Adenosine 2A receptor modulators for treatment of cancer

Abstract

The present invention relates to new drugs and pharmaceutically acceptable salts thereof useful in the treatment of cancer, in particular by immunotherapy. In particular, the present invention provides novel A2AR modulators, pharmaceutical compositions, and uses thereof.

Description

Field of the Invention

[0001] The present invention relates to adenosine 2A receptor (A2AR) modulators useful for treating cancer, particularly by immunotherapy. Background of the Invention

[0003] Patients with a variety of solid tumors often experience high mortality and reduced quality of life, indicating an urgent need for new anti-tumor therapies. An important component of successful treatment of advanced or recurrent cancer is reversal of tumor-induced immunosuppression. Current immunotherapies, including immune checkpoint inhibitor (ICI) antibodies, have so far attempted to restore anti-tumor T cell responses by directly targeting these cells or antigen-presenting cells that activate T cells. Despite clear clinical benefits in approximately 30% of patients, response rates remain limited. This is in part because relatively large biomolecule drugs (such as antibodies) have limited penetration into solid tumors and drug-mediated effects are limited to a small subset of anti-tumor immune cells.

[0004] To improve response rates, other therapies aim to address the immunosuppressive tumor microenvironment (TME) (Labani-Motlagh et al., 2020, Front Immunol.; 11:940, doi:10.3389 / fimmu.2020.00940). Immunotherapies that can more easily penetrate solid tumors, such as small molecules (Zhong et. Al., 2020, Trans. Onc. https: / / doi.org / 10.1016 / j.tranon.2019.10.001), are also promising candidates for combined cancer therapy to enhance co-administered therapies that are only partially effective.

[0005] Adenosine is a naturally occurring purine nucleoside that is present both intracellularly and extracellularly in mammalian cells. Adenosine and its phosphorylated derivatives are involved in many biological processes, including energy transfer, cell signaling, and vasodilation. Extracellular adenosine (ExAdo) and its receptors are present in healthy tissues but are also overexpressed in a variety of solid tumors (Allard et al., 2020, Nat. Immunol., Nat Rev Clin Oncol., 17(10): 611-629, doi: 10.1038 / s41571-020-0382-2) and cancer mouse models (Sidders et al., 2020, Clin Cancer Res., 1, 26(9): 2176-2187 DOI: 10.1158 / 1078-0432.CCR-19-2183). Signaling through ExAdo has been shown to lead to immunosuppression of immune cells within the tumor microenvironment (TME) (Vigano et.al., 2019 doi:10.3389 / fimmu.2019.00925). ExAdo mediates this immunosuppression by binding to the extracellular domains of adenosine receptors 2A and 2B (A2AR and A2BR). Therefore, high extracellular concentrations of adenosine are a potent cause of immunosuppression in solid tumors, as adenosine inactivates tumor-targeting immune cells expressing A2AR. Adenosine has a significantly higher affinity for A2AR, which plays a more important role in T cell immunity than A2BR (Cekic, 2013, JEM, doi: 10.1084 / jem.20130249, Lukashev, 2003, BioChem Pharmacol., doi: 10.1016 / S0006-2952(03)00158-8). Compared with A2AR and A2BR, the other two adenosine receptors, A1R and A3R, have different downstream signaling pathways, and their signals usually do not lead to immunosuppression.

[0006] A2AR is primarily found on cells of the lymphocyte lineage, such as T cells and natural killer (NK) cells, as well as myeloid cells, including dendritic cells (DCs) and macrophages (Vigano et al., 2019, supra). Given the central role of NFκB in many proinflammatory and cytotoxic antitumor immune responses, the ExAdo signaling pathway could be a therapeutic target to restore desired immune responses in cancer patients. These responses include the patient’s own cellular immunity (T lymphocytes, natural killer cells, dendritic cells, macrophages) as well as the efficacy of co-administered immunotherapies such as immune checkpoint inhibitors (e.g., anti-PD-1, anti-PD-L1, anti-TIGIT, and anti-CTLA-4 monoclonal antibodies), bispecific antibodies, adoptive cellular immunotherapy (e.g., CAR-T cell infusions), and anticancer vaccines, all of which are enhanced in a favorable (proinflammatory) TME.

[0007] Blocking immunosuppressive adenosine signaling, particularly the A2AR, therefore represents a therapeutic strategy with broad applicability in cancer treatment, including in combination with other approved cancer therapies, including radiation therapy, targeted therapies for cancers with specific mutations, and conventional chemotherapy.

[0008] Small molecule drugs that block A2AR can be classified as either “orthosteric” or “allosteric” antagonists. Orthosteric antagonists bind to the same amino acid motif on A2AR that binds the endogenous receptor ligand, adenosine. Allosteric antagonists, on the other hand, bind to a different amino acid motif that is not utilized by the endogenous ligand. To effectively block A2AR signaling, orthosteric inhibitors must be more potent than adenosine, which is present in high concentrations in many tumors. Achieving this goal requires high doses of orthosteric A2AR antagonists, which increases the potential for drug-mediated adverse effects.

[0009] Orthosteric A2AR antagonists have been developed (Zhang et al., 2020, Pharmaceuticals, 13, 237; https: / / doi.org / 10.3390 / ph13090237 ; Franco et al., 2021, Cells, 10, 2831. https: / / doi.org / 10.3390 / cells10112831, but it has not entered Phase III clinical trials so far.

[0010] Currently, there is a clear medical need for active agents that can effectively function in the immunosuppressive tumor microenvironment to provide new immunotherapeutic options. SUMMARY OF THE INVENTION

[0012] The present invention relates to the unexpected discovery that cyanopyridine compounds are able to inhibit A2AR signaling by acting as A2AR antagonists, which are classified as negative allosteric modulators (NAMs) because they achieve inhibition of target signaling regardless of extracellular adenosine concentrations, including the high micromolar concentrations present within the TME.

[0013] The object of the present invention is to provide novel therapeutic agents which can be used to elicit or enhance the immune response to immunotherapy, in particular anti-cancer immunotherapy.

[0014] The first aspect of the present invention provides a cyanopyridine compound represented by formula (I), and a pharmaceutically acceptable salt, a tautomer and a geometric isomer thereof.

[0015] Another aspect of the present invention provides a pharmaceutical composition comprising at least one compound of the present invention and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0016] The first aspect of the present invention provides a use of a cyanopyridine compound represented by formula (I) and a pharmaceutically acceptable salt thereof in treating cancer, in particular, the cancer is a solid tumor cancer or a malignant tumor that is or is susceptible to resistance to immunotherapy.

[0017] Another aspect of the present invention is to provide a use of the compound of the present invention in preparing a pharmaceutical composition, especially for treating cancer, especially solid tumor cancer or malignant tumor that is or is prone to resistance to immunotherapy.

[0018] Another aspect of the present invention relates to a pharmaceutical composition comprising at least one compound described herein and at least one pharmaceutical agent useful for treating cancer.

[0019] Another aspect of the present invention provides a method for preventing and / or treating cancer resistance against anti-cancer immunotherapy in a subject, wherein the subject has cancer or is at risk of developing resistance to anti-cancer immunotherapy, the method comprising administering the compound of the present invention or a pharmaceutical preparation thereof to the subject in need thereof.

[0020] Another aspect of the present invention provides a method for inducing or enhancing an immune response to immunotherapy, particularly anti-cancer immunotherapy, the method comprising administering an effective amount of one or more compounds described herein or their pharmaceutical preparations, in combination with one or more therapies selected from radiotherapy, chemotherapy, adoptive cell therapy (e.g., chimeric antigen receptor T cells (CAR-T); or tumor infiltrating lymphocytes (TILs)), anti-cancer vaccine therapy, targeted biological therapy (e.g., tumor-specific antibodies) or immunomodulatory therapy such as immune checkpoint inhibitors, bispecific T cell engagers or nanobodies that can bind to single or multiple drug targets.

[0021] Another aspect of the present invention is a process for preparing the compounds of formula (I) as described below.

[0022] Other objects and advantages of the present invention will be apparent from the claims and / or the following detailed description of embodiments of the present invention with reference to the accompanying drawings.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Concentration-response curves (CRCs) and Schild regression plots (B) for adenosine (A) obtained by the shift assay of Example 2, in which compounds described in the present invention were used to confirm the allosteric mode of action. A: Shift assays were performed with compounds and data of compounds of the present invention (Example 11). Largest open symbol: 30 μM of compound, and half-log dilutions were performed until submicromolar concentrations of compound - represented by the smallest open circle. Closed circles: adenosine only control. B: Schild regression plots of the indicated compounds calculated using the shift assay data.

[0025] Detailed description of the invention

[0026] An "allosteric antagonist" is an antagonist that blocks receptor signaling without binding to all or part of the amino acids that the natural ligand (adenosine) uses to bind to the receptor.

[0027] “Solid tumor cancers” refers to all cancers except hematological cancers, including but not limited to lung cancer (small cell and non-small cell), breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, melanoma, digestive system cancers, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, and brain cancer.

[0028] According to one specific embodiment, solid tumor cancers with high adenosine signaling can be characterized by validated RNA transcript signatures in tumor biopsies (Sidders et al., 2020, Clin Cancer Res., 26(9):2176-2187, and doi.org / 10.1016 / j.coph.2020.08.003), pCREB levels in the blood (Seitz https: / / doi.org / 10.1007 / s10637-018-0706-6), or can be inferred using A2AR expression levels in tumor biopsies (Allard et al., 2020, supra)) or A2AR expression levels in circulating blood immune cells (DOI: 10.3233 / JAD-131652).

[0029] Solid cancers can be considered to have a “high adenosine signature” if more than 50% of solid cancer patients have a five-fold or greater increase in adenosine signature genes compared to healthy tissue controls, see Willingham 2020 Curr. Op. Pharm https: / / doi.org / 10.1016 / j.coph.2020.08.003. These cancers include digestive system cancers such as colon, stomach and pancreatic cancers, lung cancer, cervical cancer, and head and neck cancers.

[0030] "Immunotherapeutic agents" refer to agents that support the immune system to fight diseases such as cancer. There are currently several major categories: adoptive cell therapy (such as CAR-T cells or other tumor-infiltrating immune cells), immune checkpoint inhibitor monoclonal antibodies (ICImAbs), bispecific T cell engagers (BiTEs), novel immunomodulators or molecular adjuvants, and cancer vaccines, all of which can be conceived to be used in combination with the molecules of the present invention. In addition, standard forms of cancer treatment can be used in combination with the molecules of the present invention. These treatments include chemotherapy, radiotherapy, and targeted therapy (targeting specific cancer antigens or cancer-related molecular pathways). In particular, radiotherapy has been described as an appropriate treatment combination for A2AR-targeted approaches (Allard et al., 2020, supra).

[0031] The term "bispecific T cell engager" refers to agents such as antibodies that have one binding arm that recognizes a tumor antigen and another binding arm that recognizes a T cell surface antigen, as described and exemplified by Strohl et al., 2019, Antibodies, doi:10.3390 / antib8030041.

[0032] "Nanobodies" refer to agents similar to antibodies, but typically with much lower molecular weight, as described and exemplified by Bannas et. Al., 2017 Front. Immunology 10.3389 / fimmu.2017.01603.

[0033] As used herein, "treating" generally refers to obtaining a desired pharmacological and physiological effect. The effect may be prophylactic, in terms of preventing or partially preventing a disease, disorder, or symptom thereof, and / or therapeutic, in terms of partially or completely curing a disease, symptom, disorder, or adverse effect caused by the disease.

[0034] As used herein, the "efficacy" of treatment can be measured based on changes in the disease process in response to the uses or methods of the present invention. The efficacy of the treatment of cancer of the present invention can be measured by a reduction in tumor volume and / or an increase in progression-free survival time and / or an increase in the health and well-being of the subject (e.g., inhibition of cancer). The inhibition of cancer cell growth can be, for example, by blocking cancer cells at a specific stage of the cell cycle, such as blocking at the G2 / M stage of the cell cycle. Using known imaging methods, such as magnetic resonance imaging, computerized axial tomography, PET, SPECT, photoacoustic imaging, X-ray and fluorescence imaging / detection, the inhibition of cancer cell growth can also be verified. The growth of cancer cells can also be measured visually by caliper measurement.

[0035] In particular, the efficacy of the combined treatment according to the present invention can be evaluated by a reduction in tumor size, the disappearance of the tumor, or a change in the expression of a biomarker. These biomarkers may be soluble or expressed by cancer or immune system cells. Biomarkers that can be used to demonstrate changes in A2AR signaling or biological efficacy include intracellular cAMP and pCREB (Seitz et al., 2019, Investigational New Drugs, 37, 711–721; https: / / doi.org / 10.1007 / s10637-018-0706-6), or are generally characterized as downstream effector molecules of anti-tumor immune responses, including the secretion of IL-2, TNF-α or IFN-γ cytokines (Willingham et al., 2018, Cancer Immunol. Res., 6 (10): 1136-1149; DOI: 10.1158 / 2326-6066.CIR-18-005).

[0036] As used herein, the term "subject" refers to a human or a non-human mammal, such as a non-human primate (e.g., gorillas and other apes and monkeys), a farm animal (e.g., cattle, sheep, pigs, goats, and horses), a domestic mammal (e.g., dogs and cats), or a laboratory animal (e.g., a rodent, such as mice, rats, and guinea pigs).

[0037] The "effectiveness" of a treatment according to the invention can be measured based on changes in the disease process in response to the uses or methods described herein. For example, the efficacy of a treatment according to the invention can be measured by its effect on disease signs or symptoms. A response is achieved when a subject experiences partial or complete remission, or a reduction in unwanted disease symptoms. According to a specific embodiment, efficacy can be determined by evaluating toxin target cleavage or viral replication following infection. For example, the efficacy of a toxin or antiviral treatment according to the invention can be monitored by tracking the effects on MAP kinase 1 cleavage or by improving cell survival, tissue damage, and patient survival.

[0038] The term "aryl" refers to an unsaturated aromatic carbocyclic group having 6 to 14 carbon atoms, having a single ring (eg, phenyl) or a polycyclic condensed ring (eg, indenyl, naphthyl). Aryl includes phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like.

[0039] The term "heteroaryl" refers to a monocyclic or bicyclic unsaturated aromatic group of 5 to 10 ring atoms, wherein one or more of the ring atoms are selected from N, O or S. Specific examples of heteroaryl include optionally substituted pyridyl, pyrrolyl, pyrimidinyl, furanyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuranyl, [2,3-dihydro]benzofuranyl, isobenzofuranyl, benzothienyl, benzotriazolyl, isothiazolyl, benzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyridinyl, benzothiazolyl, benzoxazolyl, quinolyl, quinazolinyl, phthalazinyl, quinoxalinyl, cinnozinyl, naphthyridinyl, pyrido[3,4-b]pyridinyl, pyrido[3,2-b]pyridinyl, pyrido[4,3-b]pyridinyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, thioxanthenyl or benzoquinolyl.

[0040] The term "heteroalkyl" refers to a C1-C 12 Alkyl, preferably C1-C6 alkyl, wherein at least one carbon is substituted by a heteroatom selected from O, N or S, including 2-methoxyethyl and the like.

[0041] The term "heterocycloalkyl" refers to a C3-C8-cycloalkyl group according to the above definition, wherein up to 3 carbon atoms are replaced by heteroatoms selected from O, S, NR, and R is defined as hydrogen or methyl. Heterocycloalkyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl, and the like.

[0042] The term "alkoxycarbonyl C1-C6 alkyl" refers to a C1-C6 alkyl group having an alkoxycarbonyl substituent, including 2-(benzyloxycarbonyl)ethyl and the like.

[0043] Unless limited to the definition of a single substituent, the term "substituted" refers to a group that is substituted with from 1 to 5 substituents selected from halogen, cyano, nitro, hydroxy, amino (-NH2, -NH-, -N-), amide (-NHC(O)-), carbonyl (-C(O)-), alkoxycarbonyl (-C(O)O-), carboxylic acid, ether (-O-), thioether (-S-), sulfoxide (-S(O)-) and sulfone (-S(O)2-), C1-C6 alkyl, aryl or heteroaryl.

[0044] A "pharmaceutical preparation" refers to a preparation that is in a form that permits the biological activity of the active ingredient to be unequivocally effective and that contains no additional ingredients that would be toxic to a subject to which the preparation would be administered.

[0045] The compounds of the present invention

[0046] According to one aspect, a cyanopyridine compound of formula (I) for use in treating cancer, particularly for use in immunotherapy to treat cancer, and a pharmaceutically acceptable salt thereof are provided;

[0047]

[0048] in,

[0049] R1 is selected from aryl (e.g., optionally substituted phenyl, such as phenyl, halophenyl, or phenyl optionally substituted by C1-C6 alkyl, such as methyl) optionally substituted by one or more halogen (e.g., fluorine), cyano, hydroxyl, or C1-C6 alkyl, and heteroaryl (e.g., optionally substituted pyridyl, optionally substituted pyrimidinyl, optionally substituted pyridazinyl, optionally substituted furanyl, optionally substituted indolyl, optionally substituted thienyl, optionally substituted pyrazolyl, optionally substituted imidazolyl, optionally substituted oxazolyl, optionally substituted thiazolyl, optionally substituted isoxazolyl, optionally substituted isothiazolyl, optionally substituted triazolyl, optionally substituted oxadiazolyl, optionally substituted thiadiazolyl, and optionally substituted tetrazolyl) optionally substituted by one or more halogen, cyano, hydroxyl, or C1-C6 alkyl;

[0050] R2 is selected from H and halogen;

[0051] R3 is selected from H, CN and optionally substituted C1-C6 alkyl (e.g., C1-C6 alkyl optionally substituted with hydroxyl, amino, ether, thioether, cyano, halogen or branched C1-C6 alkyl);

[0052] R4 is selected from XR(R'), optionally substituted aryl (e.g., optionally substituted phenyl, such as phenyl, halophenyl or phenyl optionally substituted by C1-C6 alkyl, such as methyl), and optionally substituted heteroaryl (e.g., optionally substituted pyridyl, optionally substituted pyrimidinyl, optionally substituted pyridazinyl, optionally substituted furanyl, optionally substituted indolyl, optionally substituted thienyl, optionally substituted pyrazolyl, optionally substituted imidazolyl, optionally substituted oxazolyl, optionally substituted thiazolyl, optionally substituted isoxazolyl, optionally substituted isothiazolyl, optionally substituted triazolyl, optionally substituted oxadiazolyl, any wherein X is selected from O, S, N; R and R' are each independently an optionally substituted C1-C6 alkyl group (e.g., a C1-C6 alkyl group optionally substituted with hydroxyl, amino, ether, thioether, cyano, halogen, aryl, heteroaryl or branched C1-C6 alkyl); or XRR' together forms an optionally substituted heterocycloalkyl group (e.g., an optionally substituted azetidinyl group, an optionally substituted pyrrolidinyl group, an optionally substituted piperidinyl group, an optionally substituted morpholinyl group, an optionally substituted piperazinyl group, an optionally substituted azepanyl group, an optionally substituted azacyclooctanyl group).

[0053] According to another aspect, the cyanopyridine compound of the present invention is a compound represented by formula (Ia), and a pharmaceutically acceptable salt thereof;

[0054]

[0055] in,

[0056] R1 and R2 are as defined herein;

[0057] X is selected from O and S;

[0058] R5 is an optionally substituted C1-C6 alkyl group (eg, a C1-C6 alkyl group optionally substituted by a hydroxyl group, an amino group, an ether group, a thioether group, a cyano group, a halogen group, an aryl group, a heteroaryl group, or a branched C1-C6 alkyl group).

[0059] According to another aspect, the cyanopyridine compound of the present invention is a compound represented by formula (Ib), and a pharmaceutically acceptable salt thereof;

[0060]

[0061] in,

[0062] R1 and R2 are as defined herein;

[0063] R6 and R7 are each independently an optionally substituted C1-C6 alkyl group (e.g., a C1-C6 alkyl group optionally substituted with hydroxy, amino, ether, thioether, cyano, halogen, aryl, heteroaryl or branched C1-C6 alkyl); or R6 and R7 together form an optionally substituted heterocycloalkyl group (e.g., an optionally substituted azetidinyl group, an optionally substituted pyrrolidinyl group, an optionally substituted piperidinyl group, an optionally substituted morpholinyl group, an optionally substituted piperazinyl group, an optionally substituted azepanyl group, an optionally substituted azacyclooctanyl group).

[0064] According to another aspect, the cyanopyridine compound of the present invention is a compound represented by formula (I), wherein R3 is selected from H and optionally substituted C1-C6 alkyl; R4 is selected from optionally substituted aryl (for example, optionally substituted phenyl, such as phenyl, halophenyl or phenyl optionally substituted by C1-C6 alkyl such as methyl), and optionally substituted heteroaryl (for example, optionally substituted pyridyl, optionally substituted pyrimidinyl, optionally substituted pyridazinyl, optionally substituted furanyl, optionally substituted indolyl, optionally substituted thienyl, optionally substituted pyrazolyl, optionally substituted imidazolyl, optionally substituted oxazolyl, optionally substituted thiazolyl, optionally substituted isoxazolyl, optionally substituted isothiazolyl, optionally substituted triazolyl, optionally substituted oxadiazolyl, optionally substituted thiadiazolyl and optionally substituted tetrazolyl).

[0065] According to another aspect, a cyanopyridine compound represented by formula (I) is provided:

[0066]

[0067] in,

[0068] R1 to R4 are as defined herein, provided that the compound represented by formula (I) is not a compound selected from the group consisting of:

[0069] 4-(3-(1H-pyrrol-1-yl)phenyl)-2-amino-6-(benzylthio)pyridine-3,5-dicarbonitrile (RN: 391667-62-2); 2-amino-6-(benzylthio)-4-(3-(2,5-dimethyl-1H-pyrrol-1-yl)phenyl)pyridine-3,5-dicarbonitrile (RN: 391664-25-8); 4-(3-(1H-pyrrol-1-yl)phenyl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile (RN: 391664-24-7); 4-(3-(1H-imidazol-1-yl)phenyl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile ; 4-(3-(1H-imidazol-2-yl)phenyl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile; 2-amino-4-(3'-cyano-5'-fluoro-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-41-2); 2-amino-4-(3'-(hydroxymethyl)-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-42-3); 2-amino-4-(4'-chloro-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 16461 73-50-3); 2-amino-4-(3'-chloro-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-49-0); 2-amino-4-(2'-chloro-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-48-9); 2-amino-6-phenyl-4-(4'-(trifluoromethyl)-[1,1'-biphenyl]-3-yl)pyridine-3,5-dicarbonitrile (RN: 1646173-44-5); 2-amino-6-phenyl-4-(3'-(trifluoromethyl)-[1,1' -biphenyl]-3-yl)pyridine-3,5-dicarbonitrile (RN: 1646173-46-7); 2-amino-4-(3',4'-dichloro-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-51-4); 2-amino-4-(4'-bromo-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-55-8); or 2-amino-4-(2'-methyl-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-54-7).

[0070] According to another aspect, a cyanopyridine compound of formula (I) for use as a medicament is provided;

[0071]

[0072] in,

[0073] R1 to R4 are as defined herein, with the proviso as described above.

[0074] In a particular embodiment, R1 is aryl optionally substituted with one or more halogen (eg fluorine), cyano, hydroxyl or C1-C6 alkyl (eg methyl).

[0075] In another particular embodiment, R1 is phenyl optionally substituted with one or more halogen (eg fluorine), cyano, hydroxyl or C1-C6 alkyl (eg methyl).

[0076] In another specific embodiment, R1 is phenyl optionally substituted with one or more halogen or C1-C6 alkyl, for example phenyl, halophenyl such as 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, or C1-C6 alkylphenyl such as methylphenyl (e.g. 2-methylphenyl, 3-methylphenyl, 4-methylphenyl).

[0077] In another specific embodiment, R1 is heteroaryl optionally substituted with one or more halogen, cyano, hydroxyl or C1-C6 alkyl.

[0078] In another specific embodiment, R1 is selected from optionally substituted pyridinyl (e.g. pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), optionally substituted thienyl (e.g. thien-2-yl, thien-3-yl), optionally substituted pyrazolyl (e.g. pyrazol-5-yl) and optionally substituted tetrazolyl, wherein optionally substituted means optionally substituted by one or more halogen, cyano, hydroxyl or C1-C6 alkyl.

[0079] In another specific embodiment, R1 is optionally substituted pyridinyl, wherein optionally substituted means optionally substituted by one or more halogen, cyano, hydroxyl or C1-C6 alkyl, for example pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 3-fluoro-2-pyridinyl, 2-fluoro-3-pyridinyl, 6-fluoro-2-pyridinyl, 4-fluoro-2-pyridinyl and 5-fluoro-3-pyridinyl.

[0080] In another particular embodiment, R1 is optionally substituted oxazolyl, such as 1,3,4-oxadiazolyl.

[0081] In another specific embodiment, R1 is unsubstituted heteroaryl.

[0082] In a particular embodiment, R2 is H.

[0083] In a particular embodiment, R3 is CN.

[0084] In a particular embodiment, R4 is an XR(R') group as defined herein.

[0085] In a particular embodiment, R4 is OR, wherein R is optionally substituted alkyl, such as methyl, ethyl, hydroxyethyl.

[0086] In a particular embodiment, R4 is a group SR, wherein R is optionally substituted alkyl, such as optionally substituted ethylphenyl.

[0087] In another specific embodiment, R4 is an XR(R') group, wherein XRR' together form an optionally substituted heterocycloalkyl (such as optionally substituted azetidinyl, optionally substituted pyrrolidinyl, optionally substituted piperidinyl, optionally substituted morpholinyl, optionally substituted piperazinyl, optionally substituted azepanyl, optionally substituted azocanyl).

[0088] In another specific embodiment, R4 is selected from pyridinyl, pyrrolidinyl and azetidinyl.

[0089] In another specific embodiment, R4 is optionally substituted pyridinyl.

[0090] In another specific embodiment, R4 is piperidinyl.

[0091] In another specific embodiment, X is N.

[0092] In another specific embodiment, X is O.

[0093] In another specific embodiment, X is S.

[0094] According to another aspect, the cyanopyridine compound is a compound represented by formula (Ib), wherein R6 and R7 together form an optionally substituted heterocycloalkyl group.

[0095] According to another aspect, the cyanopyridine compound is a compound represented by formula (Ib), wherein R6 and R7 together form an optionally substituted azetidine or an optionally substituted piperidinyl.

[0096] According to a specific embodiment, a negative allosteric modulator (NAMs) of A2AR is provided.

[0097] In particular, the compounds described in the present invention include compounds selected from the group consisting of:

[0098] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(azetidin-1-yl)pyridine-3,5-dicarbonitrile;

[0099] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(pyrrolidin-1-yl)pyridine-3,5-dicarbonitrile;

[0100] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0101] 2-amino-4-(2'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0102] 2-amino-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0103] 2-Amino-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile

[0104] 2-amino-4-(2'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0105] 2-Amino-4-(3'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile

[0106] 2-amino-4-(4'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0107] 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-2-yl)phenyl)pyridine-3,5-dicarbonitrile;

[0108] 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-3-yl)phenyl)pyridine-3,5-dicarbonitrile;

[0109] 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-4-yl)phenyl)pyridine-3,5-dicarbonitrile;

[0110] 2-amino-6-(piperidin-1-yl)-4-(3-(thiophen-2-yl)phenyl)pyridine-3,5-dicarbonitrile;

[0111] 2-amino-6-(piperidin-1-yl)-4-(3-(thiophen-3-yl)phenyl)pyridine-3,5-dicarbonitrile;

[0112] 4-(3-(1H-pyrazol-5-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0113] 4-(3-(1H-tetrazolyl-5-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0114] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(diethylamino)pyridine-3,5-dicarbonitrile;

[0115] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-morpholinylpyridine-3,5-dicarbonitrile;

[0116] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(bis(2-hydroxyethyl)amino)pyridine-3,5-dicarbonitrile;

[0117] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(4-methylpiperazin-1-yl)pyridine-3,5-dicarbonitrile;

[0118] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-((2-hydroxyethyl)amino)pyridine-3,5-dicarbonitrile;

[0119] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(diisopropylamino)pyridine-3,5-dicarbonitrile;

[0120] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(benzylamino)pyridine-3,5-dicarbonitrile;

[0121] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(benzylthio)pyridine-3,5-dicarbonitrile;

[0122] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile;

[0123] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-ethoxypyridine-3,5-dicarbonitrile;

[0124] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(2-hydroxyethoxy)pyridine-3,5-dicarbonitrile;

[0125] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(benzyloxy)pyridine-3,5-dicarbonitrile;

[0126] 2-amino-4-(3-(3-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0127] 2-amino-4-(3-(6-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0128] 2-amino-4-(3-(4-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0129] 2-amino-4-(3-(5-fluoropyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0130] 2-amino-4-(3-(2-fluoropyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0131] 2-amino-4-(2-fluoro-5-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0132] 2-amino-4-(2-fluoro-3-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0133] 2-amino-4-(3-fluoro-5-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile and

[0134] 4-(3-(1,3,4-oxadiazol-2-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile.

[0135] According to a specific embodiment, the compound of the present invention is selected from the following group:

[0136] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(azetidin-1-yl)pyridine-3,5-dicarbonitrile;

[0137] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(pyrrolidin-1-yl)pyridine-3,5-dicarbonitrile;

[0138] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0139] 2-amino-4-(2'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0140] 2-amino-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0141] 2-Amino-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile

[0142] 2-amino-4-(2'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0143] 2-Amino-4-(3'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile

[0144] 2-amino-4-(4'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile;

[0145] 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-2-yl)phenyl)pyridine-3,5-dicarbonitrile;

[0146] 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-3-yl)phenyl)pyridine-3,5-dicarbonitrile;

[0147] 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-4-yl)phenyl)pyridine-3,5-dicarbonitrile;

[0148] 2-amino-6-(piperidin-1-yl)-4-(3-(thiophen-2-yl)phenyl)pyridine-3,5-dicarbonitrile;

[0149] 2-amino-6-(piperidin-1-yl)-4-(3-(thiophen-3-yl)phenyl)pyridine-3,5-dicarbonitrile;

[0150] 4-(3-(1H-pyrazol-5-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; and

[0151] 4-(3-(1H-tetrazol-5-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile.

[0152] The compounds of the present invention have been prepared according to Chemdraw IUPAC standard nomenclature used in the program (product version 16.0).

[0153] Composition

[0154] The pharmaceutical composition of the present invention may comprise one or more compounds of the present invention and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0155] According to another specific aspect, the A2AR modulating effect can be achieved by delivering the pharmaceutical formulation compounds of the present invention by various routes, preferably orally.

[0156] According to a particular aspect, the composition further comprises a compound useful for preventing and / or treating cancer, in particular for cancer immunotherapy.

[0157] The compositions of the present invention may also be formulated for parenteral administration, including but not limited to injection or continuous infusion. Injectable formulations may be in the form of suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulating agents, including but not limited to suspending agents, stabilizers and dispersants. The compositions may also be provided in powder form for reconstitution with a suitable carrier, including but not limited to sterile, pyrogen-free water.

[0158] According to a particular embodiment, the composition of the invention is for oral delivery.

[0159] In another specific aspect, the compositions of the invention are suitable for delivery via single or multiple administrations.

[0160] According to a particular embodiment, the composition of the invention is a veterinary composition.

[0161] Other materials and preparation processing technologies are described in "Remington: The Science & Practice of Pharmacy, 23 rd Edition, 2020, Ed. Adeboye Adejare, Academic Press", which is incorporated herein by reference.

[0162] According to a specific aspect, a pharmaceutical composition is provided, comprising at least one compound according to the present invention and a pharmaceutically acceptable carrier, diluent or excipient.

[0163] The present invention provides compounds of the present invention, compositions thereof and methods of using the same, which can be used for preventing and / or treating medical diseases, in particular as A2AR modulators for preventing and / or treating cancer, in particular for immunotherapy.

[0164] Dosage

[0165] The compounds and compositions of the present invention can be administered or delivered in any manner, including but not limited to oral, parenteral, sublingual, transdermal, transmucosal, topical, inhaled, intratumoral or peritumoral, oral or intranasal administration, or a combination thereof. Parenteral administration includes but is not limited to intravenous, intraarterial, intraperitoneal, subcutaneous and intramuscular administration.

[0166] In another specific embodiment, the compounds of the invention are administered orally.

[0167] In another specific embodiment, the compounds of the invention are administered rectally.

[0168] In another specific embodiment, the compounds of the invention are administered intravenously.

[0169] The dose (single or multiple) administered to an individual will vary depending on a variety of factors, including pharmacokinetic properties, subject condition and characteristics (sex, age, weight, health and size), degree of symptoms, concomitant therapy, frequency of treatment and desired effect.

[0170] Combined use

[0171] According to one embodiment of the present invention, the compound and pharmaceutical preparation thereof described in the present invention can be administered alone or in combination with a pharmaceutical agent for preventing and / or treating a disease.

[0172] According to one aspect, the compounds of the present invention can be administered in combination with at least one therapeutic molecule for the treatment of cancer, in particular in cancer immunotherapy.

[0173] According to one aspect, the compounds described in the present invention will be used in combination with one or more therapies selected from radiotherapy, chemotherapy, adoptive cell therapy (e.g., chimeric antigen receptor T cells (CAR-T), or tumor infiltrating lymphocytes (TILs)), anti-cancer vaccine therapy, targeted biological therapy (e.g., tumor-specific antibodies) or immunomodulatory therapy such as immune checkpoint inhibitors, bispecific T cell engagers or nanobodies that can bind to single or multiple drug targets.

[0174] According to one aspect, the compounds described in the present invention can be administered in combination with an anti-cancer vaccine or at least one immune checkpoint inhibitor (such as at least one PD-1, PD-L1 or CTLA4 inhibitor, or a combination thereof).

[0175] According to another aspect, at least one immune checkpoint inhibitor is selected from a PD-1 inhibitor and a CTLA4 inhibitor.

[0176] According to another aspect, the compounds of the present invention will be administered in combination with an anti-cancer vaccine, wherein the anti-cancer vaccine elicits cancer-specific immunity, and the compounds of the present invention will be administered before and / or after the administration of the anti-cancer vaccine.

[0177] According to another aspect, the compounds of the present invention are used in combination with cell therapy, for example, with chimeric antigen receptor (CAR) T cells (CAR T cell therapy) or tumor infiltrating lymphocytes (TILs) (TIL cell-based bulk cell therapy) that have been expanded ex vivo. Chimeric antigen receptor (CAR) T cells are expanded in vitro according to standard methods, comprising:

[0178] - providing at least one transgenic T cell in a T cell culture medium, wherein the transgenic T cell expresses a polypeptide specific for a tumor-associated antigen on its membrane surface;

[0179] - Expanding and harvesting the genetically modified T cells in said culture medium.

[0180] TILs are expanded in vitro according to standard methods, which include:

[0181] - Isolation of polyclonal mixtures of T cells from tumor fragments;

[0182] - Expanding these polyclonal T cells in culture.

[0183] According to another aspect, the compounds described in the present invention will be administered in combination with the expanded chimeric antigen receptor (CAR) T cells or TILs via the same or different routes.

[0184] The present invention includes the administration of the compounds of the invention, wherein the compounds are administered to the subject before, simultaneously or sequentially with the treatment regimen or at least one combination agent. The compounds of the invention administered simultaneously with the at least one combination agent can be administered in the same or different compositions and in the same or different routes of administration.

[0185] Object

[0186] In one embodiment, the subject described herein suffers from cancer.

[0187] In another specific embodiment, the subject described herein is a subject suffering from a solid tumor cancer selected from, but not limited to, lung cancer (small cell and non-small cell), breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, melanoma, digestive system cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, and liver cancer.

[0188] In one embodiment, a subject according to the invention is a subject having a tumor with elevated adenosine signaling gene signature, pCREB expression or A2AR expression as described above.

[0189] In another specific embodiment, the subject according to the present invention is a subject suffering from a solid tumor cancer selected from cancers of the digestive system, in particular pancreatic cancer and colorectal cancer.

[0190] Uses of the present invention

[0191] In a specific embodiment, the present invention provides compounds, methods, uses and compositions useful for treating cancer, particularly for immunotherapy of cancer.

[0192] According to another specific embodiment, the methods, uses and compositions described herein can be used to reduce the development of tumors and / or enhance other cancer treatments (including radiotherapy, chemotherapy, adoptive cell therapy (e.g., CAR-T), anti-cancer vaccine therapy, targeted biological therapy (e.g., tumor-specific antibodies) or immunomodulatory therapy such as immune checkpoint inhibitors).

[0193] Another aspect of the present invention provides a method for preventing and / or treating cancer resistance against anti-cancer immunotherapy in a subject, wherein the subject has cancer or is at risk of developing resistance to anti-cancer immunotherapy, the method comprising administering the compound of the present invention or a pharmaceutical preparation thereof to the subject in need thereof.

[0194] Another aspect of the present invention provides a method for inducing or enhancing an immune response to immunotherapy, particularly anti-cancer immunotherapy, which comprises administering an effective amount of one or more compounds described herein or pharmaceutical preparations thereof to a subject in need thereof, in combination with an immunotherapeutic agent or radiotherapy.

[0195] In a specific embodiment, the present invention provides compounds, methods, uses and compositions for treating solid tumor cancers in combination, wherein at least one compound described herein is administered in combination with at least one anti-cancer immunotherapeutic agent.

[0196] According to a particular aspect, there is provided a method of treating a subject suffering from cancer, the method comprising administering to a subject in need thereof an effective amount of one or more compounds of the invention in combination with at least one anti-cancer treatment, particularly an anti-cancer immunotherapeutic agent.

[0197] According to another specific embodiment, the compounds, methods and compositions of the present invention are useful in combination with cell therapy, particularly for improving the immunosuppressive tumor microenvironment, thereby enhancing the infiltration of infused cell therapy into the tumor. According to a further aspect, the combination enhances the ability of T cells to kill cancer cells at the site of the disease.

[0198] References cited herein are hereby incorporated by reference in their entirety. Such modifications are intended to fall within the scope of the appended claims. Having described the present invention, the following examples are presented by way of illustration and not limitation.

[0199] Synthesis of the compounds of the present invention

[0200] The compound shown in formula (I) can use the following general method and be prepared by readily available raw materials. It should be understood that, in the case of typical or preferred experimental conditions (i.e. reaction temperature, time, reagent mole number, solvent, etc.), other experimental conditions may also be used unless otherwise indicated. Optimum reaction conditions may vary due to the specific reactants or solvents used, but those skilled in the art can use conventional optimization processes to determine these conditions.

[0201] General synthetic methods for preparing compounds of formula (I) are described in Schemes 1 to 5 below.

[0202] Solution 1

[0203]

[0204] Scheme 1 shows a single-step process for preparing compounds of the invention by displacing a cyanopyridine of formula (i) with a commercial or synthetic nucleophile of formula (ii) in the presence of a base such as trimethylamine in an aprotic solvent such as THF, wherein X, R and R' are as defined herein. The synthesis of cyanopyridines of formula (I) is well documented in the literature and can be prepared by the two-step protocol described by Murray et al., 1995 (Tetrahedron, 1995, 51, 635, DOI: 10.1016 / 0040-4020(94)00922-H) or Piper et al., 1986 (J. Med. Chem., 29, 1080, DOI: 10.1021 / jm00156a029) or the one-pot protocol described by Duindam et al., 1993 (Synthetic Commun., 23, 2605, DOI: 10.1080 / 00397919308012595).

[0205] Solution 2

[0206]

[0207] Scheme 2 shows the cross-coupling of the intermediates shown in formula (iii) and (iv) in the presence of an inorganic base (such as K2CO3) and a catalytic amount of a palladium source (such as palladium dichloride (II) or tetrakis (triphenylphosphine) palladium (0)), wherein R1, R2, R3 and R4 are as described above, and R8 and R9 are selected from halogen, boronic acid or boronic acid pinacol ester groups. The cross-coupling step can be carried out under thermal conditions in a non-polar solvent such as toluene, also known as thermal activation (as described in Pan et al., 2008, Catal. Commun., 9, 508-510), or under microwave irradiation in a polar solvent (such as methanol, water, etc.).

[0208] Solution 3

[0209]

[0210] Scheme 3 shows a specific embodiment of Scheme 2, which is used to synthesize the intermediate of formula (iiib) by cross-coupling reaction between the bromoaryl group shown in formula (iiia) and the bis(pinacol)diboron shown in commercial formula (v) under thermal activation of microwave irradiation as described in Scheme 2, wherein R3 and R4 are as described above.

[0211] Solution 4

[0212]

[0213] Scheme 4 shows the preparation of a compound of formula (Ib) in a one-step process by cyclizing a cyanopyridine with a commercial or synthetic aldehyde of formula (vi) in the presence of a cyclic amine of formula (vii), two equivalents of malononitrile (viii), a final catalyst (e.g., 4-(dimethylamino)pyridine (4-DMAP)) and an oxidant (e.g., air), in a polar protic solvent (e.g., methanol), wherein R1 and R2 are as described above, and R6 and R7 together form an optionally substituted heterocycloalkyl group as defined herein.

[0214] Solution 5

[0215]

[0216] Scheme 5 shows the preparation of the intermediate compound of formula (iiic) in a two-step process. First, the synthesis of the intermediate of formula (vi) can be achieved by reacting the commercially available bromobenzaldehyde of formula (ix) in the presence of one equivalent of malononitrile (viii), a final catalytic amount of a cyclic amine of formula (vii), an oxidant (such as air) and a polar protic solvent (such as methanol), wherein R2 is as described above, and R6 and R7 together form an optionally substituted heterocycloalkyl as defined herein. The isolated intermediate (vi) can be further reacted in the presence of one equivalent of malononitrile (iii) and one equivalent of an amine of formula (vii).

[0217] This two-step method is more suitable for obtaining gram quantities.

[0218] As described above, compound (iiic) is a specific example of compound (iiia), and thus can be used in the synthesis of the compound represented by (I) of the present invention according to the method shown in Scheme 3. Example

[0219] The following abbreviations refer to the following definitions:

[0220] CV (column void volume); cyclo (cyclohexane); DCM (dichloromethane); DHP (2,4-dihydropyran); DMF (dimethylformamide); eq. (equivalent (mol %)); ESI (electrospray ionization); HRMS (high resolution mass spectrometry); NMR (nuclear magnetic resonance (); PBS (phosphate buffered saline); rt (room temperature); TFA (trifluoroacetic acid); TLC (thin layer chromatography); TOF (time of flight); UPLC (ultra-performance liquid chromatography); UV (ultraviolet light).

[0221] Example 1: Synthesis of the compounds of the present invention

[0222] The compounds of the present invention were prepared according to Schemes 1 to 5 as follows.

[0223] General Experimental: Unless otherwise stated, all products were obtained from commercial sources and used without further purification. Anhydrous solvents such as methanol, DMF or toluene were obtained by passing through molecular sieves. Closed.

[0224] All preparative columns were flash chromatographed on a Buchi Pure C-815Flash system with UV detector. The corresponding PureFlash ID cartridges (4 g, 12 g, 24 g, 40 g or 120 g, amorphous silica gel, 35-45 μm mesh) were purchased from Buchi, and the flow rate was set according to the preset parameters (15, 30, 32, 45 and 85 ml / min, respectively). The samples were loaded as solid sediments, prepared with 40-60 μm amorphous silica gel mesh.

[0225] Unless otherwise stated, all yields reported are isolated yields.

[0226] 1 H NMR spectra were recorded on a Bruker 600 MHz spectrometer equipped with a cryoprobe and calibrated on residual protonated solvent. 13 C NMR spectra, the solvent resonance was used as an internal standard. 1 HNMR and 13 C NMR chemical shifts are expressed in parts per million based on tetramethylsilane.

[0227] Low-resolution mass spectra were recorded on an Advion PressionL instrument coupled to an ESI source and operated in both positive and negative ion modes. HRMS was recorded on a Xevo G2 TOF coupled to an ESI source and operated in either positive or negative acquisition mode.

[0228] Small-scale one-pot cyanopyridine cyclization according to Scheme 4

[0229] In a small round-bottom flask equipped with a stirrer, aldehyde (vi) (1 eq.) and malononitrile (viii) (1 eq.) are dissolved in methanol (0.2 M) (solution A). Solution A is immediately stirred vigorously. If the aldehyde contains basic sites (e.g., pyridine), no catalyst is required. Otherwise, in a small vial, a solution of the cyclic amine represented by formula (vii) (1.2 eq.) in MeOH (0.2 M) (solution B) is prepared. One or two drops of solution B are added to solution A. After stirring at room temperature for 30 min, more malononitrile (viii) (1 eq.) is added, followed by the remaining solution B dropwise. The reaction is stirred at room temperature, exposed to air, until completion; completion is determined by TLC (cyclohexane:EtOAc 8:2). The reaction mixture is washed with DCM (5×V MeOH ) was diluted, silica gel was added directly on top of the mixture, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (12 g column for 0.5 to 1 mmol starting aldehyde) using a cyclohexane:ethyl acetate gradient elution (95:5, 3CV; 95:5→8:2, 12CV; 8:2, 6CV; methanol wash).

[0230] Gram-scale cyanopyridine cyclization according to Scheme 5

[0231] In a mortar or bowl of a mechanical stirring device, aldehyde (ix) (1.05 to 1.2 equivalents) and malononitrile (viii) (1 equivalent) are dissolved in MeOH (2 to 1 M) (solution A). Solution A is immediately stirred vigorously. If the aldehyde contains basic sites (e.g., pyridine), no catalyst is required. Separately, in a vial, a solution of the cyclic amine of formula (vii) (solution B) is prepared in MeOH (0.1 M). While stirring, solution B is added dropwise to solution A until the amine content reaches 5 mol%. In less than 3 to 5 minutes, a thick white paste must be formed, which is stirred for 5 to 30 minutes before adding cold water. The precipitate is filtered, washed twice with cold water, and then washed three times with a small amount of cold ether. The powder is dried under reduced pressure for 16 to 24 hours to obtain intermediate (vi), which can be used without further purification.

[0232] In a round-bottom flask, the intermediate of formula (vi) obtained (1 equivalent) is suspended in MeOH (about 0.5M). At 0°C, malononitrile (viii) (1 equivalent) is added continuously, and then the cyclic amine (1.2 equivalents) shown in formula (vii) is added dropwise. The flask is closed with a protective device filled with CaCl2 beads (to allow air exchange), stirred at 0°C for 30min, and then stirred at room temperature until completion; completion is detected by TLC (cyclohexane: EtOAc 8:2 or DCM: MeOH 9:1), and the crude product is purified by flash chromatography.

[0233] Suzuki cross-coupling (thermal activation)

[0234] The methods used were adapted from Pan et al., 2008, supra.

[0235] Commercial or synthetic bromoaryl (1 equiv.), boronic acid or boronic acid pinacol ester (2 equiv.), potassium carbonate (3 equiv.) and palladium dichloride (0.05 equiv.) were added to toluene (0.5 M) in a 25 mL round-bottom flask equipped with a magnetic stirrer. The mixture was stirred at 110 °C for 24 h and exposed to air. The extent of the reaction was monitored by TLC (cyclohexane: DCM 8:2). The palladium and salts were removed by filtration on a celite pad and washed with DCM (2×5 mL). Finally, the solvent was removed under reduced pressure and the crude product was purified by flash chromatography (eluted with a cyclohexane-dichloromethane gradient).

[0236] Microwave-assisted Suzuki cross-coupling

[0237] Commercial or synthetic bromoaryl (1 eq.), boronic acid or boronic acid pinacol ester (1.2 to 3 eq.) and potassium carbonate (3 eq.) were dissolved in a THF:H2O 2:1 mixture (0.05 M) in a 2-5 mL microwave tube equipped with a magnetic stirrer. The solution was purged with argon for 10 min before the addition of tetrakis(triphenylphosphine)palladium(0) (0.05 eq.). The tube was sealed and heated at 80°C or 100°C for 15 to 30 min under microwave irradiation. The culture was diluted with water and the crude product was extracted with EtOAc×3. The organic phases were combined, washed with brine, dried over MgSO4, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (4 g column, less than 0.5 mmol starting bromoaryl) using a cyclohexane:MixA [toluene:acetone 8:2 mixture] gradient elution (5:5, 6CV; 5:5→0:10, 6CV; 0:10, 6CV; methanol wash).

[0238] The intermediate represented by formula (i)

[0239] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-chloropyridine-3,5-dicarbonitrile (wherein R1 is phenyl, R2 is hydrogen, and R3 is cyano)

[0240]

[0241] The intermediate was prepared according to the two-step protocol described by Murray et al., 1995 (Tetrahedron, 1995, 51, 635, DOI: 10.1016 / 0040-4020(94)00922-H) starting from 3-(trimethoxymethyl)-1,1'-biphenyl (1.29 g, 5.0 mmol, 1 eq.) and isolated as a crude powder (650 mg, 2.0 mmol, 39% isolated yield) 1 H NMR(600MHz,(CD3)2SO)δ7.91(d,J=1.8Hz,1H),7.90(dt,J=7.7,1.5Hz,1H),7.75–7.71(m,2H) ,7.68(t,J=7.7Hz,1H),7.59(dt,J=7.7,1.5Hz,1H),7.52(t,J=7.7Hz,2H),7.45–7.39(m,1H). 13 C NMR(151MHz,(CD3)2SO)δ160.3,160.2,155.3,140.5,139.1,134.1,129.5, 129.1,128.9,128.0,127.5,127.0,126.9,115.2,114.5,96.3,89.8.MS(ESI - ) Calculated value C 19 H 11 N4Cl:[MH + ]m / z=329.1, measured value m / z=329.3.

[0242] The intermediate represented by formula (vi)

[0243] 4'-Methyl-[1,1'-biphenyl]-3-carbaldehyde (wherein R1 is 4-methylphenyl and R2 is hydrogen)

[0244]

[0245] The intermediate was prepared as described above by thermal activation starting from 3-bromobenzaldehyde (0.12 mL, 1.0 mmol, 1 eq.) and p-tolylboronic acid (202 mg, 1.5 eq.) and isolated as a translucent oil (113 mg, 0.58 mmol, 58% isolated yield). 1 H NMR (600MHz, CDCl3) δ10.09 (s, 1H), 8.09 (t, J = 1.8Hz, 1H), 7.86–7.83 (m, 2H ),7.60(t,J=7.6Hz,1H),7.56–7.51(m,2H),7.32–7.27(m,2H),2.42(s,3H). 13C NMR (151MHz, CDCl3) δ192.6,142.3,138.1,136.9,133.0,129.9,129.6,128.5,128.1,127.1,21.3.

[0246] 2'-Fluoro-[1,1'-biphenyl]-3-carboxaldehyde (wherein R1 is 2-fluorophenyl and R2 is hydrogen)

[0247]

[0248] The intermediate was prepared as described above by thermal activation starting from 3-bromobenzaldehyde (0.12 mL, 1.0 mmol, 1 eq.) and 2-fluorophenylboronic acid (276 mg, 2 eq.) and isolated as a translucent oil (135 mg, 0.68 mmol, 68% isolated yield). 1 H NMR (600MHz, CDCl3) δ10.09(s,1H),8.06(q,J=1.9Hz,1H),7.90(dt,J=7.7,1.4Hz,1H),7.83(dtd,J=7.7,1.7,1.2Hz,1H),7.62(t,J=7.7Hz ,1H),7.48(td,J=7.7,1.8Hz,1H),7.38(dddd,J=8.3,7.4,5.0,1.8Hz,1H),7.25(td,J=7.5,1.2Hz,1H),7.19(ddd,J=10.8,8.3,1.2Hz,1H). 13 C NMR (151MHz, CDCl3) δ192.3 (d, J = 1.5Hz), 160.7, 159.1, 136.9 (d, J = 22.7Hz), 135.1 (d, J = 3.3Hz), 130.8 (d, J = 3.2Hz), 130.5 (d, J = 2.8 Hz), 129.9 (d, J = 8.2 Hz), 129.3, 128.9, 127.8 (d, J = 13.1 Hz), 124.8 (d, J = 3.7 Hz), 116.4 (d, J = 22.5 Hz).

[0249] 3'-Fluoro-[1,1'-biphenyl]-3-carboxaldehyde (wherein R1 is 3-fluorophenyl and R2 is hydrogen)

[0250]

[0251] The intermediate was prepared as described above by thermal activation starting from 3-bromobenzaldehyde (0.12 mL, 1.0 mmol, 1 eq.) and 3-fluorophenylboronic acid (276 mg, 2 eq.) and isolated as a translucent oil (154 mg, 0.77 mmol, 77% isolated yield). 1 H NMR (600MHz, CDCl3) δ10.10(s,1H),8.11–8.06(m,1H),7.89(dt,J=7.6,1.4Hz,1H),7.84(ddd,J=7.7,2.0, 1.2Hz,1H),7.63(t,J=7.6Hz,1H),7.46–7.40(m,2H),7.33(ddd,J=9.9,2.6,1.7Hz,1H),7.13–7.08(m,1H). 13 CNMR (151MHz, CDCl3) δ192.2 (d, J = 1.8Hz), 163.4 (d, J = 246.2Hz), 142.1 (d, J = 7.7Hz), 141.1 (d, J = 2.3Hz), 137.2 , 133.1, 130.7 (d, J = 8.4Hz), 129.8, 129.4, 128.2, 123.0 (d, J = 3.0Hz), 115.0 (d, J = 21.1Hz), 114.3 (d, J = 22.2Hz).

[0252] 4'-Fluoro-[1,1'-biphenyl]-3-carboxaldehyde (wherein R1 is 4-fluorophenyl and R2 is hydrogen)

[0253]

[0254] The intermediate was prepared as described above by thermal activation starting from 3-bromobenzaldehyde (0.12 mL, 1.0 mmol, 1 eq.) and 4-fluorophenylboronic acid (276 mg, 2 eq.) and isolated as a translucent oil (115 mg, 0.58 mmol, 58% isolated yield). 1 H NMR (600MHz, CDCl3) δ10.09(s,1H),8.06(t,J=1.8Hz,1H),7.86(dt,J=7.6,1.4H z,1H),7.82(ddd,J=7.7,2.0,1.2Hz,1H),7.63–7.57(m,3H),7.19–7.14(m,2H). 13CNMR (151MHz, CDCl3) δ192.4 (d, J = 2.0Hz), 163.0 (d, J = 247.7Hz), 141.4, 137.1 ,136.0(d,J=3.2Hz),133.0,129.7,129.0,128.9,128.0,116.1(d,J=21.7Hz).

[0255] 3-(2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazol-5-yl)benzaldehyde (wherein R1 is 2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazol-5-yl and R2 is hydrogen)

[0256]

[0257] The intermediate 3-(tetrazolyl-5-yl)benzaldehyde can be prepared according to the literature (Bianchini et al., 2021, Med. Chem., 64, 16820–16837). Then, in a dry 25 mL round-bottom flask equipped with a magnetic stirrer and a condenser, DHP (0.18 mL, 2 eq.) was dissolved in dry toluene (5 mL) and mixed with a solution of 3-(tetrazolyl-5-yl)benzaldehyde (174 mg, 1.0 mmol, 1 eq.) in dry DMF (0.79 mL). TFA (0.008 mL, 0.1 eq.) was added dropwise. The flask was purged with argon and heated at 110 ° C for 24 h. The reaction extent was determined by TLC (Rf = 0.57 in DCM: MeOH 98: 2), the reaction was stopped, and 10% Na2CO3 aqueous solution (10 mL) and water (20 mL) were added. The crude product was extracted with EtOAc (3×15 mL), the organic phases were combined, washed with brine (20 mL), dried over MgSO4, and the solvent was removed under reduced pressure. The oil (66 mg, 0.26 mmol, 26% isolated yield) was obtained by eluting with cyclohexane:DCM gradient on a 12 g silica gel column (8:2, 3 CV; 8:2→0:10, 6 CV; methanol washing). 1 HNMR (600MHz, CDCl3) δ10.11(s,1H),8.70(t,J=1.8Hz,1H),8.47(dt,J=7.7,1.5Hz,1H),8.01(dt,J=7.7,1.4Hz,1H),7.68(t,J=7.7 Hz,1H),6.10(dd,J=7.6,2.9Hz,1H),4.08–4.02(m,1H),3.88–3.80(m,1H),2.57–2.46(m,1H),2.25–2.15(m,2H),1.88–1.71(m,3H). 13CNMR (151MHz, CDCl3) δ191.81,164.1,137.1,132.7,130.8,129.9,129.0,128.6,88.2,67.1,29.2,24.7,20.9.MS(ESI + ) Calculated value C 13 H 14 N4O2:[M+Cl - ]m / z=293.1, measured value m / z=293.1.

[0258] The intermediate described by formula (iv)

[0259] 3-Bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (wherein R1 is 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole and R9 is bromine)

[0260] In a 25 mL round bottom flask equipped with a stirrer and a condenser, DHP (0.55 mL, 2 eq.) was dissolved in anhydrous toluene (3 mL). The flask was purged with argon before TFA (0.023 mL, 0.1 eq.) and pyrazole (204 mg, 3 mmol, 1 eq.) were slowly added. The solution was heated at 100 ° C for 16 h. The reaction was completed by TLC detection, which was shown by alkaline KMnO4 (Rf=0.18 in cyclohexane: ethyl acetate 8:2). The reaction was quenched with 0.5 M NaOH aqueous solution (20 mL), and the crude intermediate was extracted with EtOAc (3×15 mL). The organic phases were combined, washed with brine (20 mL), dried over MgSO4, and the solvent was removed under reduced pressure. The 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole intermediate (not shown) was purified by flash chromatography on a 12 g silica gel column with a cyclohexane:ethyl acetate gradient (9:1, 3CV; 9:1→8:2, 3CV; methanol elution) to isolate a translucent oil (421 mg, 2.8 mmol, 92% isolated yield). 3-Bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole intermediate (va) was prepared from 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole intermediate (410 mg, 2.7 mmol, 1 eq.) according to the literature protocol (Nicolaou et al., 2015, ChemMedChem., 10, 1974-1979) to isolate a light yellow oil (460 mg, 2.0 mmol, 74% isolated yield). 1H NMR(600MHz, CDCl3) δ7.50(d,J=2.4Hz,1H),6.31(d,J=2.5Hz,1H),5.32(dd,J=9.3,2.9Hz,1H),4.07 –4.03(m,1H),3.68(td,J=11.1,2.8Hz,1H),2.11–2.00(m,2H),1.70–1.65(m,3H),1.63–1.58(m,1H). 13 C NMR (151MHz, CDCl3) δ129.8,126.5,109.1,87.9,67.9,30.5,25.0,22.3.

[0261] The intermediate represented by formula (iiia)

[0262] 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (wherein R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0263]

[0264] In a 100 mL round bottom flask equipped with a stirrer, 3-bromobenzaldehyde (925 mg, 5 mmol, 1 eq.) and malononitrile (viii) (330 mg, 1 eq.) were dissolved in methanol (10 mL) (solution A). Solution A was immediately stirred vigorously. A solution of piperidine (0.59 mL, 1.2 eq.) in MeOH (5 mL) was prepared (solution B) and added dropwise to solution A over 1 h. More malononitrile (viii) (330 mg, 1 eq.) dissolved in MeOH (5 mL) was added and the reaction was stirred at room temperature and exposed to air until the reaction was complete. The reaction was detected by TLC to be complete (Rf = 0.16, cyclohexane: ethyl acetate 8:2). The medium was diluted with DCM (20 mL), silica gel was added directly to the mixture, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on a 24 g column eluting with a cyclohexane:ethyl acetate gradient (9:1, 3 CV; 9:1→8:2, 6 CV; 8:2, 8 CV; methanol). 1 H NMR (600MHz, CDCl3) δ7.65(dt,J=7.3,1.9Hz,1H),7.61(t,J=1.8Hz,1H),7.43–7.36(m,2H),5.50(s,2H),3.84–3.79(m,4H),1.76–1.68(m,6H). 13C NMR (151MHz, CDCl3) δ160.9,160.5,159.3,136.8,133.7,131.6,130.6,127.4,122.9,117.3,116.1,83.6,81.8,49.5,26.1,24.5.MS(ESI + ) Calculated value C 18 H 16 N5Br:[M+H + ]m / z=382.1&384.1, measured value m / z=382.1&384.1.HRMS(ESI + ) Calculated value C 18 H 16 N5Br:[M+H + ]m / z=382.0662, measured value m / z=382.0667.

[0265] 2-amino-4-(3-bromo-2-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (wherein R2 is 2-fluoro, R3 is cyano, and R4 is piperidinyl)

[0266]

[0267] In a small round-bottom flask equipped with a stirrer, 5-bromo-2-fluorobenzaldehyde (317 mg, 1.56 mmol, 1 eq.) and malononitrile (viii) (264 mg, 2.6 eq.) were dissolved in methanol (5 mL) (solution A). Solution A was immediately stirred vigorously. A solution of piperidine (0.24 mL, 1.5 eq.) in MeOH (1 mL) was prepared (solution B) and a few drops were added to solution A. After stirring at room temperature for 30 min, the remaining solution B was slowly added. The reaction was stirred at room temperature and exposed to air until the reaction was complete. The medium was diluted with water (10 mL) and brine (10 mL) and the crude product was extracted with AcOEt (3×15 mL). The organic phases were combined, dried over MgSO4, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on a 12 g silica column with a cyclohexane:DCM gradient (8:2, 4CV; 8:2→4:6, 8CV; 4:6, 4CV; methanol wash) to isolate a white powder (63 mg, 0.16 mmol, 10% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.60 (ddd, J=8.9, 4.5, 2.5Hz, 1H), 7.49 (dd, J=6.3, 2.5Hz ,1H),7.13(t,J=9.0Hz,1H),5.39(s,2H),3.84–3.80(m,4H),1.73–1.68(m,6H). 13C NMR (151MHz, CDCl3) δ160.2, 159.1, 158.2 (d, J = 250.9Hz), 155.2, 135.4 (d, J = 8.4Hz), 133.1 (d, J = 2.1Hz), 124 .8(d,J=16.8Hz),118.4(d,J=23.0Hz),117.2(d,J=3.6Hz),116.9,115.7,84.3,82.3,49.2,26.1,24.5.MS(ESI + ) Calculated value C 18 H 15 N5FBr:[M+H + ]m / z=400.1&402.1, measured value m / z=400.4&402.3.

[0268] 2-amino-4-(3-bromo-2-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (wherein R2 is 2-fluoro, R3 is cyano, and R4 is piperidinyl)

[0269]

[0270] In a small round-bottom flask equipped with a stirrer, 3-bromo-2-fluorobenzaldehyde (317 mg, 1.56 mmol, 1 eq.) and malononitrile (viii) (264 mg, 2.6 eq.) were dissolved in methanol (5 mL) (solution A), and solution A was immediately stirred vigorously. A solution of piperidine (0.24 mL, 1.2 eq.) in MeOH (1 mL) was prepared (solution B), and a few drops were added to solution A. After stirring at room temperature for 30 min, the remaining solution B was slowly added. The reaction was stirred at room temperature and exposed to air until the reaction was complete. The medium was diluted with water (10 mL) and brine (10 mL), and the crude product was extracted with ethyl acetate (3×15 mL). The organic phases were combined, dried over MgSO4, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on a 12 g silica column eluting with a cyclohexane:DCM gradient (8:2, 4CV; 8:2→4:6, 8CV; 4:6, 4CV; methanol wash) to isolate a white powder (63 mg, 0.16 mmol, 10% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.70 (ddd, J=8.1, 6.4, 1.6Hz, 1H), 7.32 (ddd, J=7.9, 6.2, 1.6 Hz,1H),7.18(t,J=7.8Hz,1H),5.38(s,2H),3.84–3.78(m,4H),1.76–1.66(m,6H). 13C NMR (151MHz, CDCl3) δ160.2, 159.1, 155.6, 155.5 (d, J = 250.8Hz), 135.8, 129.5 (d, J = 2.1Hz), 125.6 (d, J =4.2Hz),124.2(d,J=16.2Hz),116.8,115.7,110.3(d,J=20.9Hz),84.2,82.2,49.1,26.0,24.4.MS(ESI + ) Calculated value C 18 H 15 N5FBr:[M+H + ]m / z=400.1&402.1, measured value m / z=400.3&402.3.

[0271] 2-amino-4-(3-chloro-5-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (wherein R2 is 5-fluoro, R3 is cyano, and R4 is piperidinyl)

[0272]

[0273] In a small round-bottom flask equipped with a stirrer, 3-chloro-5-fluorobenzaldehyde (317 mg, 2 mmol, 1 eq.) and malononitrile (viii) (264 mg, 2 eq.) were dissolved in methanol (6 mL). The solution was immediately stirred vigorously and cooled to 0°C. Piperidine (0.24 mL, 1.2 eq.) dissolved in MeOH (1 mL) was slowly added. The reaction was stirred at room temperature and exposed to air until the reaction was complete. The medium was diluted with brine (20 mL) and the crude product was extracted with AcOEt (3×15 mL). The organic phases were combined, dried over MgSO4, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on a 24 g silica gel column with an isocratic gradient of cyclohexane:ethyl acetate (85:15, 12 CV; methanol elution) to isolate a white powder (136 mg, 0.38 mmol, 19% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.26–7.22(m,2H),7.09(dt,J=8.1,1.7Hz,1H),5.51(s,2H),3.84–3.80(m,4H),1.73–1.67(m,6H). 13C NMR (151MHz, CDCl3) δ163.4,161.8,160.3,159.73,159.71,159.2,137.8,137.7,136.1,136.0, 124.91,124.89,118.5,118.3,117.0,115.8,114.7,114.6,83.3,81.6,49.4,26.1,24.4.MS(ESI + ) Calculated value C 18 H 15 N5FCl:[M+H + ]m / z=356.1, measured value m / z=356.4.

[0274] The intermediate represented by formula (iiib)

[0275] (3-(2-amino-3,5-dicyano-6-(piperidin-1-yl)pyridin-4-yl)phenyl)boronic acid pinacol ester (wherein R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0276]

[0277] In a 25 mL round-bottom flask purged with argon and equipped with a magnetic stirrer, 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (iiiaa) (382 mg, 1 mmol, 1 eq.) and bis(pinacol)diboron (v) (303 mg, 1.2 eq.) were dissolved in degassed toluene (10 mL). Potassium carbonate (414 mg, 3 eq.) and tetrakis(triphenylphosphine)palladium(0) (58 mg, 0.05 eq.) were added simultaneously and heated at 110°C for 16 h. The reaction was complete by TLC (Rf = 0.06: cyclohexane: DCM 4:6). The palladium and salts were removed by filtration on a celite pad, washed with EtOAc (3 x 5 mL), and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on a 12 g column with a cyclohexane:DCM gradient (5:5, 6CV; 5:5→0:10, 6CV; 0:10, 9CV; methanol wash) to isolate an off-white powder (213 mg, 0.5 mmol, 50% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.94(dt,J=7.3,1.3Hz,1H),7.87(dt,J=1.9,0.8Hz,1H),7.54(dt,J=7.7,1. 7Hz,1H),7.50(t,J=7.5Hz,1H),5.33(s,2H),3.82–3.77(m,2H),1.72–1.66(m,5H),1.35(s,12H). 13CNMR(151MHz, CDCl3)δ162.9,160.7,159.2,137.0,134.8,134.4,131.2,128.3,117.5,116.3,84.2,82.1,49.5,27.1,26.2,25.1,24.5.MS(ESI + )C 24 H 28 N5O2B:[M+H + ]m / z=430.2, measured value m / z=430.4.

[0278] The compound represented by formula (I)

[0279] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(azetidin-1-yl)pyridine-3,5-dicarbonitrile (1) (wherein R1 is phenyl, R2 is hydrogen, R3 is cyano, and R4 is azetidine)

[0280]

[0281] The product was prepared according to the general process in Scheme 4 starting from commercial [1,1'-biphenyl]-3-carbaldehyde (55 mg, 0.30 mmol, 1 eq.) (the intermediate shown in formula (vi) wherein R1 is phenyl and R2 is hydrogen) and azetidine (0.024 mL, 1.2 eq.) (the cyclic amine shown in formula (vii)) and isolated as an off-white powder (27 mg, 0.08 mmol, 26% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.71(ddd,J=7.7,1.8,1.1Hz,1H),7.68(t,J=1.8Hz,1H),7.64–7.61(m,2H),7.57(t,J=7.7 Hz,1H),7.47–7.44(m,3H),7.38–7.35(m,1H),5.42(s,2H),4.47–4.39(m,4H),2.40(tdd,J=8.8,7.2,6.0Hz,2H). 13 C NMR (151MHz, CDCl3) δ161.0,160.1,159.3,142.0,140.6,134.8,129.40,129.37, 129.0,127.8,127.6,127.6,127.4,117.2,116.9,81.0,80.6,27.1,16.3.MS(ESI + ) Calculated value C 22 H 17 N5:[M+H +]m / z=352.2, measured value m / z=352.0.HRMS(ESI + ) Calculated value C 22 H 17 N5:[M+H + ]m / z=352.1557, measured value m / z=352.1575.

[0282] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(pyrrolidin-1-yl)pyridine-3,5-dicarbonitrile (2) (wherein R1 is phenyl, R2 is hydrogen, R3 is cyano, and R4 is pyrrolidinyl)

[0283]

[0284] The product was prepared according to the general procedure in Scheme 4 starting from commercial [1,1'-biphenyl]-3-carbaldehyde (intermediate represented by formula (vi) wherein R1 is phenyl and R2 is hydrogen) (55 mg, 0.30 mmol, 1 eq.) and pyrrolidine (0.030 mL, 1.2 eq.) (cyclic amine represented by formula (vii)) and isolated as an off-white powder (19 mg, 0.05 mmol, 17% isolated yield). 1 H NMR(600MHz, CDCl3)δ7.71(dt,J=7.8,1.5Hz,1H),7.69(t,J=1.8Hz,1H),7.65–7.62(m,2H),7.58(t,J=7 .6Hz,1H),7.48–7.44(m,3H),7.36(t,J=7.4Hz,1H),5.42(s,2H),3.87–3.82(m,4H),2.03–1.95(m,4H). 13 C NMR (151MHz, CDCl3) δ162.2,159.4,157.5,141.9,140.7,135.4,129.33,129. 30,129.0,127.8,127.6,127.5,118.4,116.9,82.2,81.2,49.9,27.1.MS(ESI + ) Calculated value C 23 H 19 N5:[M+H + ]m / z=366.2, measured value m / z=366.0. HRMS (ESI + ) Calculated value C 23 H 19 N5:[M+H + ]m / z=366.1714, measured value m / z=366.1706.

[0285] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (3) (wherein R1 is phenyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0286]

[0287] The product was prepared from commercial [1,1'-biphenyl]-3-carbaldehyde (intermediate represented by formula (vi), wherein R1 is phenyl and R2 is hydrogen) (0.080 mL, 0.5 mmol, 1 eq.) according to Sarkar et al., 2014, RSC Adv., 4, 53752–53760 and isolated as a white powder (13 mg, 0.03 mmol, 7% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.73–7.70(m,2H),7.66–7.62(m,2H),7.58(t,J=7.7Hz,1H),7.49(dt,J=7.7,1.5Hz, 1H),7.45(t,J=7.7Hz,2H),7.37(tt,J=7.4,1.3Hz,1H),5.49(s,2H),3.86–3.80(m,4H),1.73–1.70(m,6H). 13 C NMR (151MHz, CDCl3) δ162.4,161.1,159.5,142.0,140.6,135.2,129.5,129.4,129 .0,127.9,127.8,127.7,127.6,117.8,116.6,83.8,81.9,49.5,26.2,24.5.MS(ESI + ) Calculated value C 24 H 21 N5:[M+H + ]m / z=380.2, measured value m / z=380.1.HRMS(ESI + ) Calculated value C 24 H 21 N5:[M+H + ]m / z=380.1870, measured value m / z=380.1859.

[0288] 2-amino-4-(2'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (4) (wherein R1 is 2-fluorophenyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0289]

[0290] The product was prepared according to Sarkar et al., 2014, supra starting from 2'-fluoro-[1,1'-biphenyl]-3-carbaldehyde (intermediate represented by formula (vib)) (100 mg, 0.5 mmol, 1 eq.) and isolated as a white powder (45 mg, 0.11 mmol, 23% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.72–7.67(m,2H),7.59(td,J=7.6,0.7Hz,1H),7.51(dt,J=7.7,1.5Hz,1H),7.41(td,J= 4.4,3.8,2.8Hz,2H),7.35–7.31(m,1H),7.09–7.03(m,1H),5.38(s,2H),3.88–3.80(m,4H),1.78–1.63(m,6H). 13 C NMR (151MHz, CDCl3) δ163.3 (d, J = 246.0Hz), 162.1, 161.3, 159.6, 142.8 (d, J = 7.5Hz), 140.7 (d, J = 2.1Hz), 135.5, 130.5 (d, J = 8.4Hz), 1 29.5(d,J=26.6Hz),128.2,127.9,123.3,117.9,116.7,114.6(d,J=21.0Hz),114.4(d,J=22.0Hz),83.7,81.8,49.4,26.2,24.6.MS(ESI + ) Calculated value C 24 H 20 N5F:[M+H + ]m / z=398.2, measured value m / z=398.8.HRMS(ESI + ) Calculated value C 24 H 20 N5F:[M+Na + ]m / z=420.1595, measured value m / z=420.1594.

[0291] 2-amino-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (5) (wherein R1 is 3-fluorophenyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0292]

[0293] The product was prepared according to Sarkar et al., 2014, supra starting from 3'-fluoro-[1,1'-biphenyl]-3-carbaldehyde (intermediate represented by formula (vic)) (100 mg, 0.5 mmol, 1 eq.) and isolated as a white powder (34 mg, 0.08 mmol, 17% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.71(dq,J=7.7,1.6Hz,1H),7.68(q,J=1.6Hz,1H),7.58(d,J=7.7Hz,1H),7.55–7.49(m,2H),7.37–7.30( m,1H),7.23(td,J=7.5,1.2Hz,1H),7.17(ddd,J=10.9,8.2,1.2Hz,1H),5.37(s,2H),3.83–3.79(m,4H),1.71(t,J=3.1Hz,6H). 13 C NMR (151MHz, CDCl3) δ162.0, 161.4, 159.9 (d, J = 248.7Hz), 159.6, 136.5, 135.1, 131.3 (d, J = 3.3Hz), 131.1 (d, J = 3.2Hz), 129.59 (d, J = 1 .1Hz),129.55(d,J=4.0Hz),129.1,128.1,124.7(d,J=3.6Hz),117.8,116.7,116.3(d,J=22.5Hz),83.8,81.8,49.4,26.1,24.6.MS(ESI + ) Calculated value C 24 H 20 N5F:[M+H + ]m / z=398.2, measured value m / z=398.8.HRMS(ESI + ) Calculated value C 24 H 20 N5F:[M+Na + ]m / z=420.1595, measured value m / z=420.1594.

[0294] 2-amino-4-(4'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (6) (wherein R1 is 4-fluorophenyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0295]

[0296] The product was prepared according to Sarkar et al., 2014, supra starting from 4'-fluoro-[1,1'-biphenyl]-3-carbaldehyde (intermediate represented by formula (vid)) (100 mg, 0.5 mmol, 1 eq.) and isolated as a white powder (34 mg, 0.08 mmol, 17% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.68–7.64(m,2H),7.61–7.55(m,3H),7.48(dt,J=7.7,1. 5Hz,1H),7.18–7.11(m,2H),5.37(s,2H),3.84–3.79(m,4H),1.76–1.70(m,6H). 13 C NMR (151MHz, CDCl3) δ162.9 (d, J = 246.7Hz), 162.2, 161.3, 159.6, 141.0, 136.8 (d, J = 3.4Hz), 135.4, 129.5, 129.3, 129.2(d,J=8.2Hz),127.8(d,J=19.2Hz),117.9,116.8,115.9(d,J=21.5Hz),83.7,81.8,49.4,26.2,24.6.MS(ESI + ) Calculated value C 24 H 20 N5F:[M+H + ]m / z=398.2, measured value m / z=398.8.HRMS(ESI + ) Calculated value C 24 H 20 N5F:[M+Na + ]m / z=420.1595, measured value m / z=420.1594.

[0297] 2-Amino-4-(2'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (7) (wherein R1 is 2-methylphenyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0298]

[0299] As described above, the product was prepared by thermal activation starting from 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate shown in formula (iiiaa)) (60 mg, 0.16 mmol, 1 eq.) and 2-methylphenylboronic acid (intermediate shown in formula (iv) wherein R1 is 2-methylphenyl and R2 is boronic acid) (63 mg, 3 eq.) and isolated as a white powder (30 mg, 0.08 mmol, 49% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.55(t,J=7.7Hz,1H),7.47(ddt,J=7.4,5.8,1.4Hz,2H),7.43(t,J=1. 8Hz,1H),7.32–7.25(m,2H),5.35(s,2H),3.83–3.76(m,4H),2.32(s,3H),1.86–1.66(m,6H). 13 C NMR (151MHz, CDCl3) δ162.3,161.3,159.6,142.7,141.1,135.8,134.8,131.4,130.4,130.1, 129.7,128.7,127.7,127.3,126.0,117.8,116.7,83.9,81.9,49.4,26.1,24.6,20.7.MS(ESI + ) Calculated value C 24 H 21 N5:[M+H + ]m / z=394.2, measured value m / z=394.9.HRMS(ESI + ) Calculated value C 24 H 21 N5:[M+Na + ]m / z=416.1846, measured value m / z=416.1857.

[0300] 2-amino-4-(3'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (8) (wherein R1 is 3-methylphenyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0301]

[0302] As described above, the product was prepared by thermal activation starting from 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate shown in formula (iiiaa)) (60 mg, 0.16 mmol, 1 eq.) and 3-methylphenylboronic acid (intermediate shown in formula (iv) wherein R1 is 3-methylphenyl and R2 is boronic acid) (63 mg, 3 eq.) and was isolated as a white powder (47 mg, 0.12 mmol, 76% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.70(ddd,J=7.7,1.9,1.2Hz,1H),7.69(t,J=1.8Hz,1H),7.57(t,J=7.7Hz,1H),7.47–7.46(m,1H),7.43(d,J=8 .2Hz,1H),7.34(t,J=7.9Hz,1H),7.18(d,J=7.2Hz,0H),5.36(s,2H),3.82(dd,J=5.9,3.1Hz,4H),2.43(s,3H),1.72(q,J=1.5Hz,4H). 13 C NMR (151MHz, CDCl3) δ162.4,161.4,159.6,142.2,140.6,138.5,135.2,129.3,128.9,128 .5,128.3,127.9,127.5,124.7,117.9,116.8,83.8,81.9,49.4,26.2,24.6,21.7.MS(ESI + ) Calculated value C 24 H 21 N5:[M+H + ]m / z=394.2, measured value m / z=394.9.HRMS(ESI + ) Calculated value C 24 H 21 N5:[M+Na + ]m / z=416.1846, measured value m / z=416.1857.

[0303] 2-amino-4-(4'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (9) (wherein R1 is 4-methylphenyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0304]

[0305] The product was prepared according to Sarkar et al., 2014, supra starting from 4'-methyl-[1,1'-biphenyl]-3-carbaldehyde (intermediate represented by formula (via)) (98 mg, 0.5 mmol, 1 eq.) and isolated as a white powder (56 mg, 0.14 mmol, 28% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.71–7.67(m,2H),7.56(t,J=7.6Hz,1H),7.53(d,J=7.9Hz,2H),7.45(dd ,J=7.7,1.5Hz,1H),7.26(d,J=8.1Hz,2H),5.37(s,2H),3.84–3.79(m,4H),1.74–1.69(m,6H). 13 C NMR (151MHz, CDCl3) δ162.4,161.4,159.6,141.9,137.8,137.6,135.2,129.7,129.31,1 29.28,127.7,127.40,127.36,117.9,116.8,83.8,81.8,49.4,26.2,24.6,21.3.MS(ESI + ) Calculated value C 24 H 21 N5:[M+H + ]m / z=394.2, measured value m / z=394.8.HRMS(ESI + ) Calculated value C 24 H 21 N5:[M+Na + ]m / z=416.1846, measured value m / z=416.1857.

[0306] 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-2-yl)phenyl)pyridine-3,5-dicarbonitrile (10) (wherein R1 is 2-pyridyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0307]

[0308] As described above, the product was prepared using microwave-assisted Suzuk cross-coupling, reacted at 80°C for 15 min, and the starting materials were (3-(2-amino-3,5-dicyano-6-(piperidin-1-yl)pyridin-4-yl)phenyl)boronic acid pinacol ester (intermediate shown in formula (iiiba)) (64 mg, 0.15 mmol, 1 eq.) and 2-bromopyridine (intermediate shown in formula (iv) wherein R1 is 2-pyridyl and R2 is bromine) (28 mg, 1.2 eq.), and the white powder product was isolated (17 mg, 0.04 mmol, isolated yield 30%). 1 H NMR (600MHz, CDCl3) δ8.92(s,1H),8.65(s,1H),7.99(dt,J=8.0,1.8Hz,1H),7.75–7.68(m,2H),7.64(t,J=7.7 Hz,1H),7.56(dt,J=7.7,1.4Hz,1H),7.44(t,J=5.9Hz,1H),5.40(s,2H),3.86–3.80(m,4H),1.76–1.70(m,6H). 13 C NMR (151MHz, CDCl3) δ162.0,161.1,159.4,135.4,129.39,129.36,129.0,127.5,122.7,121.1,117.6,116.5,83.7,81.7,49.2,26.0,24.4.MS(ESI + ) Calculated value C 23 H 20 N6:[M+H + ]m / z=381.2, measured value m / z=381.0.HRMS(ESI + ) Calculated value for C23H20N6: [M+H+] m / z = 381.1823, measured value m / z = 381.1803.

[0309] 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-3-yl)phenyl)pyridine-3,5-dicarbonitrile (11) (wherein R1 is 3-pyridyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0310]

[0311] As described above, the product was prepared using microwave-assisted Suzuki cross-coupling, reacted at 100°C for 30 min, and the starting materials were 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate shown in formula (iiiaa)) (58 mg, 0.15 mmol, 1 eq.) and 3-pyridineboronic acid (intermediate shown in formula (iv) wherein R1 is 3-pyridyl and R2 is boronic acid) (55 mg, 3 eq.), and the white powder product was isolated (79 mg, 0.15 mmol, quantitative isolated yield). 1 H NMR (600MHz, CDCl3) δ8.92(s,1H),8.65(s,1H),7.99(dt,J=8.0,1.8Hz,1H),7.75–7.68(m,2H),7.64(t,J=7.7 Hz,1H),7.56(dt,J=7.7,1.4Hz,1H),7.44(t,J=5.9Hz,1H),5.40(s,2H),3.86–3.80(m,4H),1.76–1.70(m,6H). 13 CNMR (151MHz, CDCl3) δ161.9,161.2,159.6,149.1,148.6,138.7,136.1,135.7,134.9, 129.8,129.4,128.5,127.9,123.8,117.9,116.7,83.6,81.8,49.4,26.2,24.6.MS(ESI + ) Calculated value C 23 H 20 N6:[M+H + ]m / z=381.2, measured value m / z=381.1.HRMS(ESI + ) Calculated value C 23 H 20 N6:[M+H + ]m / z=381.1823, measured value m / z=381.1836.

[0312] 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-4-yl)phenyl)pyridine-3,5-dicarbonitrile (12) (wherein R1 is 4-pyridyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0313]

[0314] As described above, the product was prepared using microwave-assisted Suzuki cross-coupling, reacted at 80°C for 15 min, and the starting materials were 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate shown in formula (iiiaa)) (58 mg, 0.15 mmol, 1 eq.) and 4-pyridineboronic acid (intermediate shown in formula (iv) wherein R1 is 4-pyridyl and R2 is boronic acid) (55 mg, 3 eq.), and the white powder product was isolated (5 mg, 0.013 mmol, 9% isolated yield). 1 H NMR (600MHz, CDCl3) δ8.71–8.67(m,2H),7.79–7.74(m,2H),7.65(t,J=8.1Hz ,1H),7.61–7.58(m,3H),5.41(s,2H),3.86–3.80(m,4H),1.75–1.69(m,7H). 13 C NMR (151MHz, CDCl3) δ161.7,161.2,159.6,149.3,138.6,136.0,132.4,132.3,130.1, 129.4,128.8,128.7,128.1,122.6,117.9,116.8,83.6,81.8,49.5,26.3,24.6.MS(ESI + ) Calculated value C 23 H 20 N6:[M+H + ]m / z=381.2, measured value m / z=381.1.HRMS(ESI + ) Calculated value C 23 H 20 N6:[M+H + ]m / z=381.1823, measured value m / z=381.1836.

[0315] 2-amino-6-(piperidin-1-yl)-4-(3-(thiophen-2-yl)phenyl)pyridine-3,5-dicarbonitrile (13) (wherein R1 is 2-thiophene, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0316]

[0317] As described above, the product was prepared using microwave-assisted Suzuki cross-coupling at 100°C for 30 min. The starting materials were 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate shown in formula (iiiaa)) (58 mg, 0.16 mmol, 1 eq.) and 2-thiopheneboronic acid (intermediate shown in formula (iv) wherein R1 is 2-thienyl and R2 is boronic acid) (23 mg, 3 eq.), and a white powder (58 mg, 0.16 mmol, quantitative isolated yield) was isolated. 1 HNMR (600MHz, CDCl3) δ7.74(ddd,J=7.8,1.9,1.1Hz,1H),7.71(t,J=1.8Hz,1H),7.52(t,J=7.8Hz,1H),7.39(ddd,J=7.7,1.8,1.1Hz,1H), 7.37(dd,J=3.6,1.2Hz,1H),7.31(dd,J=5.1,1.2Hz,1H),7.09(dd,J=5.1,3.6Hz,1H),5.44(s,2H),3.85–3.80(m,4H),1.75–1.69(m,6H). 13 CNMR (151MHz, CDCl3) δ162.0,161.0,159.4,143.4,135.5,135.2,129.6,128.25,128 .16,127.7,126.3,125.6,124.1,117.6,116.5,83.8,81.9,49.5,26.2,24.5.MS(ESI + ) Calculated value C 22 H 19 N5S:[M+H + ]m / z=386.1, measured value m / z=386.1.HRMS(ESI + ) Calculated value C 22 H 19 N5S:[M+H + ]m / z=386.1435, measured value m / z=386.1438.

[0318] 2-amino-6-(piperidin-1-yl)-4-(3-(thien-3-yl)phenyl)pyridine-3,5-dicarbonitrile (14) (wherein R1 is 3-thienyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0319]

[0320] The product was prepared by microwave-assisted Suzuki cross-coupling at 80°C for 15 min. The starting materials were 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (the intermediate shown in formula (iiiaa)) (58 mg, 0.16 mmol, 1 eq.) and 3-thiopheneboronic acid (the intermediate shown in formula (iv) wherein R1 is 3-thienyl and R2 is boronic acid) (23 mg, 1.2 eq.) and isolated as a white powder (42 mg, 0.11 mmol, 73% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.66–7.62(m,2H),7.46(t,J=7.7Hz,1H),7.44(dd,J=2.9,1.4Hz,1H),7.3 6–7.33(m,2H),7.32(dd,J=5.0,2.9Hz,1H),5.33(s,2H),3.76–3.72(m,4H),1.69–1.62(m,6H). 13 CNMR (151MHz, CDCl3) δ162.2,161.3,159.6,141.7,136.6,135.4,129.4,128.7,127. 5,127.1,126.59,126.55,121.4,117.8,116.7,83.7,81.8,49.4,26.1,24.6.MS(ESI + ) Calculated value C 22 H 19 N5S:[M+Na + ]m / z=408.1, measured value m / z=408.0.HRMS(ESI + ) Calculated value C 22 H 19 N5S:[M+H + ]m / z=386.1435, measured value m / z=386.1438.

[0321] 4-(3-(1H-pyrazol-5-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (15) (wherein R1 is 1H-pyrazol-5-yl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0322]

[0323] The product was prepared using microwave-assisted Suzuki cross-coupling, starting with (3-(2-amino-3,5-dicyano-6-(piperidin-1-yl)pyridin-4-yl)phenyl)boronic acid pinacol ester (intermediate of formula (iiiba)) (64 mg, 0.15 mmol, 1 eq.) and 5-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (intermediate of formula (iva)) (42 mg, 1.2 eq.) for the Suzuki cross-coupling step. The deprotection reaction of pyrazole was carried out in a dry 10 ml round-bottom flask purged with argon. The intermediate (1 eq.) was dissolved in a 1:1 mixture of DCM:MeOH (3 mL). TsOH (39 mg, 1.5 eq.) was added and the solution was stirred at room temperature for 20 h. The reaction was detected to be complete by TLC (Rf=0.18 in MixA). Purification was performed twice by flash chromatography on a prepacked 4 g silica gel column using MixA 3 The product (42 mg, 0.11 mmol, 76% isolated yield) was isolated by isocratic elution with MeOH (9:1, 12 CV) and then cyclohexane:DCM gradient elution (95:5, 3 CV; 95:5→0:10, 18 CV; methanol elution). 1 H NMR (600MHz, CDCl3) δ7.88(dt,J=7.8,1.4Hz,1H),7.86(t,J=1.8Hz,1H),7.63(s,1H),7.53(t,J=7.7 Hz,1H),7.44(dt,J=7.8,1.3Hz,1H),6.65(s,1H),5.33(s,2H),3.77–3.73(m,4H),1.67–1.62(m,6H). 13 C NMR (151MHz, CDCl3) δ161.8,161.1,159.5,135.8,129.7,129.2,128.3,126.7,117.7,116.6,83.7,81.8,49.4,26.2,24.6.MS(ESI + ) Calculated value C 21 H 19 N7:[M+H + ]m / z=370.2, measured value m / z=370.3.HRMS(ESI + ) Calculated value C 21 H 19 N7:[M+H + ]m / z=370.1775, measured value m / z=370.1779.

[0324] 4-(3-(1H-tetrazolyl-5-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (16) (wherein R1 is 1H-tetrazolyl-5-yl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0325]

[0326] The product was prepared according to the general method of Scheme 4, starting from 3-(2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazolyl-5-yl)benzaldehyde (intermediate represented by formula (vie)) (60 mg, 0.23 mmol, 1 eq.) and eluted on a pre-packed 4 g silica column with a cyclohexane:DCM gradient (95:5, 3CV; 95:5→0:10, 18CV; methanol elution). + Tetrazolyl deprotection was carried out in the presence of 98:2 ethanol:water mixture (1 mL) at 80°C for 16 h (1 g resin for 100 mg compound). The resin was filtered off and washed with warm ethanol (3 x 2 mL) to isolate the product as a white powder (10 mg, 0.03 mmol, 11% isolated yield). 1 H NMR(600MHz, (CD3)2SO) δ10.20(s,1H),8.20(dt,J=7.8,1.5Hz,1H),8.15(t,J=1.8Hz,1H),7.78( t,J=7.7Hz,1H),7.72(d,J=7.7Hz,1H),7.50(s,2H),3.77–3.73(m,4H),1.68–1.58(m,6H).MS(ESI - ) Calculated value C 19 H 17 N9:[MH + ]m / z=370.2, measured value m / z=370.0.HRMS(ESI - ) Calculated value C 19 H 17 N9:[MH + ]m / z=370.1524, measured value m / z=370.1500.

[0327] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(diethylamino)pyridine-3,5-dicarbonitrile (17) (wherein R1 is phenyl, R2 is hydrogen, R3 is cyano, and R4 is diethylamino)

[0328]

[0329] The product was prepared according to the general method in Scheme 1 starting from 4-([1,1'-biphenyl]-3-yl)-2-amino-6-chloropyridine-3,5-dicarbonitrile (50 mg, 0.15 mmol) (intermediate represented by formula (i), wherein R1 is phenyl, R2 is hydrogen, and R3 is cyano) and diethylamine (0.10 ml, 6 eq.) (nucleophilic reagent represented by formula (ii)), and a white amorphous powder (22 mg, 0.06 mmol, 40% isolated yield) was isolated. 1 H NMR (600MHz, CDCl3) δ7.71(dt,J=7.8,1.4Hz,1H),7.68(t,J=1.8Hz,1H),7.66–7.61(m,2H),7.58(t,J=7.7Hz, 1H),7.48–7.42(m,3H),7.36(dd,J=7.4,1.4Hz,1H),5.50(s,2H),3.77(q,J=7.0Hz,4H),1.32(t,J=7.0Hz,6H). 13 C NMR (151MHz, CDCl3) δ162.9,159.0,158.5,141.9,140.7,135.5,129.34,129. 32,129.0,127.8,127.6,127.5,118.1,116.7,81.8,81.6,45.0,13.7.MS(ESI + ) Calculated value C 23 H 21 N5:[M+H + ]m / z=368.2, measured value m / z=368.4.HRMS(ESI + ) Calculated value C 23 H 21 N5:[M+H + ]m / z=368.1870, measured value m / z=368.1839.

[0330] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-morpholinopyridine-3,5-dicarbonitrile (18) (wherein R1 is phenyl, R2 is hydrogen, R3 is cyano, and R4 is morpholinyl)

[0331]

[0332] The product was prepared according to the general method in Scheme 1 starting from 4-([1,1'-biphenyl]-3-yl)-2-amino-6-chloropyridine-3,5-dicarbonitrile (50 mg, 0.15 mmol) (intermediate of formula (i) wherein R1 is phenyl, R2 is hydrogen and R3 is cyano) and morpholine (0.10 ml, 8 eq.) (nucleophilic reagent of formula (ii)) to obtain a white amorphous powder (12 mg, 0.03 mmol, 21% isolated yield). 1 H NMR(600MHz, CDCl3)δ7.73(dt,J=7.8,1.4Hz,1H),7.70(t,J=1.8Hz,1H),7.65–7.61(m,2H),7.59(t,J=7.7Hz,1H),7 .50–7.43(m,3H),7.37(dd,J=7.4,1.2Hz,1H),5.51(s,2H),3.88(dd,J=5.7,3.6Hz,4H),3.82(dd,J=5.6,3.7Hz,4H). 13 C NMR (151MHz, CDCl3) δ162.4,161.5,159.5,142.1,140.5,134.9,129.7,129.5,12 9.0,127.88,127.85,127.6,127.5,117.5,116.3,84.3,83.0,66.8,48.5.MS(ESI + ) Calculated value C 23 H 19 N5O:[M+H + ]m / z=382.2, measured value m / z=328.3.HRMS(ESI + ) Calculated value C 23 H 19 N5O:[M+H + ]m / z=382.1663, measured value m / z=328.1636.

[0333] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(4-methylpiperazin-1-yl)pyridine-3,5-dicarbonitrile (19) (wherein R1 is phenyl, R2 is hydrogen, R3 is cyano, and R4 is N-methylpiperazine)

[0334]

[0335] The product was prepared according to the general method in Scheme 1 starting from 4-([1,1'-biphenyl]-3-yl)-2-amino-6-chloropyridine-3,5-dicarbonitrile (50 mg, 0.15 mmol) (intermediate of formula (i) wherein R1 is phenyl, R2 is hydrogen and R3 is cyano) and N-methylpiperazine (0.10 ml, 6 eq.) (nucleophilic reagent of formula (ii)) to obtain a white amorphous powder (13 mg, 0.03 mmol, 22% isolated yield). 1 H NMR (600MHz, CD3OD) δ7.79(ddd,J=7.8,1.9,1.1Hz,1H),7.76(t,J=1.8Hz,1H),7.70–7.65(m,2H),7.62(t,J=7.7Hz,1H),7.51(ddd ,J=7.7,1.8,1.1Hz,1H),7.49–7.43(m,2H),7.38(dd,J=7.4,1.1Hz,1H),3.89(t,J=5.1Hz,4H),2.71(t,J=4.9Hz,4H),2.45(s,3H). 13 C NMR(151MHz,CD3OD)δ163.6,163.3,161.9,143.1,141.7,137.0,130.3,130.0,1 28.8,128.78,128.76,128.3,118.9,117.1,84.2,83.5,55.5,48.2,45.6.MS(ESI + ) Calculated value C 24 H 22 N6:[M+H + ]m / z=395.2, measured value m / z=395.5.HRMS(ESI + ) Calculated value C 24 H 22 N6:[M+H + ]m / z=395.1979, measured value m / z=395.1955.

[0336] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(benzylthio)pyridine-3,5-dicarbonitrile (20) (wherein R1 is phenyl, R2 is hydrogen, R3 is cyano, and R4 is benzyl mercaptan)

[0337]

[0338] The product was prepared according to the general method in Scheme 1 starting from 4-([1,1'-biphenyl]-3-yl)-2-amino-6-chloropyridine-3,5-dicarbonitrile (50 mg, 0.15 mmol) (intermediate represented by formula (i) wherein R1 is phenyl, R2 is hydrogen and R3 is cyano), benzyl mercaptan (0.03 ml, 1.5 eq.) (nucleophile represented by formula (ii)) and triethylamine (0.04 ml, 2 eq.) to obtain a white amorphous powder (19 mg, 0.04 mmol, 30% isolated yield). 1 H NMR (600MHz, CDCl3) δ7.75(dt,J=7.9,1.4Hz,1H),7.72(t,J=1.8Hz,1H),7.64–7.58(m,3H),7.50(dt,J=7.8,1.3Hz,1H),7.46(t ,J=7.7Hz,2H),7.43–7.40(m,2H),7.40–7.36(m,1H),7.34(dd,J=8.3,6.6Hz,2H),7.32–7.27(m,1H),5.70(s,2H),4.48(s,2H). 13 C NMR (151MHz, CDCl3) δ168.9,159.4,158.2,142.3,140.3,136.2,133.7,129.9,129.7,129.2, 129.1,128.9,127.99,127.87,127.65,127.55,127.3,115.6,114.9,96.8,87.0,35.1.MS(ESI - ) Calculated value C 26 H 18 N4S:[MH + ]m / z=417.1, measured value m / z=416.8.HRMS(ESI + ) Calculated value C 26 H 18 N4S:[M+Na + ]m / z=441.1145, measured value m / z=441.1118.

[0339] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile (21) (wherein R1 is phenyl, R2 is hydrogen, R3 is cyano, and R4 is methoxy)

[0340]

[0341] The product was prepared according to the general method in Scheme 1 starting from 4-([1,1'-biphenyl]-3-yl)-2-amino-6-chloropyridine-3,5-dicarbonitrile (33 mg, 0.10 mmol) (intermediate of formula (i) wherein R1 is phenyl, R2 is hydrogen and R3 is cyano), methanol (1 ml) (nucleophile of formula (ii)) and 1.8 M lithium diisopropylamine in THF solution (0.19 ml, 3 eq.), and the isolated white amorphous powder (22 mg, 0.07 mmol, 67% isolated yield). 1 H NMR(600MHz,(CD3)2SO)δ8.09(broad s,2H),7.89–7.85(m,1H),7.84(d,J=1.9Hz,1H),7.76–7.71(m,2H),7.67(t,J=7.7Hz,1H),7.55–7.50(m,3H),7.44–7.40(m,1H),4.00(s,3H). 13 C NMR (151MHz, (CD3)2SO) δ165.8,161.2,160.5,140.4,139.3,134.6,129.4,129. 1,128.6,127.9,127.4,126.90,126.87,115.6,115.2,83.4,83.3,54.8.MS(ESI + ) Calculated value C 20 H 14 N4O:[M+Na + ]m / z=349.1, measured value m / z=349.0.

[0342] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-ethoxypyridine-3,5-dicarbonitrile (22) (wherein R1 is phenyl, R2 is hydrogen, R3 is cyano, and R4 is ethoxy)

[0343]

[0344] The product was prepared according to the general method in Scheme 1 starting from 4-([1,1'-biphenyl]-3-yl)-2-amino-6-chloropyridine-3,5-dicarbonitrile (25 mg, 0.07 mmol) (intermediate of formula (i) wherein R1 is phenyl, R2 is hydrogen and R3 is cyano), ethanol (1 mL) (nucleophile of formula (ii)) and 1.8 M lithium diisopropylamine in THF (0.13 mL, 3 eq.) to isolate a white amorphous powder (19 mg, 0.055 mmol, 80% isolated yield). 1H NMR(600MHz,(CD3)2SO)δ7.97(broad s,2H),7.86(ddd,J=7.8,1.9,1.1Hz,1H),7.84(t,J=1.8Hz,1H),7.75–7.70(m,2H),7.66(t,J= 7.7Hz,1H),7.55–7.48(m,3H),7.45–7.39(m,1H),4.46(q,J=7.1Hz,2H),1.36(t,J=7.1Hz,3H). 13 C NMR(151MHz,(CD3)2SO)δ165.4,161.2,160.6,140.4,139.3,134.7,129.4,129.1, 128.5,127.9,127.4,126.91,126.87,115.7,115.2,83.5,83.1,63.4,14.3.MS(ESI + ) Calculated value C 21 H 16 N4O:[M+Na + ]m / z=363.1, measured value m / z=363.0.

[0345] 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(2-hydroxyethoxy)pyridine-3,5-dicarbonitrile (23) (wherein R1 is phenyl, R2 is hydrogen, R3 is cyano, R4 is ethoxy, 1-ol)

[0346]

[0347] The product was prepared according to the general method in Scheme 1 starting from 4-([1,1'-biphenyl]-3-yl)-2-amino-6-chloropyridine-3,5-dicarbonitrile (33 mg, 0.10 mmol) (intermediate of formula (i) wherein R1 is phenyl, R2 is hydrogen and R3 is cyano), methanol (1 ml) (nucleophile of formula (ii)) and 1.8 M lithium diisopropylamine in THF solution (0.19 ml, 3 eq.) to obtain a white amorphous powder (11 mg, 0.03 mmol, 31% isolated yield). 1H NMR(600MHz,(CD3)2SO)δ7.97(broad s,2H),7.87(dt,J=7.8,1.5Hz,1H),7.84(t,J=1.8Hz,1H),7.76–7.72(m,2H),7.66(t,J=7.7Hz,1H),7. 55–7.48(m,3H),7.46–7.39(m,1H),4.91(t,J=5.3Hz,1H),4.43(t,J=5.1Hz,2H),3.74(q,J=5.2Hz,2H). 13 C NMR(151MHz,(CD3)2SO)δ165.6,161.1,160.6,140.4,139.3,134.7,129.4,129.1, 128.6,127.9,127.4,126.90,126.86,115.6,115.2,83.6,83.2,69.0,58.9.MS(ESI + ) Calculated value C 21 H 16 N4O2:[M+Na + ]m / z=379.1, measured value m / z=379.0.

[0348] 2-Amino-4-(3-(3-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (24) (wherein R1 is 3-fluoro-2-pyridinyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0349]

[0350] As described above, the product was prepared using microwave-assisted Suzuki cross-coupling, reacted at 80°C for 15 min, and the starting materials were (3-(2-amino-3,5-dicyano-6-(piperidin-1-yl)pyridin-4-yl)phenyl)boronic acid pinacol ester (intermediate shown in formula (iiiba)) (100 mg, 0.23 mmol, 1 eq.) and 2-bromo-3-fluoropyridine (intermediate shown in formula (iv) wherein R1 is 3-fluoro-2-pyridyl and R9 is bromine) (53 mg, 1.2 eq.), and a white powder was isolated (66 mg, 0.16 mmol, 72% isolated yield). 1H NMR (600MHz, CDCl3) δ8.54(dt,J=4.6,1.6Hz,1H),8.19(dd,J=7.8,1.5Hz,1H),8.17(d,J=1.5Hz,1H),7.64(t,J=7.7Hz,1H),7.57(d t,J=7.7,1.4Hz,1H),7.53(ddd,J=11.0,8.2,1.4Hz,1H),7.31(p,J=4.2Hz,1H),5.44(s,2H),3.84–3.79(m,4H),1.73–1.70(m,6H). 13 C NMR (151MHz, CDCl3) δ162.1, 161.1, 159.5, 157.9 (d, J = 261.4Hz), 145.5 (d, J = 5.2Hz), 145.0 (d, J = 10.3Hz), 135.8 (d, J = 5.6Hz), 135.2, 131.0 (d,J=6.6Hz),129.7,129.3(d,J=6.1Hz),129.2,124.7(d,J=20.6Hz),124.1(d,J=4.1Hz),117.6,116.5,83.9,82.0,49.5,26.2,24.5.MS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.2, measured value m / z=399.1.HRMS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.1728, measured value m / z=399.1701.

[0351] 2-amino-4-(3-(6-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (25) (wherein R1 is 6-fluoro-2-pyridinyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0352]

[0353] As described above, the product was prepared using microwave-assisted Suzuki cross-coupling, reacted at 80°C for 15 min, and the starting materials were (3-(2-amino-3,5-dicyano-6-(piperidin-1-yl)pyridin-4-yl)phenyl)boronic acid pinacol ester (intermediate shown in formula (iiiba)) (100 mg, 0.23 mmol, 1 eq.) and 2-bromo-6-fluoropyridine (intermediate shown in formula (iv) wherein R1 is 6-fluoro-2-pyridyl and R9 is bromine) (53 mg, 1.2 eq.), and the white powder product was isolated (35 mg, 0.09 mmol, isolated yield 38%). 1 H NMR (600MHz, CDCl3) δ8.17–8.12(m,2H),7.86(q,J=8.0Hz,1H),7.68(dd,J=7.4,2.2Hz,1H),7.64–7.61(m,1H) ,7.55(dt,J=7.6,1.5Hz,1H),6.90(dd,J=8.1,3.0Hz,1H),5.47(s,2H),3.85–3.80(m,4H),1.75–1.69(m,6H). 13 C NMR (151MHz, CDCl3) δ163.6 (d, J = 238.9Hz), 162.1, 160.9, 159.34, 155.4 (d, J = 13.4Hz), 141.9 (d, J = 7.7Hz), 138.3, 135.5 ,129.8,129.5,129.0,127.6,117.8(d,J=4.0Hz),117.6,116.5,108.4(d,J=37.5Hz),83.8,81.9,49.5,26.2,24.5.MS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.2, measured value m / z=399.2.HRMS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.1728, measured value m / z=399.1701.

[0354] 2-amino-4-(3-(4-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (26) (wherein R1 is 4-fluoro-2-pyridinyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0355]

[0356] As described above, the product was prepared using microwave-assisted Suzuki cross-coupling at 80°C for 15 min, starting from (3-(2-amino-3,5-dicyano-6-(piperidin-1-yl)pyridin-4-yl)phenyl)boronic acid pinacol ester (intermediate of formula (iiiba)) (100 mg, 0.23 mmol, 1 eq.) and 2-bromo-4-fluoropyridine (intermediate of formula (iv) wherein R1 is 4-fluoro-2-pyridyl and R9 is bromine) (53 mg, 1.2 eq.) and isolated as a white powder (35 mg, 0.09 mmol, 38% isolated yield). 1 H NMR (600MHz, CDCl3) δ8.70(dd,J=8.5,5.6Hz,1H),8.17(d,J=7.8Hz,1H),8.14(s,1H),7.66(t,J=7.7Hz,1H),7.60(dd,J=7.7,1 .6Hz,1H),7.54(dd,J=10.1,2.4Hz,1H),7.05(ddd,J=6.5,5.1,2.6Hz,1H),5.39(s,2H),3.84–3.80(m,4H),1.74–1.70(m,6H). 13 C NMR (151MHz, CDCl3) δ169.9 (d, J = 264.1Hz), 161.8, 161.1, 159.5, 151.7, 135.7, 130.3, 129.7, 129.2, 127.8,117.7,116.6,110.8(d,J=17.0Hz),109.0(d,J=17.8Hz),83.7,81.8,49.4,26.2,24.6.MS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.2, measured value m / z=399.2.HRMS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.1728, measured value m / z=399.1701.

[0357] 2-Amino-4-(3-(5-fluoropyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (27) (wherein R1 is 5-fluoro-3-pyridinyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0358]

[0359] As described above, the product was prepared using microwave-assisted Suzuki cross-coupling at 80°C for 15 min, starting from (3-(2-amino-3,5-dicyano-6-(piperidin-1-yl)pyridin-4-yl)phenyl)boronic acid pinacol ester (intermediate of formula (iiiba)) (100 mg, 0.23 mmol, 1 eq.) and 3-bromo-5-fluoropyridine (intermediate of formula (iv) wherein R1 is 5-fluoro-3-pyridyl and R9 is bromine) (53 mg, 1.2 eq.) to isolate a white powder (76 mg, 0.19 mmol, 83% isolated yield). 1 H NMR (600MHz, CDCl3) δ8.74(d,J=1.7Hz,1H),8.51(d,J=2.2Hz,1H),7.72(ddt,J=19.1,9.9,1.6Hz,3H), 7.66(t,J=7.3Hz,1H),7.60(dd,J=7.7,1.5Hz,1H),5.43(s,2H),3.85–3.78(m,4H),1.74–1.70(m,6H). 13 C NMR (151MHz, CDCl3) δ161.5,161.0,160.8,159.5,143.4,138.3,136.7,136.2,136.1, 130.1,129.5,129.4,128.0,122.8,117.7,116.6,83.5,81.7,49.4,26.2,24.5.MS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.2, measured value m / z=399.2.HRMS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.1728, measured value m / z=399.1701.

[0360] 2-Amino-4-(3-(2-fluoropyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (28) (wherein R1 is 2-fluoro-3-pyridinyl, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0361]

[0362] As described above, the product was prepared using microwave-assisted Suzuki cross-coupling at 100°C for 30 min, starting from 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate shown in formula (iiiaa)) (100 mg, 0.23 mmol, 1 eq.) and 2-fluoro-3-pyridineboronic acid (intermediate shown in formula (iv) wherein R1 is 2-fluoro-3-pyridyl and R9 is boronic acid) (109 mg, 3 eq.) and isolated as a white powder (12 mg, 0.03 mmol, 12% isolated yield). 1 H NMR(600MHz, (CD3)2SO) δ8.29(d,J=4.6Hz,0H),8.16(ddd,J=10.3,7.4,1.9Hz,1H),7.81(dq,J=7.7,1.6Hz,1H),7.78(s,1H),7.68( t,J=7.7Hz,1H),7.60(dt,J=7.7,1.4Hz,1H),7.52(ddd,J=6.9,4.8,1.7Hz,1H),7.46(s,3H),3.76–3.71(m,4H),1.71–1.55(m,6H). 13 C NMR (151MHz, (CD3)2SO) δ 161.1, 160.7, 159.8, 159.6 (d, J = 237.7Hz), 146.9 (d, J = 14.6Hz), 141.6 (d, J = 4.2Hz), 135.8, 133.6 (d, J = 5.1Hz), 1 30.4(d,J=3.1Hz),129.1,129.0(d,J=24.0Hz),122.8(d,J=4.2Hz),122.2(d,J=28.2Hz),117.8,116.3,81.5,80.8,48.5,25.6,23.9.MS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.2, measured value m / z=399.5.HRMS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.1728, measured value m / z=399.1707.

[0363] 2-Amino-4-(2-fluoro-5-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (29) (wherein R1 is pyridinyl, R2 is 6-fluoro, R3 is cyano, and R4 is piperidinyl)

[0364]

[0365] The product was prepared as described above using a microwave-assisted Suzuki cross-coupling reaction at 100°C for 30 min, starting with 2-amino-4-(5-bromo-2-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate shown in formula (iiiab)) (58 mg, 0.15 mmol, 1 eq.) and 3-pyridineboronic acid (intermediate shown in formula (iv) wherein R1 is 3-pyridyl and R9 is boronic acid) (55 mg, 3 eq.) to isolate a white powder (53 mg, 0.13 mmol, 89% isolated yield). 1 HNMR(600MHz, CDCl3) δ8.89(d,J=2.3Hz,1H),8.66(dd,J=5.1,1.5Hz,1H),8.09(dd,J=8.0,1.9Hz,1H),7.71(ddd,J=8.6,4.6,2.4Hz,1 H),7.61(dd,J=6.5,2.4Hz,1H),7.57(dd,J=8.0,5.0Hz,1H),7.39(t,J=8.9Hz,1H),5.41(s,2H),3.86–3.81(m,4H),1.76–1.67(m,6H). 13 C NMR (151MHz, CDCl3) δ160.4,159.6(d,J=253.6Hz),159.3,155.8,145.9(d,J=82.0Hz),137.3,136.5,133.3,131.3(d,J=8.4H z),129.8(d,J=3.1Hz),124.8,124.0(d,J=15.7Hz),117.8(d,J=22.5Hz),117.2,116.1,84.3,82.4,49.2,26.2,24.5.MS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.2, measured value m / z=399.4.HRMS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.1728, measured value m / z=399.1701.

[0366] 2-amino-4-(2-fluoro-3-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (30) (wherein R1 is pyridinyl, R2 is 2-fluoro, R3 is cyano, and R4 is piperidinyl)

[0367]

[0368] As described above, the product was prepared using microwave-assisted Suzuki cross-coupling, reacting at 100°C for 30 min, starting from 2-amino-4-(3-bromo-2-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate shown in formula (iiiac)) (58 mg, 0.15 mmol, 1 eq.) and 3-pyridineboronic acid (intermediate shown in formula (iv) wherein R1 is 3-pyridyl and R9 is boronic acid) (55 mg, 3 eq.), and isolated as a paste (15 mg, 0.04 mmol, 25% isolated yield). 1 H NMR (600MHz, CDCl3) δ8.96–8.94(m,1H),8.77–8.73(m,1H),8.41(d,J=8.0Hz,1H),7.83(dd,J=8.0,5.3Hz,1H),7.63(td,J=7 .4,1.8Hz,1H),7.55(ddd,J=8.0,6.4,1.7Hz,1H),7.49(t,J=7.7Hz,1H),5.43(s,2H),3.86–3.80(m,4H),1.77–1.68(m,6H). 13 C NMR (151MHz, CDCl3) δ160.1, 159.1, 156.1 (d, J = 253.4Hz), 155.6, 143.5, 142.6, 133.9, 132.41 (d, J = 2.6H z),132.37,125.8(d,J=4.7Hz),125.7,124.4,124.3,117.1,115.9,84.1,82.1,49.1,26.0,24.4.MS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.2, measured value m / z=399.4.HRMS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.1728, measured value m / z=399.1701.

[0369] 2-amino-4-(3-fluoro-5-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (31) (wherein R1 is pyridinyl, R2 is 5-fluoro, R3 is cyano, and R4 is piperidinyl)

[0370]

[0371] As described above, the product was prepared using microwave-assisted Suzuki cross-coupling, reacting at 100°C for 30 min, starting from 2-amino-4-(3-chloro-5-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate shown in formula (iiiad)) (53 mg, 0.15 mmol, 1 eq.) and 3-pyridineboronic acid (intermediate shown in formula (iv) wherein R1 is 3-pyridyl and R9 is boronic acid) (55 mg, 3 eq.), and isolated as a white powder (56 mg, 0.14 mmol, 96% isolated yield). 1 H NMR (600MHz, CDCl3) δ8.93(s,1H),8.70(s,1H),8.15(dt,J=8.0,1.8Hz,1H),7.65–7.59(m,1H),7.49(d,J=1.6Hz, 1H),7.44(dt,J=8.9,1.9Hz,1H),7.31(dt,J=8.5,1.9Hz,1H),5.45(s,2H),3.86–3.81(m,4H),1.76–1.68(m,6H). 13 CNMR(151MHz, CDCl3)δ163.2(d,J=250.3Hz),160.8,160.1,159.4,146.3,145.4,138.0(d,J=8.4Hz),137.8,125.1, 123.8(d,J=3.1Hz),117.5,116.7(d,J=23.0Hz),116.32(d,J=22.5Hz),116.27,83.2,81.5,49.3,26.2,24.5.MS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.2, measured value m / z=399.3.HRMS(ESI + ) Calculated value C 23 H 19 N6F:[M+H + ]m / z=399.1728, measured value m / z=399.1707.

[0372] 4-(3-(1,3,4-oxadiazol-2-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (32) (wherein R1 is 1,3,4-oxadiazole, R2 is hydrogen, R3 is cyano, and R4 is piperidinyl)

[0373]

[0374] In a 25mL round-bottom flask equipped with a magnetic stirrer and a condenser, methyl 3-(2-amino-3,5-dicyano-6-(piperidin-1-yl)pyridin-4-yl)benzoate (54mg, 0.15mmol, 1eq.) was suspended in anhydrous methanol (1.5mL). Hydrazine monohydrate (37mg, 5eq.) was added to the solution, and the container was then purged with argon and sealed. The reactants were heated at 80°C for 16h. The solvent was removed under reduced pressure, and the crude product was washed with DCM:MeOH 7:3 (10mL) and dried under reduced pressure. In a 25mL round-bottom flask equipped with a magnetic stirrer and a condenser, the intermediate was suspended in excess triethyl orthoformate (1mL). The container was purged with argon and heated at 100°C for 20h. The reaction was quenched by adding water (20ml) and the crude product was extracted with ethyl acetate (3×15ml). The organic phases were combined, washed with brine, dried over MgSO4, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on a 12 g silica gel column using a cyclohexane:ethyl acetate gradient elution (8:2, 3CV; 8:2→6:4, 12CV; 6:4, 6CV) to isolate a white powder (32 mg, 0.09 mmol, total isolated yield 58%). 1 H NMR (600MHz, CDCl3) δ8.50(s,1H),8.26(dt,J=7.6,1.6Hz,1H),8.21(t,J=1.8Hz,1H),7.70(t, J=7.8Hz,1H),7.67(dt,J=7.8,1.6Hz,1H),5.66(s,2H),3.87–3.82(m,4H),1.76–1.70(m,6H). 13 C NMR (151MHz, CDCl3) δ164.1,161.2,160.2,159.2,152.9,136.0,132.2,130 .1,129.1,127.5,124.5,117.2,115.9,83.6,81.6,49.6,26.2,24.4.MS(ESI + ) Calculated value C 20 H 17 N7O:[M+H + ]m / z=372.2, measured value m / z=372.4.HRMS(ESI +) Calculated value C 20 H 17 N7O:[M+H + ]m / z=372.1568, measured value m / z=372.1565.

[0375] Example 2: Efficacy of the compounds of the present invention in CHO cells

[0376] As described below, the compounds of the invention were tested for their potency using cells expressing adenosine receptors.

[0377] a) Characterization of efficacy using cells expressing adenosine receptors

[0378] According to the present invention, Chinese hamster ovary (CHO) cells containing an internal cAMP-based biosensor (described below) and expressing recombinant A2AR are used to characterize the efficacy of the A2AR antagonists described in the present invention. The biosensor contains a cAMP binding domain that allows the measurement of activation of Gs and Gi-coupled receptors. Cells are treated with the specified concentration of A2AR antagonists in 384-well plates, and then the A2AR agonist adenosine is added. cAMP levels are measured on a luminescence / fluorescence microplate reader.

[0379] CHO cells expressing A2AR and biosensors were seeded in black 384-well plates (Nunc) and grown overnight at 37°C and 5% CO2. They were pre-incubated with compounds of the invention at different concentrations (up to 30 μM) for 6 min before adding the corresponding EC80 A2AR agonist, i.e., 400 nM adenosine (Sigma). The cAMP biosensor allows real-time monitoring of the signal using FDSS / μCELL (Hamamatsu). The assay was performed in 1X Hanks balanced salt solution (HBSS). The total reaction volume was 80 μl (45 μl cells, 15 μl antagonist and 20 μl agonist). Data analysis was performed using GraphPad Prism.

[0380] The compounds of the present invention are able to antagonize cAMP production in cell lines overexpressing A2AR in the absence of other adenosine receptors, as shown in Table 1 below, where activity represents the ability of the compound to antagonize cAMP production in response to stimulation of A2AR-expressing CHO cells with the A2AR agonist adenosine, as described in the above cAMP assay method. Therefore, these compounds can be classified as A2AR antagonists.

[0381] Table 1

[0382]

[0383]

[0384] Activity level indicates IC50 Range: +: >1000nM, ++: 200-1000nM, +++: <200nM.

[0385] b) Use “displacement assay” to verify the negative allosteric molecular action mode of small molecules

[0386] "Shift assays" using A2AR transfected cell lines are used to demonstrate the allosteric molecular pattern of A2AR inhibition mediated by the compounds of the present invention. These assays use CHO cells containing BRET-based biosensors and expressing recombinant A2AR, and "shift assays" are performed in 384-well plates, adding A2AR antagonists at specified concentrations, and then adding A2AR agonist adenosine for concentration-response curves (CRC). cAMP levels are measured on a luminescent / fluorescent microplate reader. In brief, CHO cells expressing A2AR and biosensors are seeded in black 384-well plates and grown overnight at 37°C and 5% CO2. CHO cells are pre-incubated with compounds of the present invention at different concentrations (up to 30 μM) for 6 min before adding increasing concentrations up to 1 mM adenosine (CRC). cAMP biosensor readings and measurements are as described above. Data analysis was performed using GraphPad Prism. The Schild regression graph of the compound was calculated using the shift assay data. Log (dose ratio-1) is plotted on the y-axis, relative to the Log of the antagonist concentration on the x-axis.

[0387] Negative allosteric regulation of A2AR can be identified using a "shift assay". Figure 1 As shown in A, it can be seen that as the concentration of the compound increases, the concentration-response curves (CRCs) of adenosine begin to overlap with each other (from small opening circles to larger opening circles). The data is converted into a Schild regression plot ( Figure 1 B), confirming the classification of the compounds of the invention as NAMs, since allosteric modulators lead to a leveling off of the dose ratio (DR), as shown in the upper right corner of the Schild plot. In contrast, orthosteric compounds are characterized by a completely linear DR curve in the Schild regression plot (Kenakin, 2017, Curr Protoc Pharmacol doi:10.1002 / cpph.18)

[0388] Example 3: Effect of the compounds of the present invention on the recovery of immune responses in healthy human peripheral blood immune cells Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood from healthy human donors using density centrifugation. The cells were frozen and thawed and allowed to stand for several hours in complete cell culture medium containing human albumin and antibiotics (penicillin and streptomycin; 100 units or mg / ml, respectively). In a 96-well microplate, cells were incubated with A2AR antagonists at specified concentrations of the present invention, followed by the addition of 5 μM 5'-N-ethylamidoadenosine (NECA, an adenosine analog, is a potent A2AR agonist, https: / / doi.org / 10.1371 / journal.pcbi.1007818). DMSO was used as a storage diluent for concentrated A2AR antagonists. NECA and DMSO controls (% v / v) were also tested in parallel with the therapeutic candidate drugs. After the indicated incubation times with NECA, cells were either stained to determine their intracellular pCREB levels or activated overnight with immunomagnetic beads before staining to detect intracellular cytokines as described below.

[0389] a) Flow cytometry of pCREB in cells

[0390] The ability of compounds of the invention to restore normal pCREB levels in human immune cells was tested as follows.

[0391] PBMCs cultured with A2AR antagonists in the presence of NECA were subsequently fixed and permeabilized using ice-cold paraformaldehyde and methanol-based kit reagents (ThermoFisher and Miltenyi, respectively). Cells were washed with FACS buffer and stained with fluorochrome-conjugated antibodies against the following antigens: pCREB (phosphorylated at serine 133), CD4, CD8 (all from Life Technologies), and CD3 (BD ​​Biosciences). Stained cells were acquired using a Fortessa flow cytometer (BD). Desired cell subsets were gated using FSC-A vs. FSC-H to remove doublets, and cells with lymphocyte-like morphology were identified using FSC vs. SSC parameters, followed by CD3 + T cells and CD4 + or CD8 + Subgating. The fold change of the mean fluorescence intensity (MFI) of intracellular pCREB in gated CD4 or CD8+ T cells in the condition of NECA alone was calculated and divided by the cells cultured with NECA and A2AR antagonists from the same donor. The overall activity of the test compound was calculated using the average of at least 3 donors.

[0392] The compounds described herein were tested under physiological conditions using healthy human peripheral blood mononuclear cells and in the presence of human albumin to increase the physiological relevance of the assay. The compounds of the invention were also compared to previously reported control molecules that antagonize A2AR (orthosteric antagonists) and tested in human Phase I or Phase II clinical trials. These include CPI-444, 7-(5-methylfuran-2-yl)-3-[[6-[[(3S)-tetrahydrofuran-3-yl]oxymethyl]pyridin-2-yl]methyl]triazolo[4,5-d]pyrimidin-5-amine (Ciforadenant), PBF-5091, 5-bromo-2,6-di(pyrazol-1-yl)pyrimidin-4-amine (Taminadenant), MK-38141, 4-(furan-2-yl)-10-[2-[4-[4-(2-methoxyethoxy)phenyl]piperazin-1-yl]ethyl]-3,5,6,8,10,11-hexaazatricyclo[730.0.0] 2,6 ] dodeca-1(9),2,4,7,11-pentaen-7-amine (Preladenant)) AZD4635, 6-(2-chloro-6-methylpyridin-4-yl)-5-(4-fluorophenyl)-1,2,4-triazine-3-amine (Imaradenan) and AB928, 3-[2-amino-6-[1-[[6-(2-hydroxypropane-2-yl)pyridin-2-yl]methyl]triazol-4-yl]pyrimidin-4-yl]-2-methylbenzonitrile (Etrumadenant), and two other known stereogenic compounds. Table 2 below shows the effects of the compounds on the gated human CD4 + Table 3 shows the ability of the compounds to restore pCREB to normal physiological levels (immune competence) in the above-mentioned gated human CD8 + The ability of cells to restore pCREB to normal physiological levels (immune competence) compared to a control group treated with NECA alone. The activity levels represented are the average of at least 3 donors.

[0393] Table 2

[0394]

[0395] Table 3

[0396]

[0397]

[0398] Activity ratings represent restoration of normal antitumor responses, as assessed by measuring pCREB levels in CD4+ and CD8+ T cells. Minimal antitumor responses were measured under immunocompromised conditions (NECA immunosuppression, no A2AR antagonists), and normal responses were measured under immunocompetent conditions (no NECA and A2AR antagonists); "-" indicates no reduction, "+" indicates a reduction of 0 to 40%, "++" indicates a reduction of 40 to 70%, "+++" indicates a reduction of 70 to 90%, and "++++" indicates a reduction of more than 90%. A reduction in pCREB corresponds to restoration of normal antitumor immune responses.

[0399] As shown in Table 2 and Table 3, under conditions simulating high adenosine concentrations, the compounds of the present invention can restore CD4 and CD8 + Normal physiological levels of pCREB in T cells. pCREB is a proximal (downstream) signaling element of cAMP during A2AR signaling and represents a surrogate marker of immunosuppression.

[0400] b) Assessment of intracellular cytokines using flow cytometry

[0401] As described below, compounds of the invention were tested for their ability to restore anti-tumor cytokine responses in human immune cells.

[0402] In addition, PBMCs cultured with A2AR antagonists and NECA were stimulated with 1 μl of anti-CD3 and anti-CD28 immunomagnetic beads (Life Technologies) in the presence of GolgiStop (Life Technologies) overnight to activate T cells and thus block the secretion of any newly induced cytokines. Cells were then stained with the following: fixable live-dead stain (LD Blue, Life Technologies), T cell-specific antibodies as described in the pCREB stain, followed by fixation and permeabilization using a commercial kit containing paraformaldehyde and detergent-based buffer (ThermoFisher). Intracellular staining of cytokines was performed using fluorescent dye-conjugated antibodies against tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), and interferon gamma (IFN-γ), diluted in permeabilization buffer (ThermoFisher). Cells were then washed, harvested, and gated as described for the pCREB assay with the following adjustments: dead cells were excluded using a live-dead marker, and the gated T cell subsets were then analyzed for the percentage of cytokine-positive cells, which was a percentage of total CD4 + or CD8 +Sub-gates for T cells. In each donor, the percentage of cytokine-positive cells in the immunomagnetic bead control sample alone was used as a 100% normal (immunocompetent) immune response standard, while the immunomagnetic bead plus NECA stimulation condition represented an immunosuppressive control. Using the average of at least 2 donors, the restoration of cytokine responses in the presence of A2AR antagonists was calculated as follows: 100x(1-(100-% cytokine-positive cells in the presence of A2AR antagonists / (100-% cytokine-positive cells in immunocompetent controls)).

[0403] The compounds were tested in a translational assay using in vitro activation of healthy donor human T cells in the presence of immunosuppressive concentrations of the A2AR agonist NECA as described above. The compounds were able to restore activated human blood CD4 + Normal cytokine production and activation in human blood CD8 T cells + Normal cytokine production in T cells is described below in Tables 4 and 5, respectively. Clinical stage antagonist compounds are described in Table 2.

[0404] Table 4

[0405]

[0406]

[0407] Table 5

[0408]

[0409] 2 Activity represents the recovery of immune response by considering the minimum response i

[0410] nImmunocompromised condition (NECA immunosuppression + T cell activity, no A2AR antagonist), and maximal normal (immunoenitrosomic) response (T cell activity in the absence of NECA and A2AR antagonist). ND: Not measured. Representative results show that CD4 + T cells (Table 4) and CD8 + The ability of T cells (Table 5) to secrete pro-inflammatory (anti-tumor) cytokines (TNF-α, IL-2, IFN-γ) was restored in the presence of some clinical-stage antagonist molecules or A2AR negative allosteric modulators.

[0411] Example 4: In vivo effects of the compounds of the present invention in mouse tumor models

[0412] To demonstrate the in vivo therapeutic activity of A2AR antagonists, mouse ectopic MC38 or CT26 (colon cancer) tumor models were used. Mouse colon tumor cells CT26 (FenicsBio) and MC38 (Creative-Biogene) expressing bioluminescent luciferase constructs (firefly luciferase under the control of the CMV promoter) were confirmed to be mycoplasma-free. Cells were cultured according to the manufacturer's recommendations and injected subcutaneously (0.2-1 million cells, e.g., 250,000 to 500,000 tumor cells) into immunocompetent mice (Balb / c mice for the CT26 model and C57BL / 6 mice for the MC38 model). Tumor growth was monitored until clear medium-sized tumors were established. Subsequently, mice were randomized and treated by direct intratumoral injection or systemic administration. After 7 to 14 days, the mice were randomized and the average tumor size was 50 mm 2 . Mice were treated at least once a day by the specified route and compound dose. For the combination therapy trial, mice were also treated with an existing approved immunotherapy (in vivo grade anti-PD-1 or anti-CTLA ICI monoclonal antibody, commercially purchased) by the intraperitoneal route at a specified dose and frequency. Mice were monitored daily for adverse events, and tumor burden was measured by caliper or injection of fluorescein D followed by imaging of the entire mouse using an IVIS Spectrum (PerkinElmer) instrument. Compared with vehicle control treatment under similar conditions, the compound shown in formula (Ib) of the present invention was able to significantly reduce tumor growth.

Claims

1. A compound of formula (I) for use in treating cancer, especially for use in immunotherapy to treat cancer, and a pharmaceutically acceptable salt thereof; in, R1 is selected from aryl optionally substituted by one or more halogen (e.g. fluorine), cyano, hydroxyl or C1-C6 alkyl, and heteroaryl optionally substituted by one or more halogen, cyano, hydroxyl or C1-C6 alkyl; R2 is selected from H and halogen; R3 is selected from H, CN and optionally substituted C1-C6 alkyl; R4 is selected from XR(R'), optionally substituted aryl, and optionally substituted heteroaryl; wherein X is selected from O, S, N; R and R' are each independently optionally substituted C1-C6 alkyl; or XRR' together form an optionally substituted heterocycloalkyl.

2. The compound according to claim 1, characterized in that The compound is a cyanopyridine compound represented by formula (Ia), and a pharmaceutically acceptable salt thereof; in, R1 and R2 are as described in the preceding claims; X is selected from O and S; R5 is an optionally substituted C1-C6 alkyl group (eg, a C1-C6 alkyl group optionally substituted by a hydroxyl group, an amino group, an ether group, a thioether group, a cyano group, a halogen group, an aryl group, a heteroaryl group, or a branched C1-C6 alkyl group).

3. The compound according to claim 1, characterized in that The compound is a cyanopyridine compound represented by formula (Ib), and a pharmaceutically acceptable salt thereof; in, R1 and R2 are as described in claim 1; R6 and R7 are each independently an optionally substituted C1-C6 alkyl group (e.g., a C1-C6 alkyl group optionally substituted with hydroxy, amino, ether, thioether, cyano, halogen, aryl, heteroaryl or branched C1-C6 alkyl); or R6 and R7 together form an optionally substituted heterocycloalkyl group (e.g., an optionally substituted azetidinyl group, an optionally substituted pyrrolidinyl group, an optionally substituted piperidinyl group, an optionally substituted morpholinyl group, an optionally substituted piperazinyl group, an optionally substituted azepanyl group, an optionally substituted azacyclooctanyl group).

4. A compound represented by (I), wherein R1 to R4 are as defined herein, provided that the compound represented by formula (I) is not a compound selected from the group consisting of: 4-(3-(1H-pyrrol-1-yl)phenyl)-2-amino-6-(benzylthio)pyridine-3,5-dicarbonitrile (RN: 391667-62-2); 2-amino-6-(benzylthio)-4-(3-(2,5-dimethyl-1H-pyrrol-1-yl)phenyl)pyridine-3,5-dicarbonitrile (RN: 391664-25-8); 4-(3-(1H-pyrrol-1-yl)phenyl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile (RN: 391664-24-7); 4-(3-(1H-imidazol-1-yl)phenyl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile ; 4-(3-(1H-imidazol-2-yl)phenyl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile; 2-amino-4-(3'-cyano-5'-fluoro-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-41-2); 2-amino-4-(3'-(hydroxymethyl)-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-42-3); 2-amino-4-(4'-chloro-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 16461 73-50-3); 2-amino-4-(3'-chloro-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-49-0); 2-amino-4-(2'-chloro-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-48-9); 2-amino-6-phenyl-4-(4'-(trifluoromethyl)-[1,1'-biphenyl]-3-yl)pyridine-3,5-dicarbonitrile (RN: 1646173-44-5); 2-amino-6-phenyl-4-(3'-(trifluoromethyl)-[1,1' -biphenyl]-3-yl)pyridine-3,5-dicarbonitrile (RN: 1646173-46-7); 2-amino-4-(3',4'-dichloro-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-51-4); 2-amino-4-(4'-bromo-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-55-8); or 2-amino-4-(2'-methyl-[1,1'-biphenyl]-3-yl)-6-phenylpyridine-3,5-dicarbonitrile (RN: 1646173-54-7).

5. The compound according to claim 4, characterized in that Used as medicine.

6. The compound according to any one of claims 1 to 3, characterized in that R1 is aryl optionally substituted by one or more halogen, cyano, hydroxyl or C1-C6 alkyl.

7. The compound according to any one of claims 1 to 3, characterized in that R1 is selected from phenyl, halogenated phenyl such as 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, or C1-C6 alkylphenyl such as methylphenyl, for example 2-methylphenyl, 3-methylphenyl, 4-methylphenyl.

8. The compound according to any one of claims 1 to 3, characterized in that R1 is heteroaryl optionally substituted by one or more halogen, cyano, hydroxy or C1-C6 alkyl.

9. The compound according to claim 1 or 8, characterized in that R1 is an optionally substituted heteroaryl, wherein the optionally substituted heteroaryl is selected from an optionally substituted pyridyl, an optionally substituted thienyl, an optionally substituted pyrazolyl and an optionally substituted tetrazolyl, wherein the optional substitution refers to optional substitution with one or more halogen, cyano, hydroxyl or C1-C6 alkyl.

10. The compound according to any one of claims 1 or 6 to 9, characterized in that R2 is H.

11. The compound according to any one of claims 1 or 6 to 10, characterized in that R3 is CN.

12. The compound according to any one of claims 1 or 6 to 11, characterized in that R4 is a XR(R') group, X, R and R' being as described in any of the preceding claims.

13. The compound according to any one of claims 1 or 6 to 12, characterized in that R4 is OR, wherein R is optionally substituted alkyl.

14. The compound according to any one of claims 1 or 6 to 12, characterized in that R4 is SR, wherein R is optionally substituted alkyl.

15. The compound according to any one of claims 1 or 6 to 12, characterized in that R4 is selected from pyridinyl, pyrrolidinyl and azetidinyl.

16. The compound according to any one of claims 1 or 6 to 12, characterized in that R4 is optionally substituted pyridyl.

17. The compound according to any one of claims 1 or 6 to 12, characterized in that R4 is SR, and R is as defined in any preceding claim.

18. The compound according to any one of claims 1, 2 or 6-17, characterized in that The compound is selected from the following group: 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(azetidin-1-yl)pyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(pyrrolidin-1-yl)pyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-amino-4-(2'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-amino-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-Amino-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile 2-amino-4-(2'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-Amino-4-(3'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile 2-amino-4-(4'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-2-yl)phenyl)pyridine-3,5-dicarbonitrile; 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-3-yl)phenyl)pyridine-3,5-dicarbonitrile; 2-amino-6-(piperidin-1-yl)-4-(3-(pyridin-4-yl)phenyl)pyridine-3,5-dicarbonitrile; 2-amino-6-(piperidin-1-yl)-4-(3-(thiophen-2-yl)phenyl)pyridine-3,5-dicarbonitrile; 2-amino-6-(piperidin-1-yl)-4-(3-(thiophen-3-yl)phenyl)pyridine-3,5-dicarbonitrile; 4-(3-(1H-pyrazol-5-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 4-(3-(1H-tetrazolyl-5-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(diethylamino)pyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-morpholinylpyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(bis(2-hydroxyethyl)amino)pyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(4-methylpiperazin-1-yl)pyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-((2-hydroxyethyl)amino)pyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(diisopropylamino)pyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(benzylamino)pyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(benzylthio)pyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-ethoxypyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(2-hydroxyethoxy)pyridine-3,5-dicarbonitrile; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(benzyloxy)pyridine-3,5-dicarbonitrile; 2-amino-4-(3-(3-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-amino-4-(3-(6-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-amino-4-(3-(4-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-amino-4-(3-(5-fluoropyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-amino-4-(3-(2-fluoropyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-amino-4-(2-fluoro-5-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-amino-4-(2-fluoro-3-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile; 2-amino-4-(3-fluoro-5-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile and 4-(3-(1,3,4-oxadiazol-2-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile.

19. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises at least one compound according to any one of claims 4 or 18, and a pharmaceutically acceptable carrier, diluent or excipient.

20. The compound according to any one of claims 1 or 6 to 18, characterized in that The compound will be used in combination with one or more therapies selected from radiation therapy, chemotherapy, adoptive cell therapy (e.g., CAR-T, or tumor infiltrating lymphocytes TILs), anti-cancer vaccine therapy, targeted biological therapy (e.g., tumor-specific antibodies) or immunomodulatory therapy such as anti-cancer immunotherapeutics, particularly immune checkpoint inhibitors.

21. The compound according to any one of claims 1 or 6 to 18 or 20, characterized in that The cancer is selected from lung cancer (small cell and non-small cell), breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, melanoma, digestive system cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer and brain cancer.

22. A method for preventing and / or treating cancer resistance in a subject against anti-cancer immunotherapy, characterized in that: The subject suffers from cancer or is at risk of developing resistance to anti-cancer immunotherapy, and the method comprises administering the compound or pharmaceutical preparation thereof as claimed in any one of claims 1 or 6-18 to the subject in need thereof.

23. A method for inducing or enhancing an immune response to immunotherapy, particularly anti-cancer immunotherapy, the method comprising administering an effective amount of one or more compounds as described in any one of claims 1 or 6-18 or their pharmaceutical preparations, and in combination with one or more treatments, the treatment being selected from radiotherapy, chemotherapy, adoptive cell therapy (e.g., chimeric antigen receptor T cells CAR-T; or tumor infiltrating lymphocytes TILs), anti-cancer vaccine therapy, targeted biological therapy (e.g., tumor-specific antibodies) or immunomodulatory therapy such as immune checkpoint inhibitors, bispecific T cell engagers or nanobodies that can bind to single or multiple drug targets.

24. A method for preparing a compound of formula (I) as claimed in any one of the preceding claims, characterized in that: The method comprises reacting a nucleophile represented by formula (ii) with a cyanopyridine represented by formula (i) in the presence of a base in an aprotic solvent: wherein R1 to R4, X, R and R' are as described in any of the preceding claims.

25. A method for preparing a compound of formula (I) as claimed in any one of the preceding claims, characterized in that: The method comprises cross-coupling of intermediates represented by formula (iii) and (iv) in the presence of an inorganic base and a catalytic amount of a palladium source; wherein R1, R2, R3 and R4 are as described in any of the preceding claims, and R8 and R9 are selected from halogen, boronic acid or boronic acid pinacol ester groups.

26. A method for preparing a compound of formula (I) as claimed in any one of the preceding claims, characterized in that: The method comprises reacting an aldehyde represented by formula (vi) in the presence of a cyclic amine represented by formula (vii), two equivalents of malononitrile (viii), and an oxidant (such as air) in a polar protic solvent (such as methanol); wherein R1 and R2 are as described in any of the preceding claims, and R6 and R7 together form an optionally substituted heterocycloalkyl as described in claim 1 or 3.

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