Bicyclic compound as well as pharmaceutical composition and application thereof

Abstract

The invention provides a bicyclic compound with a structure as shown in a formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a prodrug molecule thereof, and a pharmaceutical composition and application thereof. The compound provided by the invention can activate CD137, enhance the immune function of cytotoxic T lymphocytes (CTL), enhance the memory function of CD8 + T cells and CD4 + T cells, enable polarization of the CD4 + T cells to incline to a state beneficial to cellular immunity, and promote multimerization of the CD137, so that T cell immunity can be effectively activated, the volume of primary tumors can be effectively reduced, and the curative effect of the primary tumors can be improved. The compound can be used for inhibiting and treating tumors. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a bicyclic compound and a pharmaceutical composition and application thereof. Background Art

[0002] CD137 (also known as 4-1BB / TNFRSF9) is a membrane glycoprotein discovered in the late 1980s and belongs to a TNFR superfamily consisting of 28 other receptors, including CD134 (OX40 / TNFRSF4), CD40 (TNFRSF5), CD27 (TNFRSF7), CD270 (HVEM / TNFRSF14), and CD357 (GITR / TNFRSF18). CD137 is widely expressed on a variety of immune cells, including T cells, B cells, natural killer cells (NK), dendritic cells (DC), eosinophils, and mast cells. Therefore, CD137 has been considered an activation marker and has been effectively used to identify antigen-specific CD4 and CD8 T cells in both naive and memory cells. Like other members of the TNFR superfamily, CD137 initiates signaling by binding to its trimeric ligand (CD137L), which is present on all types of antigen-presenting cells. After binding to the ligand, CD137 undergoes trimerization and recruits TNF receptor-associated factors 1, 2, and 3 (TRAF1, TRAF2, and TRAF3) through two polyacidic TRAF binding consensus regions located in its cytoplasmic tail 234TTQEE238 and 246PEEEE250, thereby forming a 3CD137L:3CD137:3TRAF3 complex. Trimeric TRAF exists in different forms, namely TRAF1:(TRAF2)2, (TRAF2)3, and TRAF3:(TRAF2)2, thereby activating downstream ERK, MAPK, and NF-κB pathways.

[0003] For CD4 and CD8 T cells, activation of CD137 induces the production of interferon gamma (IFN-γ) and interleukin 2 (IL-2) as well as memory differentiation. Activation of CD137 can also enhance the cytotoxicity of NK cells and offset the inhibition of NK cells by TGFβ to exert anti-tumor function. In addition, the literature reports that activated CD137 can reprogram Tregs into cytotoxic CD4+ T cells with anti-tumor activity.

[0004] Therefore, targeting CD137 has the potential to treat cancer. Melero et al. first reported more than 20 years ago that CD137 monoclonal antibodies can inhibit tumor development by enhancing CD8+ T cell anti-tumor immunity. A large number of preclinical model studies have also confirmed the therapeutic effects of agonistic CD137 antibodies, including enhancing anti-cancer immunity and protecting mice from liver tumors, lymphomas, colon cancer, lung cancer, breast cancer, and melanoma.

[0005] Based on the advantages of small molecule drugs in terms of strong tissue penetration, low antigenicity to the human body, low manufacturing / transportation / storage costs, and convenient administration, while avoiding the toxic side effects of CD137 antibody drugs and achieving higher anti-tumor effects, it is necessary to develop small molecule CD137 agonists. Summary of the invention

[0006] Based on this, the object of the present invention is to provide a small molecule CD137 agonist, which can be used for anti-tumor.

[0007] In order to achieve the above object, the present invention includes the following technical solutions.

[0008] Use of a bicyclic compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof in the preparation of a CD137 agonist;

[0009]

[0010] Wherein, T is selected from: 0, 1, 2;

[0011] W, Y, and Z are independently selected from: O, S, N, NR 1 , CR 1 , Ring A containing W, Y, and Z is a heteroaromatic ring;

[0012] X1, X2, X3, X4, and X5 are independently selected from: N, CR 2 , Ring B including X1, X2, X3, X4, and X5 is an aromatic ring or a heteroaromatic ring;

[0013] R is selected from: R 3 Substituted or unsubstituted C1-C 12 Alkyl, R 3 Substituted or unsubstituted C3-C 12 Cycloalkyl, R 3 a substituted or unsubstituted 3-12 membered heterocyclic group, R 3 Substituted or unsubstituted C6-C 18 Aryl, R 3 a substituted or unsubstituted 5-18 membered heteroaryl group;

[0014] Each R1 Each independently selected from: hydrogen, C1-C6 alkyl;

[0015] Each R 2 are independently selected from: hydrogen, halogen, R 4 Substituted or unsubstituted C1-C6 alkyl, R 4 Substituted or unsubstituted C3-C6 cycloalkyl, R 4 Substituted or unsubstituted C1-C6 alkoxy, R 4 Substituted or unsubstituted C3-C6 cycloalkoxy, cyano, hydroxyl, amino, -C(=O)R 5 、-S(=O)R 5 、-S(=O)2R 5 、-P(=O)R 5 R 5 , tetrazolyl,

[0016] Each R 3 are independently selected from: hydrogen, halogen, R 4 Substituted or unsubstituted C1-C6 alkyl, R 4 Substituted or unsubstituted C3-C6 cycloalkyl, R 4 Substituted or unsubstituted C1-C6 alkoxy, R 4 Substituted or unsubstituted C3-C6 cycloalkoxy, cyano, hydroxy, amino, carboxyl, -C(=O)R 5 、-(CH2) m NR a R b 、-(CH2) m OCR a R b ;

[0017] Each m is independently selected from: 0, 1, 2 or 3;

[0018] Each R a and each R b are independently selected from: hydrogen, R 4 Substituted or unsubstituted C1-C6 alkyl, or R a , R b Together with the N or C to which it is connected, it forms R 4 a substituted or unsubstituted 3-12 membered heterocyclic group;

[0019] Each R 4Each of the following is independently selected from the group consisting of: deuterium, halogen, hydroxyl, amino, C1-C3 alkyl, C1-C3 alkoxy, -NH(C1-C3 alkyl), -N(C1-C3 alkyl)(C1-C3 alkyl), -C(=O)(C1-C3 alkyl), C1-C3 alkyl substituted with hydroxyl, 3-8 membered heterocyclyl, 3-8 membered heterocyclyl substituted with C1-C3 alkyl, and C1-C3 alkyl substituted with halogen;

[0020] Each R 5 Each is independently selected from the group consisting of: hydrogen, hydroxy, amino, C1-C6 alkoxy, and -NH-OH.

[0021] In some of these embodiments, T is 0.

[0022] In some embodiments, Ring A is selected from:

[0023]

[0024] In some embodiments, X1, X3, X4, and X5 are all CH, and X2 is CR 2 .

[0025] In some embodiments, X1, X2, X4, X5 are CH, X3 is CR 2 .

[0026] In some embodiments, each R 2 are independently selected from: hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, -C(=O)R 5 ; Each R 5 Each is independently selected from the group consisting of: hydrogen, hydroxy, amino, C1-C3 alkoxy, and -NH-OH.

[0027] In some embodiments, each R 2 Each is independently selected from the group consisting of hydrogen, carboxyl, methoxycarbonyl, and carbamoyl.

[0028] In some embodiments, each R 3 are independently selected from: hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, halogen-substituted C1-C3 alkyl,

[0029]

[0030] In some embodiments, each R 3 Each is independently selected from the group consisting of hydrogen, methyl, ethyl, fluorine, chlorine, bromine and methoxy.

[0031] In some embodiments, R is selected from: C1-C6 alkyl, R 3Substituted or unsubstituted C5-C 10 Cycloalkyl, R 3 a substituted or unsubstituted 5-10 membered heterocyclic group, R 3 Substituted or unsubstituted C6-C 10 Aryl, R 3 A substituted or unsubstituted 5-10 membered heteroaryl group.

[0032] In some embodiments, R is selected from: C1-C4 alkyl, R 3 Substituted or unsubstituted cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptanyl, adamantyl, spiro[2.5]octanyl, bicyclo[4.1.0]heptanyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, hexahydropyridinyl, tetrahydropyranyl, tetrahydronaphthyl, furanyl, thienyl, pyrrolyl, pyrazolyl, thiazolyl, phenyl, naphthyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, 1,3,5-triazinyl, benzofuranyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, benzopyrazolyl, quinolyl, isoquinolyl, quinoxalinyl, benzopyrimidinyl, pyridopyrimidinyl, pyridopyridinyl, purinyl, pteridinyl, imidazothiazolyl, imidazopyridazinyl, pyrazolopyrimidinyl.

[0033] In some embodiments, R is selected from: phenyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl,

[0034]

[0035] In some embodiments, R is selected from: 3 Substituted or unsubstituted C7-C 10 Spirobicycloalkyl, R 3 Substituted or unsubstituted 9-10 membered heteroaryl, R 3 Substituted or unsubstituted naphthalene-2-yl, R 3 Substituted or unsubstituted tetrahydronaphthyl; the 9-10 membered heteroaryl is a bicyclic heteroaryl.

[0036] In some embodiments, R is selected from: 3 Substituted or unsubstituted: spiro[2.5]octyl, naphth-2-yl, tetrahydronaphthyl, benzofuranyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, benzopyrazolyl, quinolyl, isoquinolyl, quinoxalinyl, benzopyrimidinyl, pyridopyrimidinyl, purinyl, pteridinyl, imidazothiazolyl, imidazopyridazinyl, pyrazolopyrimidinyl.

[0037] In some embodiments, R is selected from: 3Substituted or unsubstituted spiro[2.5]octyl.

[0038] In some embodiments, R is selected from: 3 A substituted or unsubstituted naphth-2-yl group.

[0039] In some embodiments, R is selected from: 3 A substituted or unsubstituted benzofuran-2-yl group.

[0040] In some embodiments, R is selected from: 3 Substituted or unsubstituted 1H-benzo[d]imidazol-2-yl.

[0041] In some embodiments, R is selected from: 3 A substituted or unsubstituted quinolin-3-yl group.

[0042] In some embodiments, R is selected from: 3 A substituted or unsubstituted quinoxalin-2-yl group.

[0043] In some embodiments, R is selected from: 3 Substituted or unsubstituted 1,8-naphthyridin-3-yl.

[0044] In some embodiments, R is selected from: 3 Substituted or unsubstituted 1,2,3,4-tetrahydro-2-naphthyl.

[0045] In some embodiments, R is selected from:

[0046]

[0047]

[0048] The invention discloses an application of the bicyclic compound or its pharmaceutically acceptable salt or its stereoisomer or its prodrug molecule in the preparation of an enhancer for enhancing the immune function of cytotoxic T lymphocytes.

[0049] The use of the bicyclic compound or its pharmaceutically acceptable salt or its stereoisomer or its prodrug molecule of the present invention in the preparation of an enhancer for enhancing the memory function of CD8+T cells and CD4+T cells.

[0050] The bicyclic compound or its pharmaceutically acceptable salt or its stereoisomer or its prodrug molecule of the present invention is used in the preparation of a promoter for promoting the polarization of CD4+T cells to a cellular immune state.

[0051] Use of the bicyclic compound or a pharmaceutically acceptable salt or a stereoisomer or a prodrug molecule thereof of the present invention in the preparation of an enhancer for promoting CD137 multimerization.

[0052] The use of the bicyclic compound or its pharmaceutically acceptable salt or its stereoisomer or its prodrug molecule in the preparation of drugs for preventing and / or treating tumors.

[0053] In some embodiments, the tumor is: liver cancer, lymphoma, colon cancer, lung cancer, breast cancer, melanoma, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, rectal cancer, cervical cancer, laryngeal cancer, nasopharyngeal cancer, pancreatic cancer, multiple myeloma, leukemia.

[0054] The present invention also provides a pharmaceutical composition for preventing and / or treating tumors, which is prepared from active ingredients and pharmaceutically acceptable carriers or excipients, wherein the active ingredients include the bicyclic compounds described in the present invention or pharmaceutically acceptable salts thereof or stereoisomers thereof or prodrug molecules thereof.

[0055] After long and in-depth research, the inventor of the present invention unexpectedly discovered for the first time that Ataluren (PTC124), a drug commercially available for the treatment of Duchenne muscular dystrophy and muscular atrophy, can activate CD137 and be used to treat tumors. The inventor further developed a series of new derivatives and found that these compounds can activate CD137, enhance the immune function of cytotoxic T lymphocytes (CTLs), enhance the memory function of CD8+T cells and CD4+T cells, and can tilt the polarization of CD4+T cells toward a state that is conducive to cellular immunity, and can promote the polymerization of CD137, thereby effectively activating T cell immunity, effectively reducing the size of primary tumors, and can be used to inhibit and treat tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The results of luciferase fluorescence activity determination of the tested compounds.

[0057] Figure 2 The effect of the test compound on the effector function of mouse T cells.

[0058] Figure 3 The effect of the test compound on memory cells.

[0059] Figure 4 is the effect of the test compound on CD4+T cell polarization.

[0060] Figure 5 is the effect of the test compound on CD137 multimerization.

[0061] Figure 6The test results show that the test compound inhibits the growth of mouse transplanted tumors. DETAILED DESCRIPTION

[0062] The technical scheme of the present invention is further illustrated by specific examples below. Those skilled in the art should understand that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0063] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0064] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0065] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0066] In the compounds described in the present invention, when any variable (such as R 4 , R 5 If a (, etc.) compound occurs more than once in any component, its definition at each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permitted so long as such combinations result in stable compounds. A line drawn from a substituent into the ring system indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, this means that such bonds are attached only to any suitable carbon atom of an adjacent ring. It is to be understood that one of ordinary skill in the art can select substituents and substitution patterns for the compounds of the invention to provide compounds that are chemically stable and that can be readily synthesized from readily available raw materials by techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it is to be understood that these groups may be on the same carbon atom or on different carbon atoms so long as the structure is stable.

[0067] The term "alkyl" as used herein is intended to include branched and straight chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight or branched chain. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl.

[0068] The term "cycloalkyl" used in the present invention refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic hydrocarbon group whose ring atoms are composed of carbon atoms, and the bicyclic or polycyclic rings include spirocyclic, condensed and bridged rings. For example, "cycloalkyl" includes but is not limited to the following groups: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, wait.

[0069] The term "alkoxy" used in the present invention refers to a group having an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0070] The term "heterocyclic group" as used in the present invention refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic cyclic substituent, wherein one or more ring atoms are selected from N, O or S(O)m (wherein m is an integer from 0 to 2) heteroatom, and the remaining ring atoms are carbon, and the bicyclic or polycyclic rings include spirocyclic, condensed and bridged rings. For example: morpholinyl, piperidinyl, tetrahydropyrrolyl, pyrrolidinyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, tetrahydrofuranyl, tetrahydrothienyl, etc., and N-oxides thereof. Attachment of the heterocyclic substituents may be via a carbon atom or via a heteroatom.

[0071] The term "heteroaryl" as used in the present invention refers to an aromatic ring containing one or more heteroatoms selected from O, N or S, which can be monocyclic, bicyclic or polycyclic, for example, including but not limited to: quinolyl, pyrazolyl, pyrrolyl, thienyl, furanyl, pyridyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, 1,3,5-triazinyl, benzofuranyl, benzothienyl, indolyl, indazolyl, benzimidazolyl, benzopyrazolyl, quinolyl, isoquinolyl, quinoxalinyl, benzopyrimidinyl, pyridopyrimidinyl, pyridopyridinyl, purinyl, pteridinyl, imidazothiazolyl, imidazopyridazinyl, pyrazolopyrimidinyl, etc.; "heteroaryl" is also understood to include any N-oxide derivatives of heteroaryl containing nitrogen. The connection of the heteroaryl can be achieved through a carbon atom or through a heteroatom.

[0072] As will be appreciated by those skilled in the art, "halo" or "halo" as used herein refers to chlorine, fluorine, bromine and iodine.

[0073] In one embodiment of the present invention, the present invention provides the use of a bicyclic compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof in the preparation of a CD137 agonist;

[0074]

[0075] Wherein, T is selected from: 0, 1, 2;

[0076] W, Y, and Z are independently selected from: O, S, N, NR 1 , CR 1 , Ring A containing W, Y, and Z is a heteroaromatic ring;

[0077] X1, X2, X3, X4, and X5 are independently selected from: N, CR 2 , Ring B including X1, X2, X3, X4, and X5 is an aromatic ring or a heteroaromatic ring;

[0078] R is selected from: R 3 Substituted or unsubstituted C1-C 12 Alkyl, R 3 Substituted or unsubstituted C3-C 12 Cycloalkyl, R 3 a substituted or unsubstituted 3-12 membered heterocyclic group, R 3 Substituted or unsubstituted C6-C 18 Aryl, R 3 a substituted or unsubstituted 5-18 membered heteroaryl group;

[0079] Each R 1 Each independently selected from: hydrogen, C1-C6 alkyl;

[0080] Each R 2 are independently selected from: hydrogen, halogen, R 4 Substituted or unsubstituted C1-C6 alkyl, R 4 Substituted or unsubstituted C3-C6 cycloalkyl, R 4 Substituted or unsubstituted C1-C6 alkoxy, R 4 Substituted or unsubstituted C3-C6 cycloalkoxy, cyano, hydroxyl, amino, -C(=O)R 5 、-S(=O)R 5 、-S(=O)2R 5 、-P(=O)R 5 R 5 , tetrazolyl,

[0081] Each R 3 are independently selected from: hydrogen, halogen, R 4 Substituted or unsubstituted C1-C6 alkyl, R 4 Substituted or unsubstituted C3-C6 cycloalkyl, R 4 Substituted or unsubstituted C1-C6 alkoxy, R 4 Substituted or unsubstituted C3-C6 cycloalkoxy, cyano, hydroxy, amino, carboxyl, -C(=O)R 5 、-(CH2) m NR a R b 、-(CH2) m OCR a R b ;

[0082] Each m is independently selected from: 0, 1, 2 or 3;

[0083] Each R a and each R b are independently selected from: hydrogen, R 4 Substituted or unsubstituted C1-C6 alkyl, or R a , R b Together with the N or C to which it is connected, it forms R 4 a substituted or unsubstituted 3-12 membered heterocyclic group;

[0084] Each R 4Each of the following is independently selected from the group consisting of: deuterium, halogen, hydroxyl, amino, C1-C3 alkyl, C1-C3 alkoxy, -NH(C1-C3 alkyl), -N(C1-C3 alkyl)(C1-C3 alkyl), -C(=O)(C1-C3 alkyl), C1-C3 alkyl substituted with hydroxyl, 3-8 membered heterocyclyl, 3-8 membered heterocyclyl substituted with C1-C3 alkyl, and C1-C3 alkyl substituted with halogen;

[0085] Each R 5 Each is independently selected from the group consisting of: hydrogen, hydroxy, amino, C1-C6 alkoxy, and -NH-OH.

[0086] The present invention includes the free form of the compound of formula (I), and also includes its pharmaceutically acceptable salts and stereoisomers. The term "free form" refers to a carboxylic acid or amine compound in a non-salt form. Pharmaceutically acceptable salts of the present invention can be synthesized from compounds of the present invention containing a basic part or an acidic part by conventional chemical methods. Typically, salts of basic compounds are prepared by ion exchange chromatography or by reacting a free base with a stoichiometric amount or excess of an inorganic or organic acid in the desired salt form in a suitable solvent or a combination of multiple solvents. Similarly, salts of acidic compounds are formed by reacting with a suitable inorganic or organic base.

[0087] Therefore, the pharmaceutically acceptable salts of the compounds of this invention include conventional non-toxic salts of the compounds of this invention formed by reacting alkaline compounds of this invention with inorganic or organic acids. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc., and also include salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxy-benzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, etc.

[0088] If the compound of the present invention is acidic, then suitable "pharmaceutically acceptable salts" refer to salts prepared by pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts, zinc salts, etc. Ammonium salts, calcium salts, magnesium salts, potassium salts and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic bases, including salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0089] Berg et al., "Pharmaceutical Salts," J. Pharm. Sci. '1977: 66: 1-19, describes in more detail the preparation of the above-mentioned pharmaceutically acceptable salts and other typical pharmaceutically acceptable salts.

[0090] In one embodiment, the present invention provides a method for treating tumors and other diseases in humans or other mammals using a compound having a structure represented by formula (I) and a pharmaceutically acceptable salt thereof. For example, the tumor may be liver cancer, lymphoma, colon cancer, lung cancer, breast cancer, melanoma, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, rectal cancer, cervical cancer, laryngeal cancer, nasopharyngeal cancer, pancreatic cancer, multiple myeloma, leukemia, etc.

[0091] The metabolites of the compounds and pharmaceutically acceptable salts thereof involved in the present invention, as well as prodrugs that can be converted into the structures of the compounds and pharmaceutically acceptable salts involved in the present application in vivo, are also included in the claims of the present invention.

[0092] The present invention also provides a pharmaceutical composition, which comprises an active ingredient within a safe and effective amount range, and a pharmaceutically acceptable carrier or excipient.

[0093] The "active ingredient" described in the present invention refers to the compound of formula I described in the present invention or its pharmaceutically acceptable salt or its stereoisomer or its prodrug molecule.

[0094] The "active ingredient" and pharmaceutical composition described in the present invention can be used as a CD137 agonist and can be used to prepare drugs for preventing and / or treating tumors, etc.

[0095] "Safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of active ingredient per dose, more preferably, 10-200 mg of active ingredient per dose. Preferably, the "one dose" is one tablet.

[0096] "Pharmaceutically acceptable carrier or excipient" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0097] "Compatibility" herein means that the components in the composition can be blended with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient.

[0098] Examples of pharmaceutically acceptable carriers or excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0099] In another preferred embodiment, the compound of formula I of the present invention can form a complex with a macromolecular compound or a polymer through a non-bonding reaction. In another preferred embodiment, the compound of formula I of the present invention as a small molecule can also be connected to a macromolecular compound or a polymer through a chemical bond. The macromolecular compound can be a biological macromolecule such as a high polysaccharide, protein, nucleic acid, polypeptide, etc.

[0100] There is no particular limitation on the administration of the active ingredient or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0101] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.

[0102] In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients:

[0103] (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;

[0104] (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic;

[0105] (c) humectants, for example, glycerin;

[0106] (d) disintegrating agents, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0107] (e) a buffering solvent, such as paraffin;

[0108] (f) absorption accelerators, for example, quaternary ammonium compounds;

[0109] (g) wetting agents, such as cetyl alcohol and glyceryl monostearate;

[0110] (h) adsorbents, for example, kaolin; and

[0111] (i) Lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffering agent.

[0112] The solid dosage forms can also be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They can contain opacifying agents, and the release of the active ingredient in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes.

[0113] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices.

[0114] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0115] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0116] The compounds of the present invention may be administered alone or in combination with other therapeutic agents.

[0117] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0118] The compound of formula I can be used in combination with other drugs known to treat or improve similar conditions. When administered in combination, the administration method and dosage of the original drug remain unchanged, and the compound of formula I is taken simultaneously or subsequently. When the compound of formula I is taken simultaneously with one or more other drugs, it is preferred to use a pharmaceutical composition containing one or more known drugs and the compound of formula I. Drug combination also includes taking the compound of formula I and one or more other known drugs in overlapping time periods. When the compound of formula I is used in combination with one or more other drugs, the dosage of the compound of formula I or the known drug may be lower than when they are used alone.

[0119] Drugs or active ingredients that can be used in combination with the compounds of formula I include but are not limited to:

[0120] Estrogen receptor modulators, androgen receptor modulators, retinal receptor modulators, cytotoxins / cytostatics, antiproliferative agents, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducers, cytotoxic drugs, tyrosine protein inhibitors, EGFR inhibitors, VEGFR inhibitors, serine / threonine protein inhibitors, Bcr-Abl inhibitors, c-Kit inhibitors, Met inhibitors, Raf inhibitors, MEK inhibitors, MMP inhibitors, topoisomerase inhibitors, histidine deacetylase inhibitors, proteasome inhibitors, CDK inhibitors, Bcl-2 family protein inhibitors, MDM2 family protein inhibitors, IAP family protein inhibitors, STAT family protein inhibitors, PI3K inhibitors, AKT inhibitors, integrin blockers, interferon-α, interleukin-12, COX-2 inhibitors, p53, p53 activators, VEGF antibodies, EGF antibodies, JAK inhibitors, etc.

[0121] In one embodiment, the drugs or active ingredients that can be used in combination with the compound of formula I include but are not limited to: aldesleukin, alendronic acid, interferon, atrenoin, allopurinol, allopurinol sodium, palonosetron hydrochloride, hexamethylmelamine, aminoglutethimide, amifostine, amrubicin, anacrine, anastrozole, dolasetron, aranesp, arglabin, arsenic trioxide, arnosin, 5-azacytidine, azathioprine, bacillus Calmette-Guérin or tice bacillus Calmette-Guérin, betadine, betamethasone acetate, betamethasone sodium phosphate preparation, bexarotene, bleomycin sulfate, bromourea, bortezomib, busulfan, calcitonin, alezosumab injection, capecitabine, carboplatin, casodex, cefone , cisplatin, cladribine, cladribine, clodrophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin liposome, dexamethasone, dexamethasone phosphate, estradiol valerate, denileukin 2, depomet, deslorelin, delazosen, diethylstilbestrol, diflucan, docetaxel, doxorubicin, dronabinol, chin-166-chitosan complex, eligard, rasburicase, epirubicin hydrochloride, aprepitant, epirubicin, epoetin alfa, erythropoietin, epoetin, levamisole tablets, estradiol preparations, 17-β-estradiol, estramustine sodium phosphate, ethinyl estradiol, amifostine, hydroxyphosphoric acid, vanbifu, etoposide, fadrozole, tamoxifen Oxifen preparations, filgrastim, finasteride, firaprestin, floxuridine, fluconazole, fludarabine, 5-fluorodeoxyuridine nucleoside monophosphate, 5-fluorouracil, fluoxymesterone, flutamide, formestan, 1-β-D-arabinofuranosylcytosine-5'-stearoyl phosphate, fotemustine, fulvestrant, gamma globulin, gemcitabine, gemtuzumab, imatinib mesylate, carmustine rice paper capsules, goserelin, granisiron hydrochloride, histrelin, and mexin, hydrocortisone, erythro-hydroxynonyl adenine, hydroxyurea, tentan isobetamin, idarubicin, ifosfamide, interferon α, interferon-α2, interferon α-2A, interferon α-2B, interferon α-nl, interferon α-n3, interferon β , interferon gamma-la, interleukin-2, intron A, Iressa, irinotecan, Kateri, lentinan sulfate, letrozole, leucovorin, leuprorelin acetate, levomizole, levofolinate calcium salt, levothyroxine sodium, levothyroxine sodium preparations, lomustine, lonidamine, dronabinol, nitrogen mustard, methylcobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogen, 6-mercaptopurine, mesna, methotrexate, methyl aminolevulinate, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, trilostane, doxorubicin citrate liposome, nedaplatin, pegfilgrastim, oprelleukin, neupogen, nilutamide, tamoxifen,NSC-631570, recombinant human interleukin 1-β, octreotide, ondansetron hydrochloride, dehydrocortisone oral solution, oxaliplatin, paclitaxel, prednisone sodium phosphate preparation, pegaspargase, PEGASYS, pentostatin, lysin, pilocarpine hydrochloride, pilocarpine, plicamycin, porfimer sodium, prednimustine, stiprenisolone, prednisone, premarin, procarbazine, recombinant human erythropoietin, raltitrexed, libi, etidronate rhenium-186, rituximab, doxycycline-A, romotide, pilocarpine hydrochloride tablets, octreotide, sarmustine, semustine, sizoran, sobuzosine, sodium methylprednisolone, pafosic acid, stem cell therapy, streptozocin, strontium-89 chloride, levothyroxine sodium, tamoxifen Fen, tamsulosin, tasofenamin, tastolactone, taxotere, tesifazines, temozolomide, teniposide, testosterone propionate, methyltestosterone, thioguanine, thiotepa, thyroid stimulating hormone, tiludronic acid, topotecan, toremifene, tositumomab, trastuzumab, treosulfan, retinoic acid, methotrexate tablets, trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin pamoate, eufodine, uridine, valrubicin, vesirinone, vinblastine, vincristine, vinamide, vinorelbine, virulizine, dexrazoxane, statins ester, zofran, paclitaxel protein stabilized preparation, acolbifene, interferon r-lb, affinitak, aminopterin, arzoxifene, as oprisnil, atamestane, atrasentan, BAY43-9006, Avastin, CCI-779, CDC-501, Celebrex, cetuximab, clenatol, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin, eflornithine, exitecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implant, holmium-166DOTMP, ibandronic acid, interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, libra, lonafamib, miproxifene, minocycline, MS-2 09. Liposomal MTP-PE, MX-6, nafarelin, nemorubicin, nevastatin, noratript, oblimersen, onco-TCS, osidem, paclitaxel polyglutamate, sodium bromoglutamate, PN-401, QS-21, quasi-sea, R-1549, raloxifene, ranpirnase, 13-cis retinoic acid, satraplatin, ciocalcitol, T-138067, tarceva, paclitaxel docosahexaenoic acid, thymosin αl, gazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, trans-MID-lo7R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100 and zolelinic acid or a combination thereof.

[0122] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0123] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0124] The starting materials in the following examples can be obtained from commercial sources, or prepared by methods known in the art, or prepared according to the methods described herein.

[0125] Example 1 Synthesis of 3-(5-(naphthalen-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-25, JNU-0921)

[0126] a Reaction conditions: (a) hydroxylamine (50% wt aqueous solution), ethanol, reflux, 2h; (b) 2-naphthoic acid, EDCI, HOBt, DMF, 120-140℃, 2h; (c) 2NNaOH (aq), methanol, room temperature, 2h.

[0127] Step 1: Synthesis of intermediate (4)

[0128] Methyl 3-cyanobenzoate (3,15 g, 93 mmol) was dissolved in 150 ml of ethanol, and then 10 ml of hydroxylamine aqueous solution (50% wt. in water) was added. The reaction was refluxed for 2 hours. After the reaction was completed, the solvent in the reaction system was decompressed and dried to obtain intermediate 4. The intermediate did not need to be purified and was directly used for the next step reaction. 1 H NMR (400MHz, DMSO-d6) δ9.77(s,1H),8.29(t,J=1.5Hz,1H),8.03-7.85(m,2H),7.53(t,J=7.8Hz,1H),5.94(s,2H),3.87(s,3H).

[0129] Step 2: Synthesis of methyl 3-(5-(naphthalen-2-yl)-1,2,4-oxadiazol-3-yl)benzoate (5)

[0130] 2-Naphthoic acid (93 mmol) was dissolved in 20 ml of DMF, and then EDCI (21.4 g, 111.7 mmol) and HOBt (15.1 g, 111.7 mmol) were added to the system. After stirring at room temperature for 10 minutes, intermediate 4 (93 mmol) was added to the reaction system, and the temperature was raised to 120-140°C and stirred for 2 hours until the reaction was completed. The reaction system was cooled, and water was added to the reaction system until no solid precipitated. Intermediate 5 was obtained by suction filtration. Intermediate 5 did not need to be purified and was directly used for the next step reaction. 1 H NMR(500MHz,DMSO-d6)δ8.92(s,1H),8.67(s,1H),8.40(d,J=7.8Hz,1H),8.22(dt,J=13.5,8.2H z, 4H), 8.08 (d, J = 8.0Hz, 1H), 7.79 (t, J = 7.7Hz, 1H), 7.71 (dt, J = 13.8, 6.7Hz, 2H), 3.94 (s, 3H).

[0131] Step 3: Synthesis of 3-(5-(naphthalen-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-25, JNU-0921)

[0132] Intermediate 5 (6.6 g, 20 mmol) was dissolved in 50 mL of methanol, and then 20 mL of 2N NaOH (aq) aqueous solution was added to the reaction system. The reaction system was vigorously stirred at room temperature for 2 hours. After the reaction was complete, most of the organic solvent was dried, and 4N HCl solution was added dropwise until no solid precipitated. The final product 2-25 was obtained by suction filtration as a white solid. 1 H NMR(500MHz,DMSO-d6)δ13.40(s,1H),8.90(s,1H),8.67(t,J=1.4Hz,1H),8.38-8.31(m, 1H),8.25-8.14(m,4H),8.06(d,J=8.0Hz,1H),7.75(t,J=7.7Hz,1H),7.73-7.64(m,2H). 13 C NMR(126MHz,DMSO-d6)δ176.25,168.21,167.03,135.30,132.81,132.67,132.33,131.49,130.27, 129.81,129.75,129.64,129.35,128.35,128.29,127.90,127.02,123.97,120.93.HRMS(ESI)calcd for C 19 H 13 N2O3[M+H]+ 317.0921; found 317.0912.

[0133] Example 2 Synthesis of 3-(5-phenyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-1)

[0134]

[0135] The synthesis method is the same as that of Example 1, except that benzoic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0136] 1 H NMR (400MHz, DMSO-d6) δ13.38 (s, 1H), 8.68-8.63 (m, 1H), 8.34 (d, J = 7.8Hz, 1H), 8.28 -8.20(m,2H),8.19-8.12(m,1H),7.75(dd,J=10.5,4.7Hz,2H),7.68(t,J=7.4Hz,2H).

[0137] Example 3 Synthesis of 4-(5-phenyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-2)

[0138]

[0139] The synthesis method is the same as that of Example 1, except that benzoic acid is used instead of 2-naphthoic acid, and methyl 4-cyanobenzoate is used instead of methyl 3-cyanobenzoate to participate in the reaction.

[0140] 1 H NMR (400MHz, DMSO-d6) δ13.30 (s, 1H), 8.29-8.18 (m, 4H), 8.15 (d, J = 8.3Hz, 2H), 7.75 (d, J = 7.3Hz, 1H), 7.68 (t, J = 7.5Hz, 2H).

[0141] Example 4 Synthesis of 3-(5-phenyl-1,2,4-oxadiazol-3-yl)benzoic acid methyl ester (2-3)

[0142]

[0143] The synthesis method is the same as step 2 of Example 1, except that benzoic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0144] 1H NMR (400MHz, DMSO-d6) δ8.64(s,1H),8.37(d,J=7.8Hz,1H),8.29-8.13(m,3H),7.77(dd,J=16.3,7.8Hz,2H),7.68(t,J=7.5Hz,2H),3.93(s,3H).

[0145] Example 5 Synthesis of 3-(5-phenyl-1,2,4-oxadiazol-3-yl)benzamide (2-4)

[0146]

[0147] The synthesis method is the same as step 2 of Example 1, except that benzoic acid is used instead of 2-naphthoic acid, and 3-cyanobenzamide is used instead of methyl 3-cyanobenzoate to participate in the reaction.

[0148] 1 H NMR (400MHz, DMSO-d6) δ8.60 (s, 1H), 8.34-8.16 (m, 4H), 8.11 (d, J = 7.8Hz, 1H), 7.82-7.62 (m, 4H), 7.56 (s, 1H).

[0149] Example 6 Synthesis of 3-(5-phenyl-1,3,4-oxadiazol-2-yl)benzoic acid (2-5)

[0150]

[0151] Benzoyl hydrazide (1.4g, 10mmol) and monomethyl m-dibenzoate (1.8g, 10mmol) were added to 20ml of phosphorus oxychloride, and the reaction system was heated to 120 degrees Celsius under nitrogen protection for 18 hours. After cooling to room temperature, the reaction solution was poured into 500ml of ice water. The reaction solution was adjusted to a pH value of about 8-9 with solid sodium bicarbonate, and the precipitate was precipitated and filtered. The solid obtained by filtration was dissolved in 100ml of methanol, and then 10ml of 2N NaOH (aq) aqueous solution was added to the system. The reaction system was vigorously stirred at room temperature for 2 hours. When the reaction was complete, most of the organic solvent was dried, and 4N HCl solution was added dropwise until the solid no longer precipitated. The target product was obtained by filtration.

[0152] 1 H NMR (400MHz, DMSO-d6) δ13.44(s,1H),8.62(s,1H),8.38(d,J=7.6Hz,1H),8.23-8.09(m,3H),7.79(t,J=7.8Hz,1H),7.70-7.59(m,3H).

[0153] Example 7 Synthesis of 3-(3-phenyl-1,2,4-oxadiazol-5-yl)benzoic acid (2-6)

[0154]

[0155] The synthesis method is the same as that of Example 1, except that monomethyl isophthalate is used instead of 2-naphthoic acid, and benzonitrile is used instead of m-cyanobenzoate to participate in the reaction.

[0156] 1 H NMR (400MHz, DMSO-d6) δ13.50 (s, 1H), 8.68 (s, 1H), 8.42 (d, J = 7.7Hz, 1H), 8.26 ( d,J=7.8Hz,1H),8.12(d,J=7.7Hz,2H),7.81(t,J=7.8Hz,1H),7.68-7.57(m,3H).

[0157] Example 8 Synthesis of 3-(5-tert-butyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-7)

[0158]

[0159] The synthesis method is the same as that of Example 1, except that pivalic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0160] 1 H NMR (400MHz, DMSO-d6) δ13.34(s,1H),8.54(t,J=1.5Hz,1H),8.23(d,J=7.8Hz,1H),8.17-8.10(m,1H),7.70(t,J=7.8Hz,1H),1.46(s,9H).

[0161] Example 9 Synthesis of 3-(5-cyclopentyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-8)

[0162]

[0163] The synthesis method is the same as that of Example 1, except that cyclopentylcarboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0164] 1H NMR (600MHz, DMSO-d6) δ13.33(s,1H),8.53(s,1H),8.23(d,J=7.8Hz,1H),8.13(d,J=7.8Hz,1H),7.70(t,J=7.8Hz,1H) ,3.55-3.47(m,1H),2.15(dt,J=12.3,7.5Hz,2H),1.91(td,J=14.9,7.6Hz,2H),1.81-1.73(m,2H),1.74-1.62(m,2H).

[0165] Example 10 Synthesis of 3-(5-cyclohexyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-9)

[0166]

[0167] The synthesis method is the same as that of Example 1, except that cyclohexylcarboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0168] 1 H NMR (400MHz, DMSO-d6) δ13.33(s,1H),8.54(d,J=1.6Hz,1H),8.23(d,J=7.8Hz,1H),8.13(d,J=7.8Hz,1H),7.70(t,J=7.8Hz,1H ),3.13(tt,J=10.9,3.6Hz,1H),2.18-1.98(m,2H),1.85-1.71(m,2H),1.70-1.54(m,3H),1.50-1.35(m,2H),1.35-1.20(m,1H).

[0169] Example 11 Synthesis of (5-(bicyclo[2.2.1]heptane-2-yl)-1,2,4-oxadiazole-3-yl)benzoic acid (2-10)

[0170]

[0171] The synthesis method is the same as that of Example 1, except that bicyclo[2.2.1]heptane-2-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0172] 1H NMR (400MHz, DMSO-d6) δ13.95-12.11(m,1H),8.53(d,J=5.1Hz,1H),8.23(t,J=7.0 Hz,1H),8.13(dd,J=7.8,1.2Hz,1H),7.70(td,J=7.8,3.6Hz,1H),3.56-3.50(m,1H ),3.14(dd,J=9.0,5.0Hz,1H),2.72-2.56(m,1H),2.42-2.32(m,1H),2.14-1.93(m ,1H),1.87-1.72(m,1H),1.60-1.48(m,2H),1.45-1.37(m,1H),1.29-1.03(m,2H).

[0173] Example 12 Synthesis of 3-(5-adamantyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-11)

[0174]

[0175] The synthesis method is the same as that of Example 1, except that adamantanecarboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0176] 1 H NMR (400MHz, DMSO-d6) δ13.28(s,1H),8.54(s,1H),8.23(d,J=8.0Hz,1H),8.13(d,J=7.8Hz,1H),7.70(t,J=7.8Hz,1H),2.09(s,9H),1.77(s,6H).

[0177] Example 13 Synthesis of 3-(5-(spiro[2.5]octane-6-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-12)

[0178]

[0179] The synthesis method is the same as that of Example 1, except that spiro[2.5]octane-6-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0180] 1H NMR (400MHz, DMSO-d6) δ13.75-12.95(m,1H),8.55(s,1H),8.24(d,J=7.8Hz,1H),8.13(d,J=7.8Hz,1H),7.71(t,J= 7.8Hz,1H),3.23-3.12(m,1H),2.09(t,J=8.7Hz,2H),1.85-1.71(m,4H),1.07(d,J=5.9Hz,2H),0.39-0.20(m,4H).

[0181] Example 14 Synthesis of 3-(5-(bicyclo[4.1.0]heptane-7-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-13)

[0182]

[0183] The synthesis method is the same as that of Example 1, except that bicyclo[4.1.0]heptane-7-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0184] 1 H NMR (400MHz, DMSO-d6) δ13.93-12.81(m,1H),8.49(t,J=1.5Hz,1H),8.25-8.14(m,1H),8.15-8.05(m,1H), 7.68(t,J=7.8Hz,1H),2.24(t,J=4.4Hz,1H),2.03-1.88(m,2H),1.89-1.70(m,4H),1.29(t,J=3.0Hz,4H).

[0185] Example 15 Synthesis of 3-(5-(thiazol-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-14)

[0186]

[0187] The synthesis method is the same as that of Example 1, except that thiazole-2-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0188] 1 H NMR (400MHz, DMSO-d6) δ13.41(s,1H),8.62(t,J=1.5Hz,1H),8.42-8.26(m,3H),8.23-8.15(m,1H),7.76(t,J=7.8Hz,1H).

[0189] Example 16 Synthesis of 3-(5-(5-methyl-1H-pyrazol-3-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-15)

[0190]

[0191] The synthesis method is the same as that of Example 1, except that 5-methyl-1H-pyrazole-3-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0192] 1 H NMR(400MHz,DMSO-d6)δ13.56(s,1H),13.36(s,1H),8.62(t,J=1.5Hz,1H),8.42- 8.26(m,1H),8.23-8.12(m,1H),7.74(t,J=7.8Hz,1H),6.84(s,1H),2.35(s,3H).

[0193] Example 17 Synthesis of 3-(5-(pyridin-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-16)

[0194]

[0195] The synthesis method is the same as that of Example 1, except that pyridine-2-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0196] 1 H NMR (400MHz, DMSO-d6) δ13.37(s,1H),8.87(d,J=4.1Hz,1H),8.66(t,J=1.5Hz ,1H),8.36(ddd,J=8.8,7.8,4.6Hz,2H),8.22-8.10(m,2H),7.81-7.71(m,2H).

[0197] Example 18 Synthesis of 3-(5-(pyridin-3-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-17)

[0198]

[0199] The synthesis method is the same as that of Example 1, except that pyridine-3-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0200] 1 H NMR (400MHz, DMSO-d6) δ9.37(d,J=1.7Hz,1H),8.90(dd,J=4.8,1.6Hz,1H),8.65(s,1H),8. 59(dt,J=8.0,1.9Hz,1H),8.24(d,J=7.5Hz,1H),8.15(d,J=7.7Hz,1H),7.76-7.60(m,2H).

[0201] Example 19 Synthesis of 3-(5-(pyridin-4-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-18)

[0202]

[0203] The synthesis method is the same as that of Example 1, except that pyridine-4-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0204] 1 H NMR(400MHz, DMSO-d6)δ13.40(s,1H),8.92(dd,J=4.4,1.6Hz,2H),8.65(t,J=1.6Hz,1H), 8.38-8.31(m,1H),8.23-8.17(m,1H),8.14(dd,J=4.4,1.7Hz,2H),7.77(t,J=7.8Hz,1H).

[0205] Example 20 Synthesis of 3-(5-(pyrimidin-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-19)

[0206]

[0207] The synthesis method is the same as that of Example 1, except that pyrimidine-2-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0208] 1 H NMR (400MHz, DMSO-d6) δ13.38(s,1H),9.14(d,J=4.9Hz,2H),8.66(t,J=1.5Hz,1H), 8.40-8.30(m,1H),8.21-8.14(m,1H),7.84(t,J=4.9Hz,1H),7.76(t,J=7.8Hz,1H).

[0209] Example 21 Synthesis of 3-(5-(3-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-20)

[0210]

[0211] The synthesis method is the same as that of Example 1, except that m-fluorobenzoic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0212] 1H NMR (400MHz, DMSO-d6) δ13.37(s,1H),8.64(t,J=1.5Hz,1H),8.40-8.26(m,1H),8.23-8.13(m,1 H),8.09-8.04(m,1H),8.04-7.89(m,1H),7.74(ddd,J=9.5,7.9,4.5Hz,2H),7.68-7.48(m,1H).

[0213] Example 22 Synthesis of 3-(5-(4-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-21)

[0214]

[0215] The synthesis method is the same as that of Example 1, except that p-fluorobenzoic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0216] 1 H NMR (400MHz, DMSO-d6) δ13.41(s,1H),8.64(t,J=1.5Hz,1H),8.38-8.24(m,3H),8.21-8.13(m,1H),7.75(t,J=7.8Hz,1H),7.57-7.47(m,2H).

[0217] Example 23 Synthesis of 3-(5-(Benzofuran-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-22)

[0218]

[0219] The synthesis method is the same as that of Example 1, except that benzofuran-2-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0220] 1 H NMR (400MHz, DMSO-d6) δ13.39 (s, 1H), 8.65 (t, J = 1.5Hz, 1H), 8.39-8.31 (m, 1H), 8.24-8.1 3(m,2H),7.94-7.81(m,2H),7.76(t,J=7.8Hz,1H),7.64-7.54(m,1H),7.49-7.40(m,1H).

[0221] Example 24 Synthesis of 3-(5-(1H-benzo[d]imidazol-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-23)

[0222]

[0223] The synthesis method is the same as that of Example 1, except that 1H-benzo[d]imidazole-2-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0224] 1 H NMR (400MHz, DMSO-d6) δ14.14(s,1H),13.41(s,1H),8.74(t,J=1.5Hz,1H),8.40-8.34(m,1H),8.23-8.17 (m,1H),7.87(d,J=7.2Hz,1H),7.79(t,J=7.8Hz,1H),7.66(d,J=7.5Hz,1H),7.41(dd,J=20.7,7.0Hz,2H).

[0225] Example 25 Synthesis of 3-(5-(naphthyl-1-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-24)

[0226]

[0227] The synthesis method is the same as that of Example 1, except that 1-naphthoic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0228] 1 H NMR (400MHz, DMSO-d6) δ13.69-13.23(m,1H),9.14(d,J=8.6Hz,1H),8.71(t,J=1.5Hz,1H),8.47(dd,J=7.3,1.2 Hz,1H),8.45-8.39(m,1H),8.35(d,J=8.2Hz,1H),8.25-8.17(m,1H),8.15(d,J=8.1Hz,1H),7.94-7.62(m,4H).

[0229] Example 26 Synthesis of 3-(5-(quinolin-3-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-26)

[0230]

[0231] The synthesis method is the same as that of Example 1, except that quinoline-3-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0232] 1H NMR (400MHz, DMSO-d6) δ9.60 (s, 1H), 9.37 (s, 1H), 8.71 (s, 1H), 8.40 (d, J = 7.6Hz, 1H), 8.3 2(d,J=7.6Hz,1H),8.19(t,J=8.5Hz,2H),8.03-7.96(m,1H),7.79(dd,J=12.5,7.6Hz,2H).

[0233] Example 27 Synthesis of 3-(5-(quinoxaline-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-27)

[0234]

[0235] The synthesis method is the same as that of Example 1, except that 2-quinoxalinecarboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0236] 1 H NMR(400MHz,DMSO-d6)δ9.77(s,1H),8.72(s,1H),8.41(d,J=7.8Hz,1H),8.39-8.31(m,1H ),8.27(d,J=7.3Hz,1H),8.21(d,J=7.7Hz,1H),8.12-8.01(m,2H),7.79(t,J=7.7Hz,1H).

[0237] Example 28 Synthesis of 3-(5-(1,8-naphthyridin-3-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-28)

[0238]

[0239] The synthesis method is the same as that of Example 1, except that 1,8-naphthyridine-3-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0240] 1 H NMR (400MHz, DMSO-d6) δ13.40(s,1H),9.74(d,J=2.2Hz,1H),9.44(d,J=2.3Hz,1H),9.25(d,J=2.2Hz,1H), 8.77(dd,J=8.1,1.5Hz,1H),8.68(s,1H),8.36(d,J=7.8Hz,1H),8.18(d,J=7.7Hz,1H),7.88-7.70(m,2H).

[0241] Example 29 Synthesis of 3-(5-(1-bromonaphthyl-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-29)

[0242]

[0243] The synthesis method is the same as that of Example 1, except that 1-bromonaphthyl-2-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0244] 1 H NMR (400MHz, DMSO-d6) δ13.38(s,1H),8.68(s,1H),8.45(d,J=8.3Hz,1H),8.38(d,J=7.7Hz,1H) ,8.24(d,J=8.6Hz,1H),8.18(dd,J=13.7,7.9Hz,2H),8.11(d,J=8.5Hz,1H),7.91-7.71(m,3H).

[0245] Example 30 Synthesis of 3-(5-(5-bromonaphthyl-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-30)

[0246]

[0247] The synthesis method is the same as that of Example 1, except that 5-bromonaphthyl-2-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0248] 1 H NMR (400MHz, DMSO-d6) δ13.33(s,1H),8.99(s,1H),8.68(t,J=1.5Hz,1H),8.43-8.26(m,4 H),8.21-8.14(m,1H),8.07(d,J=7.5Hz,1H),7.76(t,J=7.8Hz,1H),7.60(t,J=8.0Hz,1H).

[0249] Example 31 Synthesis of 3-(5-(7-bromonaphthyl-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-31)

[0250]

[0251] The synthesis method is the same as that of Example 1, except that 7-bromonaphthyl-2-carboxylic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0252] 1 H NMR(400MHz,DMSO-d6)δ13.35(s,1H),9.00-8.80(m,1H),8.78-8.60(m,1H), 8.58-8.48(m,1H),8.45-8.11(m,4H),8.11-7.94(m,1H),7.89-7.61(m,2H).

[0253] Example 32 Synthesis of 3-(5-naphthylethyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-32)

[0254]

[0255] The synthesis method is the same as that of Example 1, except that 2-naphthylacetic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0256] 1 H NMR (400MHz, DMSO-d6) δ13.46(s,1H),8.52(s,1H),8.22(d,J=7.8Hz,1H),8.13(d,J= 7.8Hz,1H),7.96-7.90(m,4H),7.69(t,J=7.8Hz,1H),7.58-7.49(m,3H),4.64(s,2H).

[0257] Example 33 Synthesis of 3-(5-(1,2,3,4-tetrahydro-2-naphthyl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-33)

[0258]

[0259] The synthesis method is the same as that of Example 1, except that 1,2,3,4-tetrahydro-2-naphthoic acid is used instead of 2-naphthoic acid to participate in the reaction.

[0260] 1 H NMR (400MHz, DMSO-d6) δ13.28(s,1H),8.56(t,J=1.5Hz,1H),8.25(dd,J=7.8,1.2Hz,1H),8.19-8.11(m,1H),7.71(t,J=7.8 Hz,1H),7.27-7.04(m,4H),3.65-3.56(m,1H),3.26-3.15(m,2H),3.00-2.86(m,2H),2.43-2.35(m,1H),2.10-1.97(m,1H).

[0261] Example 34 Activity Determination of Compounds

[0262] Construction of cell line: IL-2 promoter-driven Luciferase gene was transferred into human Jurkat T cells (named J-luc cells), and CD137 was overexpressed on this basis, and the cells were named Jurkat-CD137-luciferase (JC-luc); CRISPR / Cas9 was used to knock out endogenous CD137 in J-luc cells to obtain J-luc-sgCD137 cells. CD137 is activated by binding to its ligand CD137L or agonist antibody, and promotes the expression of IL-2 by activating the downstream signal NF-κB. Therefore, the activation ability of CD137 can be characterized by measuring the activity of Luciferase.

[0263] Determination of luciferase activity: JC-luc and J-luc-sgCD137 cells were placed in 96-well plates, and 1 μg / mL CD3 antibody stimulation and 5 μg / mL CD137 agonist antibody (aCD137) or test compounds were added for 6 hours. Luciferase activity was detected.

[0264] EC 50 Determination of EC value: Different concentrations of the tested drugs were placed in 96-well plates with JC-luc cells, and 1 μg / mL CD3 antibody was added for stimulation for 6 hours. Luciferase activity was detected and its EC value was calculated by Graphpadprism 50 .

[0265] Experimental results and analysis: The results of the Luciferase experiment showed that (1) aCD137 antibody could significantly activate JC-luc cells, but had no effect on J-luc-sgCD137 cells ( Figure 1 A in the figure, which proves that the constructed test model is feasible. (2) Most of the tested compounds have the function of significantly activating CD137 ( Figure 1 Compound 2-25 (JNU-0921) has the best activity in activating CD137, and its EC 50 The value is 64.07nM ( Figure 1 In the figure, AT refers to Ataluren (PTC124), and its structural formula is:

[0266] Example 35 Effect of Compounds on Mouse T Cell Effector Function

[0267] (A) Detect the effect of the test compound on the division and proliferation of mouse CD8+T cells: Wild-type (WT) mice and CD137 knockout (CD137KO) mice were killed by cervical dislocation, immersed in 75% ethanol for 5 minutes, and the mice were taken out. The left abdomen of the mice was cut open with sterile surgical scissors and surgical forceps, and the spleen was taken out, ground, and filtered. This process needs to be sterile. The ground single cell suspension was transferred to a 15mL tube, 3-5 times the volume of red blood cell lysis solution was added for 5 minutes, centrifuged, and the supernatant was discarded. Resuspend with 1mL PBS, filter into a new 15mL tube, centrifuge, discard the supernatant, add culture medium to resuspend into a single cell suspension, count, and take 1×10 7 The cells were stained with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) and stained in a 37°C incubator for 10 minutes, then placed in 4°C for 5 minutes, and then added with complete medium to terminate the culture. The cells were centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, PBS was added for resuspending, and the cells were centrifuged again, the supernatant was discarded, and the cells were counted. 5 The stained cells were inoculated in a 96-well plate containing 1 μg / mL CD3 antibody, and aCD137 antibody (positive control) or 2 μM compound 2-25 (JNU-0921) was added for treatment. After 24 hours, the cells were collected and stained with CD8 and CD4 flow cytometry antibodies, and finally the fluorescence intensity of CFSE in CD8 and CD4 positive T cells was detected by flow cytometry. The fluorescence was diluted, indicating that the cells had divided.

[0268] (B) Detection of the effect of the test compound on mouse CD8+ T cell activation: single cell suspensions of spleens from WT mice and CD137KO mice were prepared according to the above method. 5 The cells were placed in a 96-well plate containing 1 μg / mL CD3 antibody, and 5 μg / mL aCD137 and 2 μM test compound JNU-0921 were added for treatment. After 24 hours, the cells were collected and the expression of CD25 and CD69 on the surface of CD8+ and CD4+ T cells was detected by flow cytometry.

[0269] (C) Detection of the effect of the test compound on the killing function of CD8+ T cells in mice: single cell suspensions of spleens from WT and CD137KO mice were prepared according to the above method. 1×10 7The cells were placed in a 10 cm dish, 1 μg / mL CD3 / CD28 antibody, 55 μM mercaptoethanol and 10 ng / mL IL-2 were added to the culture medium, and induced in a 37°C 5% CO2 cell culture incubator for 3 days. Generally, after 3 days of induction, they are all mature CTLs and can be used for killing experiments. The mature CTLs induced for three days were used as effector cells, and the P815-aCD3 cells expressing CD3 antibodies were used as target cells. First, the P815-aCD3 cells were collected and CFSE stained according to the method (A). CTL and CFSE-stained P815-aCD3 cells were placed in a U-shaped 96-well in a ratio of 5:2 and co-incubated. At the same time, 2 μM of the test compound JNU-0921 and 5 μg / mL aCD137 agonist antibody were added as positive controls. After 6 hours, the cells were collected and stained with 10 μg / mL PI, and the proportion of PI-positive cells in CFSE-positive P815-aCD3 cells was detected by flow cytometry.

[0270] (D) The effects of the test compounds on the cytokine secretion function of mouse CD8+T and CD4+T cells were detected. Single cell suspensions of spleens from WT mice and CD137KO mice were prepared according to the above method. 1×10 5 The cells were placed in a 96-well plate containing 1 μg / mL CD3 antibody, and 5 μg / mL aCD137 and 100 nM test compound JNU-0921 were added for treatment. 0.5 μM protein transport inhibitor GolgiPlug was added two hours before the cells were harvested 48 hours later. TM Finally, the expression of cytokines on the surface of CD8+ and CD4+ T cells was detected by flow cytometry.

[0271] Experimental results and analysis:

[0272] The flow cytometry results showed that (1) by detecting the fluorescence intensity of CFSE in CD8+ and CD4+ T cells by flow cytometry, it was found that the addition of CD137 agonist antibody (aCD137) could significantly promote the proliferation of CD8+ and CD4+ T cells. The addition of the test compound JNU-0921 treatment could also significantly promote the proliferation and division of CD8+ and CD4+ T cells. This phenomenon did not occur in CD137KO mice ( Figure 2 AC in the figure). (2) Both aCD137 and the test compound JNU-0921 treatment significantly increased the expression of activation markers CD25 and CD69 in CD8+ and CD4+ T cells, while there was no change in CD137KO cells ( Figure 2 (3) Both aCD137 and the test compound JNU-0921 treatment significantly enhanced the killing ability of CD8+ on target cells, while no change was observed in CD137KO cells ( Figure 2 (4) aCD137 and the test compound JNU-0921 treatment can significantly enhance the expression of cytokines IL-2, IFNγ and Perforin in CD8+ and CD4+ T cells ( Figure 2 JO in.

[0273] Example 36 Detection of the effect of the test compound on memory cells

[0274] (A) Establishment of memory cell model: OT1 mice and OT1 / CD137KO mice were subjected to the experimental method of Example 35 (A) to obtain single cell suspension, and 1×10 7 The cells were placed in a 10 cm dish, 10 nM OVA257-264, 55 μM mercaptoethanol and 10 ng / mL IL-2 were added to the culture medium, and the cells were induced in a 37°C 5% CO2 cell culture incubator for 3 days. Generally, mature CTLs were obtained after 3 days of induction. CTL and EG7 were mixed and cultured at a ratio of 5:2, EG7 cells were added every three days, and memory cells were generated after repeated stimulation three times. During this process, 5 μg / mL aCD137 and 100 nM test compound JNU-0921 were added at the same time. The proportion of memory cells in CD8+T cells and the expression of surface exhaustion factors were detected by flow cytometry.

[0275] (B) Establishment of LM-OVA mouse model: 2000 LM-OVA bacteria were transfused into CD45.1WT mice through the tail vein. The next day, OT1 mice were transfused with 1×10 6 After adding 50 mg / kg of the test compound JNU-0921 for 45 days, the proportion of memory cells in the blood and spleen of mice was detected by flow cytometry to evaluate the effect of the test compound JNU-0921 on memory cells in the LM-OVA mouse model.

[0276] Experimental results and analysis:

[0277] Flow cytometry showed that (1) the addition of the CD137 agonist antibody aCD137 or the compound JNU-0921 significantly promoted the formation of CD44 / CD62L double positive memory cells, but this was not the case in CD137KO cells ( Figure 3 (2) Adding agonist antibody aCD137 or compound JNU-0921 can significantly reduce the expression of exhaustion markers PD-1, CTLA4 and TIM-3 on the surface of CD8+T cells ( Figure 3(3) Adding compound JNU-0921 to the mouse blood and spleen can increase the proportion of memory cells ( Figure 3 in IK).

[0278] Example 37 Detection of the effect of the test compound on CD4+ T cell polarization

[0279] (A) For Th1 cell polarization, use CD4+T cell isolation kit (Biolegend, No. 480005) was used to isolate CD4+T cells from the spleens of WT mice and CD137KO mice. 5×10 5 CD4+T cells were incubated in 96-well plates containing 1μg / mL CD3 / CD28 antibody, 10ng / mL IL-2, 10μg / mL anti-IL-4, 10ng / mL IL-12 and 500nM test compound JNU-0921 or 5μg / mL aCD137 for 5 days. On the sixth day, the cells were harvested, washed once with PBS, and restimulated for 5 hours in a medium containing 50ng / mL PMA, 1μg / mL ionomycin and monensin. The percentage of IFNγ+CD4+T cells was detected by flow cytometry.

[0280] (B) For Th2 cell polarization, use CD4+ T cell isolation kit (Biolegend, No. 480005) was used to isolate CD4+ T cells from the spleens of WT mice and CD137KO mice. 5×10 5 CD4+T cells were placed in a 96-well plate containing 5μg / mL Con A, 20ng / mL IL-2, 50ng / mL IL-4 and 500nM test compound JNU-0921 or 5μg / mL aCD137 and incubated for 6 days. On the seventh day, the cells were harvested, washed once with PBS, and restimulated for 5 hours in a medium containing 1μg / mL CD3 / CD28 antibody and monensin. The percentage of IL-4+CD4+T cells was detected by flow cytometry.

[0281] (C) For Th9 cell polarization, use CD4+ T cell isolation kit (Biolegend, No. 480005) was used to isolate CD4+ T cells from the spleens of WT mice and CD137KO mice. 5×10 5CD4+T cells were incubated in 96-well plates containing 10ng / mL TGF-β1, 10ng / mL IL-4, 10ng / mL IL-2, 10μg / mL anti-IFNγ and 500nM test compound JNU-0921 or 5μg / mL aCD137 for 3 days. On the fourth day, the cells were harvested, washed once with PBS, and then restimulated in a medium containing 500ng / mL PMA and 500ng / mL ionomycin and monensin for 5 hours. The percentage of IL-9+CD4+T cells was detected by flow cytometry.

[0282] (D) For Treg cell polarization, use CD4+T cell isolation kit (Biolegend, No. 480005) was used to isolate CD4+T cells from the spleens of WT mice and CD137KO mice. 5×10 5 CD4+T cells were placed in a 96-well plate containing 1 μg / mL CD3 / CD28 antibody, 10 ng / mL IL-2, 5 ng / mL TGF-β and 500 nM test compound JNU-0921 or 5 μg / mL LaCD137 and incubated for 5 days. The percentage of Foxp3+CD4+T cells was detected by flow cytometry.

[0283] Experimental results and analysis:

[0284] The flow cytometry results showed that (1) both the CD137 agonist antibody aCD137 and the test compound JNU-0921 could increase the proportion of Th1 cells and inhibit the proportion of Th2 cells, but this phenomenon was not seen in CD137KO mice ( Figure 4 (2) aCD137 and the test compound JNU-0921 can both increase the proportion of Th9 cells and inhibit the proportion of Treg cells ( Figure 4 The EH in CD137 cells was also inhibited, and the expression of Treg cell activation markers ICOS and CTLA-4 was suppressed, while this phenomenon was not seen in CD137KO mice ( Figure 4 in IL).

[0285] Example 38 Detection of the effect of the test compound on CD137 multimerization

[0286] (A) Bimolecular fluorescence complementation experiment: CD137-fused Nluc and Cluc plasmids were constructed, respectively, and these plasmids were co-transfected into 293T cells. After adding different concentrations of the test compound (500 nM, 1 μM) JNU-0921 and treating for 6 hours, the activity of luciferase was detected.

[0287] (B) Immunoprecipitation experiment: CD137-fused Flag and MYC plasmids were constructed and co-transfected into 293T cells. After addition of 1 μM of the test compound JNU-0921 for 6 hours, the cells were precipitated with FLAG antibody and finally developed with MYC antibody.

[0288] (C) Immunofluorescence experiment: CD137-fused GFP and mcherry plasmids were constructed and co-transfected into 293T cells. After treatment with 1 μM of the test compound JNU-0921 for 6 hours, the cells were fixed with 4% paraformaldehyde solution for 30 minutes at room temperature in the dark. The cells were washed 3 times with PBS and stained with DAPI for 2 minutes. The images were taken using a laser scanning confocal microscope.

[0289] Experimental results and analysis: (1) Luciferase activity experiments showed that the test compound JNU-0921 could significantly promote the activity of Luciferase after treatment, indicating that the test compound JNU-0921 could promote the polymerization of CD137 ( Figure 5 (2) The confocal microscopy results showed that the test compound JNU-0921 significantly promoted the co-localization of CD137-GFP and CD137-mcherry after treatment ( Figure 5 BC in the sample). (3) Immunoprecipitation experiments showed that the test compound JNU-0921 could promote the polymerization of CD137 ( Figure 5 D).

[0290] Example 39 Detection of the effect of the test compound on the growth of transplanted tumors in mice

[0291] MC38 cells were resuspended in PBS and Matrigel at a ratio of 1:1. 100 μL of cell suspension contained 2×10 6 The cells were inoculated on both sides of WT and Rag1 mice. After the tumor was fixed, the mice were randomly divided into two groups, one group was the control group, and the other group was treated with the test compound JNU-0921. The treatment was carried out by oral gavage at a dose of 50 mg / kg for two weeks. After two weeks of administration, the mice were euthanized, the tumors were removed, photographed, and the volume and weight of the tumors were recorded.

[0292] Experimental results and analysis: In WT mice, compared with the control group, the test compound JNU-0921 can significantly inhibit tumor growth and reduce tumor volume and weight. However, in Rag1 mice, there is no therapeutic effect ( Figure 6 ).

[0293] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the following embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0294] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a bicyclic compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof in the preparation of a CD137 agonist; in, T is selected from: 0, 1, 2; W, Y, and Z are independently selected from: O, S, N, NR 1 , CR 1 , Ring A containing W, Y, and Z is a heteroaromatic ring; X1, X2, X3, X4, and X5 are independently selected from: N, CR 2 , Ring B including X1, X2, X3, X4, and X5 is an aromatic ring or a heteroaromatic ring; R is selected from: R 3 Substituted or unsubstituted C1-C 12 Alkyl, R 3 Substituted or unsubstituted C3-C 12 Cycloalkyl, R 3 a substituted or unsubstituted 3-12 membered heterocyclic group, R 3 Substituted or unsubstituted C6-C 18 Aryl, R 3 a substituted or unsubstituted 5-18 membered heteroaryl group; Each R 1 Each independently selected from: hydrogen, C1-C6 alkyl; Each R 2 are independently selected from: hydrogen, halogen, R 4 Substituted or unsubstituted C1-C6 alkyl, R 4 Substituted or unsubstituted C3-C6 cycloalkyl, R 4 Substituted or unsubstituted C1-C6 alkoxy, R 4 Substituted or unsubstituted C3-C6 cycloalkoxy, cyano, hydroxyl, amino, -C(=O)R 5 、-S(=O)R 5 、-S(=O)2R 5 、-P(=O)R 5 R 5 , tetrazolyl, Each R 3 are independently selected from: hydrogen, halogen, R 4 Substituted or unsubstituted C1-C6 alkyl, R 4 Substituted or unsubstituted C3-C6 cycloalkyl, R 4 Substituted or unsubstituted C1-C6 alkoxy, R 4 Substituted or unsubstituted C3-C6 cycloalkoxy, cyano, hydroxy, amino, carboxyl, -C(=O)R 5 、-(CH2) m NR a R b 、-(CH2) m OCR a R b ; Each m is independently selected from: 0, 1, 2 or 3; Each R a and each R b are independently selected from: hydrogen, R 4 Substituted or unsubstituted C1-C6 alkyl, or R a , R b Together with the N or C to which it is connected, it forms R 4 a substituted or unsubstituted 3-12 membered heterocyclic group; Each R 4 Each of the following is independently selected from the group consisting of: deuterium, halogen, hydroxyl, amino, C1-C3 alkyl, C1-C3 alkoxy, -NH(C1-C3 alkyl), -N(C1-C3 alkyl)(C1-C3 alkyl), -C(=O)(C1-C3 alkyl), C1-C3 alkyl substituted with hydroxyl, 3-8 membered heterocyclyl, 3-8 membered heterocyclyl substituted with C1-C3 alkyl, and C1-C3 alkyl substituted with halogen; Each R 5 Each is independently selected from the group consisting of: hydrogen, hydroxy, amino, C1-C6 alkoxy, and -NH-OH.

2. The use according to claim 1, characterized in that: Ring A is selected from:

3. The use according to claim 1, characterized in that: X1, X3, X4, X5 are all CH, X2 is CR 2 ; Or X1, X2, X4, X5 are all CH, and X3 is CR 2 .

4. The use according to claim 1, characterized in that: Each R 2 are independently selected from: hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, -C(=O)R 5 ; Each R 5 Each is independently selected from the group consisting of: hydrogen, hydroxy, amino, C1-C3 alkoxy, and -NH-OH.

5. The use according to claim 4, characterized in that: Each R 2 Each is independently selected from the group consisting of hydrogen, carboxyl, methoxycarbonyl, and carbamoyl.

6. The use according to claim 1, characterized in that: Each R 3 are independently selected from: hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, halogen-substituted C1-C3 alkyl, 7. The use according to claim 6, characterized in that: Each R 3 Each is independently selected from the group consisting of hydrogen, methyl, ethyl, fluorine, chlorine, bromine and methoxy.

8. The use according to any one of claims 1 to 7, characterized in that: R is selected from: C1-C6 alkyl, R 3 Substituted or unsubstituted C5-C 10 Cycloalkyl, R 3 a substituted or unsubstituted 5-10 membered heterocyclic group, R 3 Substituted or unsubstituted C6-C 10 Aryl, R 3 A substituted or unsubstituted 5-10 membered heteroaryl group.

9. The use according to claim 8, characterized in that: R is selected from: C1-C4 alkyl, R 3 Substituted or unsubstituted cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptanyl, adamantyl, spiro[2.5]octanyl, bicyclo[4.1.0]heptanyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, hexahydropyridinyl, tetrahydropyranyl, tetrahydronaphthyl, furanyl, thienyl, pyrrolyl, pyrazolyl, thiazolyl, phenyl, naphthyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, 1,3,5-triazinyl, benzofuranyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, benzopyrazolyl, quinolyl, isoquinolyl, quinoxalinyl, benzopyrimidinyl, pyridopyrimidinyl, pyridopyridinyl, purinyl, pteridinyl, imidazothiazolyl, imidazopyridazinyl, pyrazolopyrimidinyl.

10. The use according to claim 9, characterized in that: R is selected from: phenyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, 11. The use according to any one of claims 1 to 7, characterized in that: R is selected from: R 3 Substituted or unsubstituted C7-C 10 Spirobicycloalkyl, R 3 Substituted or unsubstituted 9-10 membered heteroaryl, R 3 Substituted or unsubstituted naphthalene-2-yl, R 3 Substituted or unsubstituted tetrahydronaphthyl; the 9-10 membered heteroaryl is a bicyclic heteroaryl.

12. The use according to claim 11, characterized in that: R is selected from: R 3 Substituted or unsubstituted: spiro[2.5]octyl, naphth-2-yl, tetrahydronaphthyl, benzofuranyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, benzopyrazolyl, quinolyl, isoquinolyl, quinoxalinyl, benzopyrimidinyl, pyridopyrimidinyl, purinyl, pteridinyl, imidazothiazolyl, imidazopyridazinyl, pyrazolopyrimidinyl.

13. The use according to claim 12, characterized in that: R is selected from:

14. The use according to claim 1, characterized in that: The bicyclic compound is selected from the following compounds:

15. The use according to claim 1, characterized in that: The bicyclic compound is selected from the following compounds:

16. Use of the bicyclic compound described in any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof, in the preparation of an enhancer for enhancing the immune function of cytotoxic T lymphocytes, an enhancer for enhancing the memory function of CD8+T cells and CD4+T cells, an enhancer for promoting the polarization of CD4+T cells to a cellular immune state, or an enhancer for promoting CD137 multimerization.

17. Use of the bicyclic compound or a pharmaceutically acceptable salt or a stereoisomer or a prodrug molecule thereof as claimed in any one of claims 1 to 15 in the preparation of a drug for preventing and / or treating tumors; Preferably, the tumor is: liver cancer, lymphoma, colon cancer, lung cancer, breast cancer, melanoma, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, rectal cancer, cervical cancer, laryngeal cancer, nasopharyngeal cancer, pancreatic cancer, multiple myeloma, leukemia.

18. The bicyclic compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof.

19. A pharmaceutical composition for preventing and / or treating tumors, characterized in that: The invention is prepared from active ingredients and pharmaceutically acceptable carriers or excipients, wherein the active ingredients include the bicyclic compound described in any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof.

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