a single bond; A represents O, NH or N; B represents O or NH ₂ ; R represents hydrogen, halogen, nitro, hydroxyl or amino _; R ₂ represents hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₁₀ alkyl C(O)R ₃ , -C(O)NR ₄ R ₅ , -C(O)R ₃ , C ₆ -C ₁₀ Aryl, -(C ₁ -C ₅ )alkyl(C ₆ -C ₁₀ )aryl, C ₁ -C ₁₀ aromatic heterocyclic group or -(C ₁ -C ₅ )alkyl(C ₁ -C ₁₀ ) Aromatic heterocyclic group; wherein said aromatic heterocyclic group may optionally contain one or more other heteroatoms selected from O, S or N; said alkyl, aryl or aromatic heterocyclic group may optionally is optionally monosubstituted to pentasubstituted by the following identical or different substituents selected from the group consisting of: hydrogen, halogen, hydroxyl, methoxy or R ₃ ; R ₃ , R ₄ , R ₅ can be the same or different, representing hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, (C ₁ -C ₅ ) alkyl (C ₆ -C ₁₀ ) aryl, C ₁ -C ₁₀ aromatic heterocyclic group or (C ₁ -C ₅ ) alkyl (C ₁ -C ₁₀ ) aromatic heterocyclic group; wherein said aromatic heterocyclic group Can optionally contain one or more other heteroatoms selected from O, S or N; said alkyl, aryl or aromatic heterocyclic group can be optionally monosubstituted by the same or different substituents below to five substitutions, the substituents are selected from: hydrogen, halogen, hydroxyl or methoxy. 3. The benzo five-membered heterocyclic compound or its pharmaceutically acceptable salt according to claim 2, characterized in that: α and β represent a single bond or a double bond; when α is a single bond, β is a double bond; when α is a double bond, β is a single bond; A represents O, NH or N; B represents O or NH ₂ ; R represents hydrogen, chlorine, fluorine, nitro or amino _; R is selected from _: 4. The benzo five-membered heterocyclic compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein said compound is selected from: 5. the preparation method of benzo five-membered heterocyclic compound according to claim 1, is characterized in that: a) When A is O and B is O, the preparation method of the compound shown in general formula (I) is: the condensation reaction between different substituted o-aminophenols and CDI produces intermediate 1, 1 and different substituted α-chloro Substituted acetophenone was reacted to obtain 2a-f, 2a-f was reduced by sodium borohydride to obtain 3a-f; 1 was reacted with different substituted aralkyl chlorides to obtain 4a-j, 4i was reduced to obtain 5; 1 and Different acyl chlorides were reacted to prepare 6a-c; 1 was reacted with different amine compounds to obtain 7a-c; the synthetic route was as follows: Wherein, R ₁ and R ₂ are as defined in claim 1; b) when A is NH, and B is O, the preparation method of the compound shown in general formula (I) is: 2-benzimidazolone reacts with α-chloro-4'-fluoroacetophenone to prepare 8,8 9 was prepared by sodium borohydride reduction; its synthetic route is as follows: Wherein, R ₁ and R ₂ are as defined in claim 1; c) When A is N and B is NH ₂ , the preparation method of the compound shown in general formula (I) is: 1H-2-aminobenzimidazole is protected by Boc to obtain 10, and 10 reacts with acid chloride to obtain 11,11 The Boc substituent is removed to obtain 12; its synthetic route is as follows: Wherein, R ₁ and R ₂ are as defined in claim 1. 6. A pharmaceutical composition, which is made up of a therapeutically effective amount of an active component and a pharmaceutically acceptable adjuvant; said active component comprises benzopentapenta as described in any one of claims 1-4 A membered heterocyclic compound (I) or a pharmaceutically acceptable salt thereof; the pharmaceutically acceptable adjuvant includes a pharmaceutically acceptable carrier, diluent and/or excipient. 7. The compound described in any one of claims 1-4, its stereoisomer, its pharmaceutically acceptable salt or the pharmaceutical composition described in claim 6 are used in the preparation of indoleamine 2,3-bis Application of oxygenase 1 inhibitor, said indoleamine 2,3-dioxygenase 1 inhibitor is used for the treatment of indoleamine 2,3-dioxygenase 1-mediated immunosuppressive related diseases In a patient, the disease related to the immunosuppression mediated by indoleamine 2,3-dioxygenase 1 is cancer, viral infection, neurodegenerative disease, cataract, organ transplant rejection, depression or autoimmune disease. 8. Use of the compound described in any one of claims 1-4 or its pharmaceutically acceptable salt or the composition described in claim 6 in the preparation of medicines, the medicine is used to treat cancer, virus in patients Infection, neurodegenerative disease, cataracts, organ transplant rejection, depression, or autoimmune disease. 9. The application according to claim 7-8, wherein said cancer is malignant melanoma, lung cancer, breast cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, lymphoma, leukemia, prostate cancer, testicular cancer, One or more of kidney cancer, brain cancer, head and neck cancer, ovarian cancer, cervical cancer, endometrial cancer, mesothelioma, thyroid cancer, liver cancer and esophageal cancer; the virus infection is human immunodeficiency virus, One of the hepatitis B virus, hepatitis C virus, influenza virus, polio virus, cytomegalovirus, coxsackie virus, human papilloma virus, Epstein-Barr virus, and varicella-zoster virus or multiple infections; the neurodegenerative disease is one of memory impairment, Alzheimer's disease, cognitive impairment, senile dementia, Parkinson's disease, Parkinson's syndrome and movement disorders or multiple; the autoimmune disease is 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. 10. The application according to claims 7-9, characterized in that: one or more chemotherapeutic agents, targeted anti-tumor drugs, immune checkpoint inhibitors, immune checkpoint agonists, Antitumor vaccines, antiviral agents, antiviral vaccines, cytokine therapy, adoptive cellular immunotherapy or radiotherapy; the chemotherapeutic agents are alkylating agents, tubulin inhibitors, topoase inhibitors, platinum drugs, Antimetabolite drugs or hormonal anti-tumor drugs; the targeted anti-tumor drugs are protein kinase inhibitors, proteasome inhibitors, isocitrate dehydrogenase inhibitors, anti-tumor drugs based on epigenetics or cell Cyclic signaling pathway inhibitors; the immune checkpoint inhibitors are CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, TIM-3 inhibitors, VISTA inhibitors, LAG3 Inhibitors, TIGIT inhibitors, A2AR inhibitors or VTCN1 inhibitors; the immune checkpoint agonists are STING agonists, 4-1BB agonists, OX40 agonists, RORγ agonists or ICOS agonists.