Bicyclic compounds, pharmaceutical compositions thereof, and uses thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a bicyclic compound, its pharmaceutical composition, and its applications. Background Technology

[0002] CD137 (also known as 4-1BB / TNFRSF9) is a membrane glycoprotein discovered in the late 1980s, belonging to a superfamily of TNFR receptors 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 (NK) cells, dendritic cells (DCs), eosinophils, and mast cells. Therefore, CD137 has been recognized as 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 trimer ligand (CD137L), which is present in all types of antigen-presenting cells. Upon ligand binding, CD137 trimers and recruits TNF receptor-associated factors 1, 2, and 3 (TRAF1, TRAF2, and TRAF3) through two polyacidic TRAF-binding consensus regions located at its cytoplasmic tails 234TTQEE238 and 246PEEEE250, forming the 3CD137L:3CD137:3TRAF3 complex. The trimer 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-γ (IFN-γ) and interleukin-2 (IL-2), as well as memory differentiation. Activation of CD137 can also enhance the cytotoxicity of NK cells and counteract the inhibitory effect of TGFβ on NK cells to exert anti-tumor functions. Furthermore, it has been reported 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 could inhibit tumor development by enhancing CD8+ T cell anti-tumor immunity. Numerous preclinical model studies have also confirmed the therapeutic effects of agonist CD137 antibodies, including enhancing anti-cancer immunity and protecting mice from liver tumors, lymphomas, colon cancers, lung cancers, breast cancers, and melanomas.

[0005] Given the advantages of small molecule drugs, such as 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 purpose of the present invention is to provide a small molecule CD137 agonist that can be used for anti-tumor purposes.

[0007] To achieve the above objectives, the present invention includes the following technical solutions.

[0008] The use of bicyclic compounds having the structure shown in formula (I), or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or prodrug molecules thereof, in the preparation of CD137 agonists;

[0009]

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

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

[0012] X1, X2, X3, X4, and X5 are independently selected from: N, CR 2 The ring B containing 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 C3-C, whether substituted or not 12 cycloalkyl, R 3 Substituted or unsubstituted 3-12 membered heterocyclic groups, R 3 Replaced or unreplaced C6-C 18 Aryl, R 3 Substituted or unsubstituted 5-18 heteroaryl groups;

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

[0015] Each R 2 Each is independently selected from: hydrogen, halogen, R 4 Substituted or unsubstituted C1-C6 alkyl groups, R 4 Substituted or unsubstituted C3-C6 cycloalkyl, R 4 Substituted or unsubstituted C1-C6 alkoxy groups, 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 Each is independently selected from: hydrogen, halogen, R 4 Substituted or unsubstituted C1-C6 alkyl groups, R 4 Substituted or unsubstituted C3-C6 cycloalkyl, R 4 Substituted or unsubstituted C1-C6 alkoxy groups, R 4 Substituted or unsubstituted C3-C6 cycloalkoxy, cyano, hydroxyl, 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 Selected independently from: hydrogen, R 4 Substituted or unsubstituted C1-C6 alkyl groups, or R a R b Together with the N or C connected to it, they form R 4 Substituted or unsubstituted 3-12 membered heterocyclic groups;

[0019] Each R 4Each is independently selected from: 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), hydroxyl-substituted C1-C3 alkyl, 3-8 membered heterocyclic group, C1-C3 alkyl-substituted 3-8 membered heterocyclic group, halogen-substituted C1-C3 alkyl;

[0020] Each R 5 Each of the following is independently selected from: hydrogen, hydroxyl, 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, and X5 are all CH, and X3 is CR. 2 .

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

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

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

[0029]

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

[0031] In some embodiments, R is selected from: C1-C6 alkyl groups, R3 Replaced or unreplaced C5-C 10 cycloalkyl, R 3 Substituted or unsubstituted 5-10 membered heterocyclic groups, R 3 Replaced or unreplaced C6-C 10 Aryl, R 3 Substituted or unsubstituted 5-10 heteroaryl groups.

[0032] In some embodiments, R is selected from: C1-C4 alkyl groups, R 3 The following groups, whether substituted or unsubstituted, are: cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, adamantyl, spiro[2.5]octyl, bicyclo[4.1.0]heptyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, hexahydropyridyl, tetrahydropyranyl, tetrahydronaphthyl, furanyl, thiophene, pyrroleyl, pyrazolyl, thiazolyl, phenyl, naphthyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, 1,3,5-triazinyl, benzofuranyl, benzothiophene, indolyl, inzolyl, benzimidazolyl, benzopyrazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, benzopyrimidinyl, pyridopyrimidinyl, purine, pteridinyl, imidazothiazolyl, imidazopyridazinyl, pyrazolopyrimidinyl.

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

[0034]

[0035] In some embodiments, R is selected from: R 3 Replaced or unreplaced C7-C 10 Spirobicycloalkyl, R 3 Substituted or unsubstituted 9-10 aryl groups, R 3 Substituted or unsubstituted naphth-2-yl, R 3 The substituted or unsubstituted tetrahydronaphthyl group; the 9-10 membered heteroaryl group is a bicyclic heteroaryl group.

[0036] In some embodiments, R is selected from: R 3 The following groups may be substituted or unsubstituted: spiro[2.5]octyl, naphth-2-yl, tetrahydronaphthyl, benzofuranyl, benzothiopheneyl, indolyl, inzolyl, benzimidazolyl, benzopyrazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, benzopyrimidinyl, pyridinylpyrimidinyl, purine, pteridinyl, imidazothiazolyl, imidazopyridazinyl, pyrazolylpyrimidinyl.

[0037] In some embodiments, R is selected from: R3 Substituted or unsubstituted spiro[2.5]octyl.

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

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

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

[0041] In some embodiments, R is selected from: R 3 Substituted or unsubstituted quinoline-3-yl.

[0042] In some embodiments, R is selected from: R 3 Substituted or unsubstituted quinoxaline-2-yl.

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

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

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

[0046]

[0047]

[0048] The use of the bicyclic compounds, pharmaceutically acceptable salts thereof, stereoisomers thereof, or prodrug molecules thereof described in this invention in the preparation of enhancers for enhancing the immune function of cytotoxic T lymphocytes.

[0049] The use of the bicyclic compounds, pharmaceutically acceptable salts thereof, stereoisomers thereof, or prodrug molecules thereof described in this invention in the preparation of enhancers for enhancing the memory function of CD8+ T cells and CD4+ T cells.

[0050] The application of the bicyclic compound, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule described in this invention, in the preparation of a promoter for stimulating the polarization of CD4+ T cells to a cellular immune state.

[0051] The use of the bicyclic compound, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule described in this invention, in the preparation of promoters for promoting CD137 polymerization.

[0052] The use of the bicyclic compounds, pharmaceutically acceptable salts thereof, stereoisomers thereof, or prodrug molecules thereof described in this invention in the preparation of medicaments for the prevention and / or treatment of 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, or leukemia.

[0054] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of tumors, which is prepared from an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient includes the bicyclic compound described in this invention or its pharmaceutically acceptable salt or its stereoisomer or its prodrug molecule.

[0055] Through long-term and in-depth research, the inventors of this invention unexpectedly discovered for the first time that Ataluren (PTC124), a commercially available drug used to treat Duchenne muscular dystrophy and muscle atrophy, can activate CD137 for the treatment of tumors. The inventors further developed a series of new derivatives, finding that these compounds can all activate CD137, enhance the immune function of cytotoxic T lymphocytes (CTLs), enhance the memory function of CD8+ T cells and CD4+ T cells, tilt CD4+ T cell polarization towards a state favorable to cellular immunity, and promote CD137 polymerization. This effectively activates T cell immunity, effectively reduces the size of primary tumors, and can be used to inhibit and treat tumors. Attached Figure Description

[0056] Figure 1 The results show the luciferase fluorescence activity assay of the test compound.

[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 The effect of the test compound on CD4+ T cell polarization.

[0060] Figure 5 The effect of the test compound on the polymerization of CD137.

[0061] Figure 6The results show the test results of the test compound inhibiting the growth of xenografts in mice. Detailed Implementation

[0062] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are merely illustrative of the present invention and should not be construed as limiting the invention.

[0063] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0064] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0065] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0066] In the compounds described in this invention, when any variable (e.g., R) 4 R 5 If a component (e.g., a substituent) appears more than once in any component, the definition of each occurrence is independent of the definition of each subsequent occurrence. Similarly, combinations of substituents and variables are permitted, provided such combinations stabilize the compound. A line drawn from a substituent into the ring system indicates that the bond referred to can be attached to any substituted ring atom. If the ring system is polycyclic, it means that such a bond is attached only to any suitable carbon atom of a neighboring ring. It should be understood that those skilled in the art can select the substituents and substitution patterns of the compounds of this invention to provide chemically stable compounds that can be readily synthesized from readily available starting materials using techniques in the art and the methods described below. If a substituent is itself substituted by more than one group, it should be understood that these groups can be on the same carbon atom or on different carbon atoms, as long as the structure is stable.

[0067] As used in this invention, the term "alkyl" refers to both 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. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

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

[0069] The term "alkoxy" as used in this 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] As used in this invention, the term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic, bicyclic, or polycyclic cyclic substituent, wherein one or more ring atoms are selected from heteroatoms of N, O, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Bicyclic or polycyclic groups include spirocyclic, fused-ring, and bridged-ring groups. Examples include: morpholinyl, piperidinyl, tetrahydropyrrolyl, pyrrolylalkyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazoleyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophenyl, dihydrotriazolyl, dihydroazacyclobutane, tetrahydrofuranyl, tetrahydrothiophenyl, etc. And so on, and their N-oxides. The connection of heterocyclic substituents can be achieved through carbon atoms or through heteroatoms.

[0071] As used in this invention, the term "heteroaryl" refers to an aromatic ring containing one or more heteroatoms selected from O, N, or S. This aromatic ring can be monocyclic, bicyclic, or polycyclic, and includes, but is not limited to: quinolinyl, pyrazolyl, pyrroloyl, thiophene, furanyl, pyridinyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, 1,3,5-triazinyl, benzofuranyl, benzothiophene, indoleyl, inzolyl, benzimidazolyl, benzopyrazolyl, quinolinyl, isoquinolinyl, quinoxalolinyl, benzopyrimidinyl, pyridopyridinyl, purine, pteridinyl, imidazothiazolyl, imidazopyridazinyl, pyrazolopyrimidinyl, etc. "Heteroaryl" is also understood to include any N-oxide derivative of a nitrogen-containing heteroaryl group. The linkage of heteroaryl groups can be achieved through carbon atoms or through heteroatoms.

[0072] As will be understood by those skilled in the art, the term "halogen" or "halogen" as used in this invention 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 the structure shown in 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] Where T is selected from: 0, 1, 2;

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

[0077] X1, X2, X3, X4, and X5 are independently selected from: N, CR 2 The ring B containing 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 C3-C, whether substituted or not 12 cycloalkyl, R 3 Substituted or unsubstituted 3-12 membered heterocyclic groups, R 3 Replaced or unreplaced C6-C 18 Aryl, R 3 Substituted or unsubstituted 5-18 heteroaryl groups;

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

[0080] Each R 2 Each is independently selected from: hydrogen, halogen, R 4 Substituted or unsubstituted C1-C6 alkyl groups, R 4 Substituted or unsubstituted C3-C6 cycloalkyl, R 4 Substituted or unsubstituted C1-C6 alkoxy groups, 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 Each is independently selected from: hydrogen, halogen, R 4 Substituted or unsubstituted C1-C6 alkyl groups, R 4 Substituted or unsubstituted C3-C6 cycloalkyl, R 4 Substituted or unsubstituted C1-C6 alkoxy groups, R 4 Substituted or unsubstituted C3-C6 cycloalkoxy, cyano, hydroxyl, 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 Selected independently from: hydrogen, R 4 Substituted or unsubstituted C1-C6 alkyl groups, or R a R b Together with the N or C connected to it, they form R 4 Substituted or unsubstituted 3-12 membered heterocyclic groups;

[0084] Each R 4Each is independently selected from: 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), hydroxyl-substituted C1-C3 alkyl, 3-8 membered heterocyclic group, C1-C3 alkyl-substituted 3-8 membered heterocyclic group, halogen-substituted C1-C3 alkyl;

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

[0086] This invention includes the free form of compounds of formula (I), as well as their 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 this invention can be synthesized from compounds of this invention containing a basic or acidic moiety using conventional chemical methods. Typically, salts of basic compounds are prepared by ion-exchange chromatography or by reacting a free base with a stoichiometric or excess amount of the desired salt form of an inorganic or organic acid in a suitable solvent or a combination of solvents. Similarly, salts of acidic compounds are formed by reacting with a suitable inorganic or organic base.

[0087] Therefore, pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts of the compounds of the present invention formed by reacting an alkaline compound of the present invention with an inorganic or organic acid. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic 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, pyric acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethylsulfonic acid, trifluoroacetic acid, etc.

[0088] If the compounds of this invention are acidic, then a suitable "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic alkali, including inorganic 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, manganese salts, manganese 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, wherein substituted amines include 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, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, aminobutanetriol, etc.

[0089] Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci. '1977: 66: 1–19, describe in more detail the preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts.

[0090] In one embodiment, the present invention provides a method for treating diseases such as tumors in humans or other mammals using a compound having the structure shown in Formula (I) and its pharmaceutically acceptable salts, for example, the tumors 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] Metabolites of the compounds and pharmaceutically acceptable salts involved in this invention, as well as prodrugs that can be converted in vivo into structures of the compounds and pharmaceutically acceptable salts involved in this application, are also included in the claims of this invention.

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

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

[0094] The "active ingredient" and pharmaceutical composition described in this invention can be used as CD137 agonists and can be used to prepare drugs for the prevention and / or treatment of tumors, etc.

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

[0096] "Pharmaceutically acceptable carriers or excipients" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0097] "Compatibility" here refers to the ability of the components in the composition to interact with and blend with the active ingredients of the present invention without significantly reducing the efficacy of the active ingredients.

[0098] Pharmaceutically acceptable examples of 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, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as...). Wetting agents (such as sodium dodecyl 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 polymer through non-bonding interaction. In another preferred embodiment, the compound of formula I of the present invention, as a small molecule, can also be linked to a macromolecular compound or polymer through chemical bonds. The macromolecular compound can be a biological macromolecule such as a polysaccharide, protein, nucleic acid, polypeptide, etc.

[0100] There are no particular limitations on the administration of the active ingredients or pharmaceutical compositions 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 components:

[0103] (a) Fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol and silica;

[0104] (b) Adhesives, such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0105] (c) Moisturizers, such as glycerin;

[0106] (d) Disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginate, certain complex silicates, and sodium carbonate;

[0107] (e) Slow solvents, such as paraffin;

[0108] (f) Absorption accelerators, such as quaternary ammonium compounds;

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

[0110] (h) Adsorbents, such as kaolin; and

[0111] (i) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer.

[0112] The solid dosage form can also be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active ingredient from this composition can be delayed in a portion of the digestive tract. Examples of suitable encapsulating components are polymers and waxes.

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

[0114] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0115] Compositions for parenteral injection may comprise 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 this invention can 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 the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 20–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0118] Compounds of Formula I can be used in combination with other known drugs for treating or improving similar symptoms. When used in combination, the original drug's administration method and dosage remain unchanged, while Compound I is taken simultaneously or subsequently. When Compound I is taken concurrently with one or more other drugs, a pharmaceutical composition containing one or more known drugs and Compound I is preferred. Drug combination also includes taking Compound I with one or more other known drugs during overlapping time periods. When Compound I is used in combination with one or more other drugs, the dosage of Compound I or the known drug may be lower than the dosage when they are taken alone.

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

[0120] Estrogen receptor modulators, androgen receptor modulators, retinal-like receptor modulators, cytotoxins / cell inhibitors, 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 drug or active ingredient that can be used in combination with the compound of Formula I includes, but is not limited to, interleukin, alendronate, interferon, atranoin, allopurinol, allopurinol sodium, palonosetron hydrochloride, hexamethylmelamine, aminoglucopyranoside, amifostine, amrubicin, azithromycin, anatozol, dolasetron, aranesp, arglabin, arsenic trioxide, arnoxin, 5-azacytidine, azathioprine, BCG or TICE BCG, betamethasone acetate, betamethasone sodium phosphate preparation, bexarotine, bleomycin sulfate, bromouridine, bortezomib, busulfan, calcitonin, alectozimumab injection, capecitabine, carboplatin, Casper, cefesone Interleukin, daunorubicin, chlorambucil, cisplatin, cladribine, cladribine, chlorodronate, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin liposomes, dexamethasone, dexamethasone phosphate, estradiol valerate, diethylstilbestrol, desmopressin, docetaxel, desoxyfluorouridine, doxorubicin, dronabinol, 166-chitosan complex, eligard, raburicase, epirubicin hydrochloride, aprepitant, epirubicin, alfa-ebertheline, erythropoietin, etoposide, levamisole tablets, estradiol preparations, 17-β-estradiol, estradiol sodium phosphate, ethinylestradiol, amifostine, hydroxyphosphate, vanbex, etoposide, fazodazole, tamoxifen Filafen preparations, filgrastim, phenacetin, feracetam, fluorouridine, fluconazole, fludarabine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil, flumethasone, flutamide, formestan, 1-β-D-arasulfofuranocytidine-5'-stearoyl phosphate, formustin, fulvestrant, gamma globulin, gemcitabine, gemtuzumab, imatinib mesylate, carmustine rice paper capsules, goserelin, granisilone hydrochloride, histamine relin, and omeprazole, hydrocortisone, erythro-hydroxynonyladenine, hydroxyurea, tetanisoprostol, idarubicin, ifosfamide, interferon α, interferon-α2, interferon α-2A, interferon α-2B, interferon α-n1, interferon α-n3, interferon β Interferon γ-1a, Interleukin-2, Intron A, Iressa, Irinotecan, Keterene, Lentinan sulfate, Letrozole, Levofloxacin, Levofloxacin acetate, Levotetraimidazole, Levofolinate calcium salt, Levothyroxine sodium, Levothyroxine sodium preparations, Lomustine, Clonidamine, Drowanesol, Nitrogen mustard, Mecobalamin, Medroxyprogesterone acetate, Medroxyprogesterone acetate, Melphalan, Esterified estrogen, 6-Mercaptopurine, Mesna, Methotrexate, Methylaminolevulinate, Mitofosine, Minocycline, Mitomycin C, Mitotane, Mitoxantrone, Tralostertan, Doxorubicin citrate liposomes, Nedaplatin, Pegylated filgrastim, Olepipril, Interleukin, Neupogen, Nilumethicone, TamoxifenNSC-631570, Recombinant Human Interleukin-1-β, Octreotide, Odanciron Hydrochloride, Dehydrocortisone Oral Solution, Oxaliplatin, Paclitaxel, Prednisone Sodium Phosphate Preparation, Pegaspargase, Pegasys, Pentostatin, Streptomycin Preparation, Pilucarpine Hydrochloride, Pirarubicin, Prucalomycin, Porphyrom Sodium, Prednisone, Steprednisolone, Prednisone, Premarin, Procarbazine, Recombinant Human Erythropoietin, Raltitrexate, Ribeye, Rhenium-186 Etidronate, Rituximab, Rituximab-A, Romotide, Pilocarpine Hydrochloride Tablets, Octreotide, Samosine, Semustine, Cizonan, Sobuzosen, Methylprednisolone Sodium, Paphosphatase, Stem Cell Therapy, Levozocin, Strontium Chloride-89, Levothyroxine Sodium, Tamoxifen Tansulosin, Tastolactone, Dosotericin, Tecidiazole, Temozolomide, Teniposide, Testosterone Propionate, Methyltestosterone, Thioguanine, Thiotepa, Thyroid Stimulating Hormone, Tiludronate, Topotecan, Toremifene, Tosimozab, Trastuzumab, Triostazol, Retinoic Acid, Methotrexate Tablets, Trimethylmelamine, Trimethoprim, Triptorelin Acetate, Triptorelin Dihydroxynaphthyl Naphthyl Acetate, Ufodin, Ureidine, Pentorubicin, Visorcinol, Vincristine, Vincristine, Vincrylein, Vinorelin, Verrucidium, Dextromethorphan, Nettostatin Ester, Scyproterone, Paclitaxel Protein Stabilizer, Acolbifene, Interferon R-LB, Affinitak, Aminopterin, Azoxifen, As Oprisnil, Atamitan, Atrasentan, BAY43-9006, Avastin, CCI-779, CDC-501, Celebrex, Cetuximab, Crinatorta, Cyproterone Acetate, Decitabine, DN-101, Doxorubicin-MTC, dSLIM, Dutasteride, Edootecarin, Eflunomide, Ecinotecan, Fenivel Aamine, Histamine Dihydrochloride, Histamine Relin Hydrogel Implant, Holmium-166DOTMP, Ibandronate, Interferon Gamma, Intron-PEG, Ixabepilone, Keyhole Hemocyanin, L-651582, Lanleptide, Lasoxifene, Libra, Lonafamib, Miprexifene, Minotriol, MS-2 09. Liposomes MTP-PE, MX-6, Nafarelin, Nemorubicin, Neovastatin, Noratropide, Olimex, Onco-TCS, Osidem, Paclitaxel Polyglutamate, Sodium Pormylate, PN-401, QS-21, Quasi-Yan, R-1549, Raloxifene, Leopoldin, 13-cis-retinoic acid, Saplatin, Ciocalcitriol, T-138067, Tarceva, Docosahexaenoic acid paclitaxel, Thymosin α1, Gazofuran, Tipifanib, Tirazamine, TLK-286, Toremifene, Trans-MID-lo7R, Vasoprothiolane, Vatalanib, Vertepofen, Vinpocetine, Z-100, and Zoledronic acid or combinations thereof.

[0122] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0123] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0124] The raw materials used in the following examples may be commercially available, or prepared by methods known in the art, or prepared according to the methods described herein.

[0125] Example 13: Synthesis of 5-(naphthyl-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, 2 h; (b) 2-Naphthoic acid, EDCI, HOBt, DMF, 120-140℃, 2 h; (c) 2NNaOH (aq), methanol, room temperature, 2 h.

[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 complete, the solvent in the reaction system was evaporated to dryness under reduced pressure to obtain intermediate 4. The intermediate did not require purification and was directly added to the next reaction step. 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-(naphth-2-yl)-1,2,4-oxadiazol-3-yl)benzoate (5)

[0130] 2-Naphthoic acid (93 mmol) was dissolved in 20 mL of DMF. 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 complete. The reaction system was cooled, and water was added until no more solid precipitated. The mixture was then filtered to obtain intermediate 5. Intermediate 5 did not require purification and was directly added to the next reaction step. 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-(naphth-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 stirred vigorously at room temperature for 2 hours until the reaction was complete. Most of the organic solvent was evaporated, and 4N HCl solution was added dropwise until no more solid precipitated. The final product 2-25, a white solid, was obtained by filtration. 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 23 Synthesis of (5-phenyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-1)

[0134]

[0135] The synthesis method is the same as in Example 1, except that benzoic acid is used instead of 2-naphthoic acid 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 34: Synthesis of 4-(5-phenyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-2)

[0138]

[0139] The synthesis method is the same as in Example 1, except that benzoic acid is used instead of 2-naphthoic acid and methyl 4-cyanobenzoate is used instead of methyl 3-cyanobenzoate 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 43 Synthesis of methyl 5-(5-phenyl-1,2,4-oxadiazol-3-yl)benzoate (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 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 53 Synthesis of (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-cyanobenzoamide is used instead of methyl 3-cyanobenzoate 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 63 Synthesis of (5-phenyl-1,3,4-oxadiazol-2-yl)benzoic acid (2-5)

[0150]

[0151] Benzoyl hydrazide (1.4 g, 10 mmol) and monomethyl m-benzoate (1.8 g, 10 mmol) were added to 20 mL of phosphorus oxychloride. The reaction system was heated to 120 °C for 18 hours under nitrogen protection. After cooling to room temperature, the reaction solution was poured into 500 mL of ice water. The pH of the reaction solution was adjusted to approximately 8-9 with sodium bicarbonate solid, and a precipitate was formed and filtered. The obtained solid was dissolved in 100 mL of methanol, and then 10 mL of 2N NaOH(aq) aqueous solution was added to the system. The reaction system was stirred vigorously at room temperature for 2 hours until the reaction was complete. Most of the organic solvent was evaporated, and 4N HCl solution was added dropwise until no more solid 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 73 Synthesis of (3-phenyl-1,2,4-oxadiazol-5-yl)benzoic acid (2-6)

[0154]

[0155] The synthesis method is the same as in Example 1, except that monomethyl isophthalate is used instead of 2-naphthoic acid, and benzonitrile is used instead of m-cyanobenzoate 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 83 - Synthesis of (5-tert-butyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-7)

[0158]

[0159] The synthesis method is the same as in Example 1, except that pentovalic acid is used instead of 2-naphthoic acid 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 93 - Synthesis of (5-cyclopentyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-8)

[0162]

[0163] The synthesis method is the same as in Example 1, except that cyclopentylcarboxylic acid is used instead of 2-naphthoic acid 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 103: Synthesis of (5-cyclohexyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-9)

[0166]

[0167] The synthesis method is the same as in Example 1, except that cyclohexylcarboxylic acid is used instead of 2-naphthoic acid 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-oxadiazol-3-yl)benzoic acid (2-10)

[0170]

[0171] The synthesis method is the same as in Example 1, except that bicyclic [2.2.1]heptane-2-carboxylic acid is used instead of 2-naphthoic acid 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 123 Synthesis of (5-adamantyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-11)

[0174]

[0175] The synthesis method is the same as in Example 1, except that adamantane carboxylic acid is used instead of 2-naphthoic acid 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 133 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 in Example 1, except that spiro[2.5]octane-6-carboxylic acid is used instead of 2-naphthoic acid 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 143 Synthesis of 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 in Example 1, except that bicyclic [4.1.0]heptane-7-carboxylic acid is used instead of 2-naphthoic acid 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 153 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 in Example 1, except that thiazole-2-carboxylic acid is used instead of 2-naphthoic acid 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 163 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 in Example 1, except that 5-methyl-1H-pyrazole-3-carboxylic acid is used instead of 2-naphthoic acid 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 173 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 in Example 1, except that pyridine-2-carboxylic acid is used instead of 2-naphthoic acid 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 183 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 in Example 1, except that pyridine-3-carboxylic acid is used instead of 2-naphthoic acid 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 193 Synthesis of 5-(pyridin-4-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-18)

[0202]

[0203] The synthesis method is the same as in Example 1, except that pyridine-4-carboxylic acid is used instead of 2-naphthoic acid 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 203 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 in Example 1, except that pyrimidine-2-carboxylic acid is used instead of 2-naphthoic acid 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 213 Synthesis of 5-(3-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-20)

[0210]

[0211] The synthesis method is the same as in Example 1, except that m-fluorobenzoic acid is used instead of 2-naphthoic acid 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 223 Synthesis of 5-(4-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-21)

[0214]

[0215] The synthesis method is the same as in Example 1, except that p-fluorobenzoic acid is used instead of 2-naphthoic acid 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 233 Synthesis of 5-(benzofuran-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-22)

[0218]

[0219] The synthesis method is the same as in Example 1, except that benzofuran-2-carboxylic acid is used instead of 2-naphthoic acid 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 243 Synthesis of 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 in Example 1, except that 1H-benzo[d]imidazol-2-carboxylic acid is used instead of 2-naphthoic acid 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 253 Synthesis of 5-(naphthyl-1-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-24)

[0226]

[0227] The synthesis method is the same as in Example 1, except that 1-naphthoic acid is used instead of 2-naphthoic acid 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 263 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 in Example 1, except that quinoline-3-carboxylic acid is used instead of 2-naphthoic acid 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 273 Synthesis of 3-(5-(quinoxalo-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-27)

[0234]

[0235] The synthesis method is the same as in Example 1, except that 2-quinoxalocarboxylic acid is used instead of 2-naphthoic acid 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 283 Synthesis of 5-(1,8-naphthid-3-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-28)

[0238]

[0239] The synthesis method is the same as in Example 1, except that 1,8-naphthidine-3-carboxylic acid is used instead of 2-naphthoic acid 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 293 Synthesis of 5-(1-bromonaphthyl-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (2-29)

[0242]

[0243] The synthesis method is the same as in Example 1, except that 1-bromonaphthyl-2-carboxylic acid is used instead of 2-naphthoic acid 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 303 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 in Example 1, except that 5-bromonaphthyl-2-carboxylic acid is used instead of 2-naphthoic acid 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 313 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 in Example 1, except that 7-bromonaphthyl-2-carboxylic acid is used instead of 2-naphthoic acid 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 323 Synthesis of (5-naphthylethyl-1,2,4-oxadiazol-3-yl)benzoic acid (2-32)

[0254]

[0255] The synthesis method is the same as in Example 1, except that 2-naphthoic acid is used instead of 2-naphthoic acid 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 333: Synthesis of 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 in Example 1, except that 1,2,3,4-tetrahydro-2-naphthoic acid is used instead of 2-naphthoic acid 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 the compound

[0262] Cell line construction: The IL-2 promoter-driven Luciferase gene was transfected into human Jurkat T cells (named J-luc cells), and CD137 was overexpressed. These cells were named Jurkat-CD137-luciferase (JC-luc). Endogenous CD137 in J-luc cells was knocked out using CRISPR / Cas9 to obtain J-luc-sgCD137 cells. CD137 is activated upon binding to its ligand CD137L or an agonist antibody, and promotes IL-2 expression by activating the downstream signaling NF-κB. Therefore, the activation capacity of CD137 in these cells can be characterized by measuring Luciferase activity.

[0263] Luciferase activity assay: JC-luc and J-luc-sgCD137 cells were placed in 96-well plates and treated with 1 μg / mL CD3 antibody and 5 μg / mL CD137 agonist antibody (aCD137) or the test compound for 6 hours. Luciferase activity was then detected.

[0264] EC 50 Determination of Luciferase activity: Different concentrations of the test drug were placed in 96-well plates with JC-luc cells, and 1 μg / mL of CD3 antibody was added for stimulation for 6 hours. Luciferase activity was detected, and its EC50 was calculated using GraphPadprism. 50 .

[0265] Experimental results and analysis: The results of the Luciferase experiment showed that (1) the aCD137 antibody could significantly activate JC-luc cells, but had no response to J-luc-sgCD137 cells. Figure 1 (A) This result proves that the constructed test model is feasible. (2) Most of the test compounds have the function of significantly activating CD137. Figure 1 (B in the text). Compound 2-25 (JNU-0921) showed the best activation activity against CD137, with its EC50... 50 The value is 64.07 nM ( Figure 1 (C) In the diagram, AT refers to Ataluren (PTC124), whose structural formula is:

[0266] Example 35: Experiment on the effect of compounds on mouse T cell effector function

[0267] (A) Detection of the effect of the test compound on the proliferation of mouse CD8+ T cells: Wild-type (WT) mice and CD137 knockout (CD137KO) mice were euthanized by cervical dislocation and immersed in 75% ethanol for 5 minutes. The mice were then removed, and their spleens were harvested by cutting open the left abdomen with sterile surgical scissors and forceps. The spleens were ground and filtered, maintaining aseptic technique throughout the process. The ground single-cell suspension was transferred to a 15 mL tube, and 3-5 times the volume of erythrocyte lysis buffer was added. After 5 minutes of centrifugation, the supernatant was discarded. The cells were resuspended in 1 mL PBS, filtered into a new 15 mL tube, centrifuged, and the supernatant was discarded. Culture medium was added to resuspend the cells in a single-cell suspension. The cells were counted, and 1 × 10⁻⁶ cells were collected. 7 Cells were stained with 5 μM carboxyfluorescein diacetate succinimide (CFSE) at 37°C for 10 minutes, then at 4°C for 5 minutes. The staining was terminated by adding complete culture medium. Cells were centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in PBS. The cells were then centrifuged again, the supernatant was discarded, and the cells were counted. 1×10⁻⁶ cells were then... 5 The stained cells were seeded in 96-well plates containing 1 μg / mL CD3 antibody, and treated with either aCD137 antibody (positive control) or 2 μM compound 2-25 (JNU-0921). Cells were harvested after 24 hours and stained with flow cytometry antibodies against CD8 and CD4. Finally, the fluorescence intensity of CFSE in CD8 and CD4 positive T cells was detected by flow cytometry. Diluted fluorescence indicated cell division.

[0268] (B) Detection of the effect of the test compound on the activation of mouse CD8+ T cells: Single-cell suspensions of spleen cells were prepared from WT mice and CD137KO mice according to the method described above. 1×10 5 Cells were placed in 96-well plates containing 1 μg / mL CD3 antibody, and 5 μg / mL aCD137 and 2 μM of the 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 spleen cells from WT and CD137KO mice were prepared according to the above method. Counting was performed using 1×102 cells. 7Cells were placed in 10cm dishes, and 1 μg / mL CD3 / CD28 antibody, 55 μM mercaptoethanol, and 10 ng / mL IL-2 were added to the culture medium. The cells were induced and cultured at 37°C in a 5% CO2 incubator for 3 days. After 3 days of induction, the cells were generally mature CTLs, which could be used for killing experiments. The mature CTLs induced for three days were used as effector cells, and P815-aCD3 cells expressing CD3 antibody were used as target cells. First, P815-aCD3 cells were collected and CFSE stained according to method (A). CTLs and CFSE-stained P815-aCD3 cells were placed in U-shaped 96-well plates at a ratio of 5:2 and co-incubated. 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 10 μg / mL PI was added for staining. Flow cytometry was used to detect the proportion of PI-positive cells among the CFSE-positive P815-aCD3 cells.

[0270] (D) Detection of the effect of the test compound on cytokine secretion function in mouse CD8+T and CD4+T cells. Single-cell suspensions of spleen cells were prepared from WT mice and CD137KO mice according to the above method. 1×10 5 Cells were placed in 96-well plates containing 1 μg / mL CD3 antibody, along with 5 μg / mL aCD137 and 100 nM of the test compound JNU-0921. Two hours before harvesting the cells, 0.5 μM of the protein transport inhibitor GolgiPlug was added. TM Processing. Finally, flow cytometry was used to detect the expression of cytokines on the surface of CD8+ and CD4+ T cells.

[0271] Experimental results and analysis:

[0272] 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) significantly promoted the proliferation of CD8+ and CD4+ T cells. Treatment with the test compound JNU-0921 also significantly promoted the proliferation and division of CD8+ and CD4+ T cells. This effect was not observed in CD137KO mice. Figure 2 (2) Treatment with aCD137 and the tested compound JNU-0921 significantly increased the expression of activation markers CD25 and CD69 in CD8+ and CD4+ T cells, while no change was observed in CD137KO cells. Figure 2 (3) Treatment with aCD137 and the tested compound JNU-0921 significantly enhanced the killing ability of CD8+ against target cells, while no change was observed in CD137KO cells. Figure 2 (4) Treatment with aCD137 and the tested compound JNU-0921 significantly enhanced the expression of cytokines IL-2, IFNγ, and perforin in CD8+ and CD4+ T cells. Figure 2 (JO in the middle).

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

[0274] (A) Establishment of the memory cell model: Single-cell suspensions were obtained from OT1 mice and OT1 / CD137KO mice according to the experimental method in Example 35(A), and 1×10⁻⁶ cells were counted. 7 Cells were placed in 10 cm dishes, and 10 nM MOVA257-264, 55 μM mercaptoethanol, and 10 ng / mL IL-2 were added to the culture medium. The cells were induced and cultured at 37°C in a 5% CO2 incubator for 3 days. Generally, mature CTLs were induced after 3 days. CTLs were then mixed with EG7 cells at a ratio of 5:2, and EG7 cells were added every three days. This stimulation was repeated three times to generate memory cells. During this process, 5 μg / mL aCD137 and 100 nM of the test compound JNU-0921 were added simultaneously. Flow cytometry was used to detect the proportion of memory cells and the expression of depletion factors on the surface of CD8+ T cells.

[0275] (B) Establishment of the LM-OVA mouse model: 2000 LM-OVA bacteria were reinfused into CD45.1WT mice via the tail vein. The next day, OT1 mice were used to obtain a single-cell suspension according to the experimental method in Example 35(A), and 1×10⁻⁶ cells were reinfused into the mice. 6 The cells were introduced into CD45.1WT mice. After treatment with 50 mg / kg of the test compound JNU-0921 for 45 days, the proportion of memory cells in the blood and spleen of the 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 analysis showed that (1) treatment with the CD137 agonist antibody aCD137 or the compound JNU-0921 significantly promoted the formation of CD44 / CD62L double-positive memory cells, while this effect was not observed in CD137KO cells. Figure 3 (2) Treatment with the agonist antibody aCD137 or compound JNU-0921 significantly reduced the expression of depleted markers PD-1, CTLA4, and TIM-3 on the surface of CD8+ T cells. Figure 3(3) Treatment with compound JNU-0921 increased the proportion of memory cells in mouse blood and spleen. Figure 3 (IK in the middle).

[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 × 10⁶ cells were used... 5 CD4+ T cells were incubated for 5 days in 96-well plates containing 1 μg / mL LCD3 / CD28 antibody, 10 ng / mL IL-2, 10 μg / mL anti-IL-4, 10 ng / mL IL-12, and 500 nM of the test compound JNU-0921 or 5 μg / mL aCD137. On day 6, the cells were harvested, washed once with PBS, and restimulated for 5 hours in medium containing 50 ng / mL LPMA, 1 μg / mL ionomycin, and monensin. The percentage of IFNγ+CD4+ T cells was determined 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 × 10⁶ cells were used... 5 CD4+ T cells were incubated for 6 days in 96-well plates containing 5 μg / mL Con A, 20 ng / mL IL-2, 50 ng / mL IL-4, and 500 nM of the test compound JNU-0921 or 5 μg / mL aCD137. On day 7, the cells were harvested, washed once with PBS, and restimulated for 5 hours in medium containing 1 μg / mL CD3 / CD28 antibody and monensin. The percentage of IL-4+ CD4+ T cells was determined by flow cytometry.

[0281] (C) For Th9 cell polarization, using 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 × 10⁶ cells were used... 5CD4+ T cells were incubated for 3 days in 96-well plates containing 10 ng / mL LTGF-β1, 10 ng / mL IL-4, 10 ng / mL IL-2, 10 μg / mL anti-IFNγ, and 500 nM of the test compound JNU-0921 or 5 μg / mL aCD137. On the fourth day, the cells were harvested, washed once with PBS, and then restimulated for 5 hours in medium containing 500 ng / mL PMA and 500 ng / mL ionomycin and monensin. The percentage of IL-9+ CD4+ T cells was determined 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 × 10⁶ cells were used... 5 CD4+ T cells were incubated for 5 days in 96-well plates containing 1 μg / mClC3 / CD28 antibody, 10 ng / mL IL-2, 5 ng / mL TGF-β, and 500 nM of the test compound JNU-0921 or 5 μg / mLaCD137. The percentage of Foxp3+CD4+ T cells was determined by flow cytometry.

[0283] Experimental results and analysis:

[0284] Flow cytometry results showed that (1) both the CD137 agonist antibody aCD137 and the test compound JNU-0921 increased the proportion of Th1 cells and inhibited the proportion of Th2 cells, but this effect was not observed in CD137KO mice. Figure 4 (2) Both aCD137 and the tested compound JNU-0921 can increase the proportion of Th9 cells and inhibit the proportion of Treg cells. Figure 4 The expression of EH in Treg cells was inhibited, as was the expression of activation markers ICOS and CTLA-4 in Treg cells, but this effect was not observed in CD137KO mice. Figure 4 (IL in the middle).

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

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

[0287] (B) Immunoprecipitation assay: Plasmids containing CD137 fusion Flag and MYC were constructed and co-transformed into 293T cells. After treatment with 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 assay: CD137-fused GFP and mcherry plasmids were constructed and co-transformed 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 at room temperature in the dark for 30 minutes. The cells were washed three times with PBS and stained with DAPI for 2 minutes. Images were taken using a laser scanning confocal microscope.

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

[0290] Example 39: Detection of the effect of the test compound on the growth of xenografts in mice.

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

[0292] Experimental Results and Analysis: In WT mice, compared with the control group, the test compound JNU-0921 significantly inhibited tumor growth and reduced tumor volume and weight. However, in Rag1 mice, there was no therapeutic effect. Figure 6 ).

[0293] The technical features of the above embodiments can be combined in any way. For the sake of brevity, 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 embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The use of bicyclic compounds having the structure shown in Formula (I), or pharmaceutically acceptable salts thereof, or stereoisomers thereof, in the preparation of CD137 agonists; in, T is selected from: 0, 1, 2; W, Y, and Z are independently selected from O and N, respectively, and the ring A containing W, Y, and Z is a heteroaromatic ring; Ring B containing X1, X2, X3, X4, and X5 is an aromatic ring; X1, X3, X4, and X5 are all CH, and X2 is CR. 2 Alternatively, X1, X2, X4, and X5 are all CH, and X3 is CR. 2 ; R is selected from: R 3 The following groups, whether substituted or unsubstituted: cyclohexyl, bicyclo[2.2.1]heptyl, adamantyl, spiro[2.5]octyl, bicyclo[4.1.0]heptyl, hexahydropyridyl, tetrahydropyranyl, tetrahydronaphthyl, furanyl, thiophene, pyrrole, pyrazolyl, thiazolyl, phenyl, naphthyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, 1,3,5-triazinyl, benzofuranyl, quinolinyl, isoquinolinyl, quinoxalinyl, benzopyrimidinyl, pyridopyrimidinyl, pyridopyridinyl, pteridinyl; Each R 2 Each is independently selected from: -C(=O)R 5 ; Each R 3 Each of the following is independently selected from: hydrogen, halogen, C1-C6 alkyl, and C1-C6 alkoxy; Each R 5 Each of the following is independently selected from: hydroxyl, amino, C1-C6 alkoxy, -NH-OH.

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

3. The application according to claim 1, characterized in that, Each R 2 Each is independently selected from: -C(=O)R 5 Each R 5 Each of the following is independently selected from: hydroxyl, amino, C1-C3 alkoxy, -NH-OH.

4. The application according to claim 3, characterized in that, Each R 2 Each group is independently selected from: carboxyl, methoxycarbonyl, and carbamoyl.

5. The application according to claim 1, characterized in that, Each R 3 Each of the following is independently selected from: hydrogen, halogen, C1-C3 alkyl, and C1-C3 alkoxy.

6. The application according to claim 5, characterized in that, Each R 3 Each of the following is independently selected from: hydrogen, methyl, ethyl, fluorine, chlorine, bromine, and methoxy.

7. The application according to any one of claims 1-6, characterized in that, R is selected from: phenyl, cyclohexyl, 。 8. The application according to any one of claims 1-6, characterized in that, R is selected from: R 3 The following groups may be substituted or unsubstituted: spiro[2.5]octyl, naphth-2-yl, tetrahydronaphthyl, benzofuranyl, quinolinyl, isoquinolinyl, quinoxalinyl, benzopyrimidinyl, pyridinylpyrimidinyl, pteridinyl.

9. The application according to claims 1-6, characterized in that, R is selected from: 。 10. The application according to claim 1, characterized in that, The bicyclic compounds are selected from the following compounds: 。 11. The application according to claim 1, characterized in that, The bicyclic compounds are selected from the following compounds: 。 12. The use of the bicyclic compound of any one of claims 1-11, or a pharmaceutically acceptable salt thereof, or a stereoisomer 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, a promoter for promoting the polarization of CD4+ T cells to a cellular immune state, or a promoter for promoting CD137 polymerization.

13. The use of the bicyclic compound of any one of claims 1-11, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a medicament for the prevention and / or treatment of tumors; wherein the medicament is a CD137 agonist.

14. The application according to claim 13, characterized in that, The drug can enhance the immune function of cytotoxic T lymphocytes, enhance the memory function of CD8+ T cells and CD4+ T cells, promote the polarization of CD4+ T cells to a cellular immune state, or promote CD137 polymerization.

15. The application according to claim 13, characterized in that, The tumors mentioned are: 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, and leukemia.

16. A bicyclic compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that, The bicyclic compounds are selected from the following compounds: 。 17. A pharmaceutical composition for the prevention and / or treatment of tumors, characterized in that, The pharmaceutical composition is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient includes the bicyclic compound of claim 16 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof; and the pharmaceutical composition for the prevention and / or treatment of tumors is a CD137 agonist.

Images