Adenosine 2A receptor modulators and their use

Abstract

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

Description

Technical Field

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

Background Art

[0002] High mortality and reduced quality of life are common among patients with various types of solid tumors, indicating an urgent need for new antitumor therapies. An important element in successfully treating progressive or recurrent cancer involves reversing tumor-induced immunosuppression. Current immunotherapies, including immune checkpoint inhibitor (ICI) antibodies, have hitherto attempted to restore antitumor T cell responses either by directly targeting these cells or by targeting antigen-presenting cells that activate T cells. Despite clear clinical benefits in approximately 30% of patients, the response rate remains limited. This is due in part to the limited penetration of relatively large biomolecular drugs (e.g., antibodies) into solid tumors and the fact that the effects of the drugs only reach a small subset of antitumor immune cell subsets.

[0003] To improve the response rate, 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 use in combination cancer therapies to reinforce combination therapies that are only partially effective when used alone.

[0004] Adenosine is a naturally occurring purine nucleoside found in mammals both intracellularly and extracellularly. 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 many types 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 mouse models of cancer (Sidders et al., 2020, Clin Cancer Res., 1, 26(9):2176-2187 DOI:10.1158 / 1078-0432.CCR-19-2183). Signaling via ExAdo has been demonstrated to cause immunosuppression of immune cells found 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). Thus, high extracellular adenosine concentrations are effective as a cause of immunosuppression in solid tumors because they inactivate tumor-targeted immune cells that express A2AR. Adenosine has a significantly higher affinity for A2AR, and A2AR plays a more important role in T cell immunity compared to A2BR (Cekic, 2013, JEM, doi:10.1084 / jem.20130249, Lukashev, 2003, BioChem Pharmacol., doi:10.1016 / S0006-2952(03)00158-8). The remaining two adenosine receptors, A1R and A3R, have different downstream signaling pathways compared to A2AR and A2BR, and their signaling usually does not result in immunosuppression.

[0005] A2AR is mainly found in lymphoid lineage cells such as T cells and natural killer (NK) cells, as well as in the myeloid lineage including dendritic cells (DC) and macrophages (Vigano et al., 2019, supra). Due to the central role of NFκB in many pro-inflammatory and cytotoxic anti-tumor immune responses, the ExAdo signaling pathway can be targeted by therapies to restore the desired immune response in cancer patients. These responses include the patient's own cellular immunity (T lymphocytes, natural killer cells, dendritic cells, macrophages), as well as the effectiveness of co-administered immunotherapies such as immune checkpoint inhibitors (such as anti-PD-1, anti-PD-L1, anti-TIGIT, and anti-CTLA-4 monoclonal antibodies), bispecific antibodies, adoptive cell immunotherapy (such as CAR-T cell infusion), and anti-cancer vaccines, all of which will be enhanced within the favorable (inflammatory-inducing) TME.

[0006] Therefore, the blockade of immunosuppressive adenosine signaling, particularly A2AR, is a treatment strategy with broad applicability in cancer therapy, including combination therapies with radiotherapy, targeted therapies for cancers with specific mutations, and other approved cancer treatments such as conventional chemotherapy.

[0007] 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 is engaged by adenosine, the endogenous receptor ligand. In contrast, allosteric antagonists bind to a distinct amino acid motif not utilized by the endogenous ligand. To effectively block A2AR signaling, orthosteric inhibitors must be more potent than adenosine, which is present at high concentrations in many tumors. Achieving this requires high doses of orthosteric A2AR antagonists, which increases the potential for unwanted drug-mediated effects.

[0008] 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 have not yet entered Phase III clinical trials.

[0009] Currently, there is clearly a medical need for active agents that act effectively in the immunosuppressive tumor microenvironment to provide new options for immunotherapy.

Summary of the Invention

[0010] The present invention relates to the unexpected finding of a cyanopyridine compound that can inhibit A2AR signaling as an A2AR antagonist classified as a negative allosteric modulator (NAM) because it achieves inhibition of target signaling regardless of the extracellular adenosine concentration containing a high micromolar concentration present in the TME.

[0011] One object of the present invention is to provide a novel therapeutic agent useful for inducing or increasing an immune response against immunotherapy, particularly anti-cancer immunotherapy.

[0012] The first aspect of the present invention provides a cyanopyridine compound of formula (I), as well as pharmaceutically acceptable salts, tautomers, and geometric isomers thereof.

[0013] Another aspect of the present invention provides a pharmaceutical composition comprising at least one compound according to the present invention, as well as pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier, diluent, or excipient thereof.

[0014] A first aspect of the present invention provides a cyanopyridine compound of formula (I), and pharmaceutically acceptable salts thereof, which are used in the treatment of solid tumor cancers or malignant tumors that are particularly resistant to cancer treatment, especially immunotherapy, or sensitive to the presence of a large number.

[0015] Another aspect of the present invention pertains to the use of a compound according to the present invention for the preparation of a pharmaceutical composition for the treatment of solid tumor cancers or malignant tumors that are particularly resistant to cancer treatment, especially immunotherapy, or sensitive to the presence of a large number.

[0016] Another aspect of the present invention relates to a pharmaceutical composition comprising at least one compound according to the present invention in combination with at least one agent useful in the treatment of cancer.

[0017] Another aspect of the present invention is a method for preventing resistance to anti-cancer immunotherapy and / or treating cancer in a subject suffering from cancer or at risk of having resistance to anti-cancer immunotherapy, the method comprising administering a compound according to the present invention or a pharmaceutical formulation thereof to a subject in need thereof.

[0018] Another aspect of the present invention is a method for inducing or increasing an immune response to immunotherapy, especially anti-cancer immunotherapy, the method comprising administering an effective amount of one or more compounds according to the present invention or a pharmaceutical formulation thereof in combination with one or more of the following treatments: radiotherapy, chemotherapy, adoptive cell therapy (e.g., chimeric antigen receptor T cells; CAR-T, or tumor-infiltrating lymphocytes; TIL), anti-cancer vaccine therapy, targeted biological therapy (e.g., tumor-specific antibodies), or immunomodulatory therapy, e.g., immune checkpoint inhibitors, bispecific T cell engagers, or nanobodies capable of binding to single or multiple drug targets.

[0019] Another aspect of the present invention is a method for the preparation of a compound according to formula (I) as defined below.

[0020] Further objects and advantageous aspects of the present invention will be apparent from the appended drawings, from the claims, and / or from the following detailed description of embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

Figure 1

[0022] The term "allosteric antagonist" means an antagonist that blocks receptor signaling without binding to all or part of the amino acids used by the natural ligand (adenosine) to bind to the receptor.

[0023] The expression "solid tumor cancer" refers to all cancers except blood 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, cancers of the digestive system, hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer and brain cancer.

[0024] According to one particular embodiment, solid tumor cancers with high adenosine signaling can be characterized by an RNA transcription signature verified in a tumor biopsy (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 optionally, estimated using A2AR expression levels in a tumor biopsy (Allard et al., 2020, supra), or circulating blood immune cells (DOI: 10.3233 / JAD-131652).

[0025] The solid cancer state is considered to be characterized as "high adenosine signaling" when more than 50% of patients with this type of cancer have more than a 5-fold 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 cancer states include cancers of the digestive system such as colon, stomach, and pancreatic cancers, as well as lung, cervical, and head and neck cancers.

[0026] The expression "immunotherapeutic agent" means an agent that supports the immune system in fighting diseases such as cancer. Currently, there are several major categories: adoptive cell therapy (e.g., CAR-T cells or other tumor-infiltrating immune cells), immune checkpoint inhibitor monoclonal antibodies (ICI mAb), bispecific T cell engagers (BiTE), novel immunomodulators or molecular adjuvants, and cancer vaccines, all of which are envisioned to be used in combination with this molecule. In addition, standard forms of cancer therapy can be used in combination with this molecule. These include chemotherapy, radiation therapy, and targeted therapy (specific to a particular cancer antigen or cancer-related molecular pathway). Radiation therapy in particular has been described as an appropriate combination therapy with an A2AR-targeted approach (Allard et al., 2020, supra).

[0027] The term "bispecific T cell engager" means an agent such as an antibody that has one binding arm that recognizes a tumor antigen and another binding arm that recognizes an antigen on the surface of a T cell, as outlined and exemplified in Strohl et al., 2019, Antibodies, doi:10.3390 / antib8030041.

[0028] The term "nanobody" means an agent similar to an antibody, but typically of much lower molecular weight, as outlined and exemplified in Bannas et al., 2017 Front. Immunol. 10.3389 / fimmu.2017.01603.

[0029] As used herein, "treatment" and "treating", etc., generally mean obtaining the desired pharmacological and physiological effects. The effects can be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, and / or therapeutic in terms of partially or completely curing a disease, condition, symptom, or adverse effect resulting from a disease.

[0030] The term "efficacy" of a treatment according to the present invention can be measured based on changes in the course of a disease in response to the use or method according to the present invention. The efficacy of the treatment of cancer according to the present invention can be measured by reduction of tumor volume, and / or increase in progression-free survival, and / or increase in the health and well-being of the subject (e.g., suppressing cancer). Inhibition of cancer cell growth can be demonstrated, for example, by arrest of cancer cells at a particular stage of the cell cycle, e.g., arrest at the G2 / M phase of the cell cycle. Inhibition of cancer cell growth can also be demonstrated using well-known imaging methods, such as magnetic resonance imaging, computed tomography, PET, SPECT, photoacoustic imaging, X-ray and fluorescence imaging / detection. Cancer cell growth can also be determined macroscopically via measurement using calipers.

[0031] In particular, the efficacy of the combination therapy according to the present invention can be evaluated by reduction of tumor size, disappearance of tumors, or altered expression of biomarkers. These biomarkers can be soluble or can be expressed by cancer or immune system cells. Biomarkers that can be used to demonstrate modification or biological efficacy of A2AR signaling 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 downstream effector molecules generally characterized as anti-tumor immune responses including IL-2, TNF-α or IFN-γ cytokine secretion (Willingham et al., 2018, Cancer Immunol.Res., 6(10):1136 - 1149; DOI:10.1158 / 2326-6066.CIR-18-0056).

[0032] As used herein, the term "subject" means a human or non-human mammal, such as a non-human primate (e.g., chimpanzees and other apes and monkey species), livestock animals (e.g., cows, sheep, pigs, goats and horses), domesticated mammals (e.g., dogs and cats), or laboratory animals (e.g., rodents, e.g., mice, rats and guinea pigs).

[0033] The term "efficacy" of the treatment according to the present invention can be measured based on changes in the course of a disease in response to the use or method according to the present invention. For example, the efficacy of the treatment according to the present invention can be measured by its effect on the signs or symptoms of the disease. A response is achieved when the subject experiences a partial or complete alleviation or reduction of the unwanted symptoms of the disease. According to certain embodiments, efficacy can be measured through the evaluation of toxin target cleavage or viral replication after infection. For example, the efficacy of a toxin or antiviral treatment according to the present invention can be monitored by tracking the effect on cleavage of kinase 1 of map kinase, or by improvement in cell survival, tissue damage and patient survival.

[0034] The term "aryl" means an unsaturated aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple fused rings (e.g., indenyl, naphthyl). Examples of aryl include phenyl, naphthyl, anthryl, phenanthrenyl, and the like.

[0035] The term "heteroaryl" refers to a monocyclic or bicyclic unsaturated aromatic moiety of 5 to 10 ring atoms, wherein one or more of the ring atoms is selected from N, O, or S. Specific examples of heteroaryl groups include pyridyl, pyrrolyl, pyrimidinyl, furyl, 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, benzofuryl, [2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolidinyl, quinazolinyl, phthalazinyl, quinoxalinyl, cinnolinyl, naphthyridinyl, pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl or benzquinolyl, which may optionally be substituted.

[0036] The term "heteroalkyl" refers to C 1 -C 12 -alkyl, preferably C 1 -C 6 -alkyl, wherein at least one carbon is replaced by a heteroatom selected from O, N, or S, and includes 2-methoxyethyl and the like.

[0037] The term "heterocycloalkyl" refers to C 3 -C 8-Refers to a cycloalkyl group, where up to 3 carbon atoms are replaced by a heteroatom selected from the group consisting of O, S, and NR, and R is defined as hydrogen or methyl. Examples of heterocycloalkyl include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl, etc.

[0038] "Alkoxycarbonyl C 1 -C 6 alkyl" refers to a C 1 -C 6 alkyl group having an alkoxycarbonyl substituent, including 2-(benzyloxycarbonyl)ethyl, etc.

[0039] Unless otherwise restricted by the definition of each substituent, the term "substituted" means a group substituted with 1 to 5 substituents selected from the group consisting of 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 -), C 1 -C 6 alkyl, aryl, or heteroaryl.

[0040] The term "pharmaceutical preparation" means a preparation in such a form that it tolerates the biological activity of the active ingredient that is clearly effective and does not contain additional components that would be toxic to the subject to whom the preparation will be administered.

[0041] Compound according to the invention According to one aspect, a cyanopyridine compound of formula (I) for use in the treatment of cancer, particularly in immunotherapy

Chemical formula

[0042] According to a further aspect, the cyanopyridine compound according to the present invention is a compound of formula (Ia):

Chemical formula

[0043] According to a further aspect, the cyanopyridine compound according to the present invention is a compound of formula (Ib):

Chemical formula

[0044] According to a further aspect, the cyanopyridine compound according to the invention has R 3 which is selected from H and optionally substituted C 1 -C 6 alkyl, and R 4 which is optionally substituted aryl (for example, optionally substituted phenyl, for example, phenyl, halogenophenyl, or phenyl optionally substituted by C 1 -C 6 alkyl, for example, methyl), and optionally substituted heteroaryl (for example, optionally substituted pyridinyl, optionally substituted pyrimidinyl, optionally substituted pyridazinyl, optionally substituted furanyl, optionally substituted indolyl, optionally substituted thiophenyl, optionally substituted pyrazolo, optionally substituted imidazolo, optionally substituted oxazolo, optionally substituted thiazolo, optionally substituted isoxazolo, optionally substituted isothiazolo, optionally substituted triazolo, optionally substituted oxadiazolo, optionally substituted thiadiazolo, optionally substituted tetrazolyl), and is a compound of formula (I).

[0045] According to another aspect, the cyanopyridine compound according to formula (I) [Chemical formula] [wherein R 1 to R 4 are as defined herein], provided that the compound of formula (I) is the following compound: 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: 1646173-50-3); 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); There is provided a cyanopyridine compound that is not one of these.

[0046] According to another aspect, there is provided a cyanopyridine compound of formula (I) for use as a medicament [Chemical formula] [wherein R 1 ~R 4 are as defined herein subject to the conditions defined above], and there is provided a cyanopyridine compound subject to the conditions defined above.

[0047] In certain embodiments, R 1 is aryl optionally substituted by one or more halogens (e.g., fluoro), cyano, hydroxy or C 1 -C 6 alkyl (e.g., methyl).

[0048] In further certain embodiments, R 1 is phenyl optionally substituted by one or more halogens (e.g., fluoro), cyano, hydroxy or C 1 -C 6 alkyl (e.g., methyl).

[0049] In further certain embodiments, R 1 is phenyl optionally substituted by one or more halogens or C 1 -C 6 alkyl, e.g., phenyl, halogenophenyl, e.g., 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, or C 1 -C6 Alkylphenyl, such as methylphenyl like 2-methylphenyl, 3-methylphenyl, 4-methylphenyl.

[0050] In another specific embodiment, R 1 is heteroaryl optionally substituted by one or more halogens, cyano, hydroxy or C 1 -C 6 alkyl.

[0051] In a further specific embodiment, R 1 is optionally substituted pyridinyl (e.g., pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), optionally substituted thiophenyl (e.g., thiophen-2-yl, thiophen-3-yl), optionally substituted pyrazolyl (e.g., pyrazol-5-yl), and optionally substituted tetrazolyl, where optionally substituted means substitution by one or more halogens, cyano, hydroxy or C 1 -C 6 alkyl.

[0052] In a further specific embodiment, R 1 is optionally substituted pyridinyl, where optionally substituted means substitution by one or more halogens, cyano, hydroxy or C 1 -C 6 alkyl, such as 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.

[0053] In a further specific embodiment, R 1 is optionally substituted oxazolyl, such as 1,3,4-oxadiazolyl.

[0054] In another specific embodiment, R 1 is an unsubstituted heteroaryl.

[0055] In a specific embodiment, R 2 is H.

[0056] In a specific embodiment, R 3 is CN.

[0057] In a specific embodiment, R 4 is an XR(R’) group as defined herein.

[0058] In a specific embodiment, R 4 is an OR group (wherein R is optionally substituted alkyl, for example, methyl, ethyl, hydroxyethyl).

[0059] In a specific embodiment, R 4 is an SR group (wherein R is optionally substituted alkyl, for example, optionally substituted ethylphenyl).

[0060] In a further specific embodiment, R 4 is an XR(R’) group (wherein XRR’ together form an optionally substituted heterocyclic alkyl (optionally substituted azetidine, optionally substituted pyrrolidine, optionally substituted piperidine, optionally substituted morpholinyl, optionally substituted piperazine, optionally substituted azepane, optionally substituted azocane, etc.)).

[0061] In a further specific embodiment, R 4 is selected from pyridine, pyrrolidine and azetidine.

[0062] In a further specific embodiment, R 4is a pyridine which may be optionally substituted.

[0063] In a further specific embodiment, R 4 is piperidine.

[0064] In a further specific embodiment, X is N.

[0065] In a further specific embodiment, X is O.

[0066] In a further specific embodiment, X is S.

[0067] According to a further aspect, the cyanopyridine compound is a compound of formula (Ib) in which R 6 and R 7 together form an optionally substituted heterocyclic alkyl.

[0068] According to a further aspect, the cyanopyridine compound is a compound of formula (Ib) in which R 6 and R 7 together form an optionally substituted azetidine or an optionally substituted piperidine.

[0069] According to a specific embodiment, a negative allosteric modulator (NAM) of A2AR is provided.

[0070] The compounds of the present invention are in particular 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-Tetrazol-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-morpholinopyridine-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 selected therefrom.

[0071] According to certain embodiments, the compounds of the present invention are 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; and 4-(3-(1H-Tetrazol-5-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile selected from

[0072] The compounds of the present invention are named according to the IUPAC standards used in the program Chemdraw Professional® (product version 16.0).

[0073] Composition The pharmaceutical composition of the present invention can comprise one or more compounds according to the present invention, and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0074] According to another specific embodiment, the A2AR modulation effect can be achieved through the delivery of the formulated compounds of the present invention via various routes, preferably orally.

[0075] According to a specific embodiment, the composition further comprises a compound useful in the prevention and / or treatment of cancer, particularly in cancer immunotherapy.

[0076] The composition of this invention may also be formulated for parenteral administration, including, but not limited to, by 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, stabilizing and dispersing agents. The composition may also be provided in powder form for reconstitution with a suitable vehicle including, but not limited to, sterile pyrogen-free water.

[0077] According to a specific embodiment, the composition according to the present invention is for oral delivery.

[0078] In another specific embodiment, the composition according to the present invention is adapted for delivery by single or multiple administrations.

[0079] According to a specific embodiment, the composition of the present invention is a veterinary composition.

[0080] Additional materials and formulation processing techniques, etc., are shown in Remington: Science & Practice of Pharmacy, 23rd Edition, 2020, edited by Adeboye Adejare, Academic Press, which is hereby incorporated by reference as part of the disclosure of this specification.

[0081] According to certain aspects, there is provided a pharmaceutical composition comprising at least one compound according to the present invention, and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0082] The present invention provides compounds, compositions thereof, and methods of using them that are useful as A2AR modulators for the prevention and / or treatment of medical disorders, particularly for the prevention and / or treatment of cancer, particularly for use in immunotherapy.

[0083] Route of administration The compounds and compositions of this invention can be administered or delivered in any manner including, but not limited to, oral, parenteral, sublingual, transdermal, transmucosal, topical, intratumoral or peritumoral, buccal, or intranasal administration, or combinations thereof. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous and intramuscular.

[0084] In another specific embodiment, the compound according to the present invention is administered orally.

[0085] In another specific embodiment, the compound according to the present invention is administered rectally.

[0086] In another specific embodiment, the compound according to the present invention is administered intravenously.

[0087] The dosage administered to an individual as a single or multiple doses varies according to various factors including pharmacokinetic properties, the condition and characteristics of the subject (gender, age, weight, health, and size), the degree of symptoms, concomitant therapy, treatment frequency, and the desired effect.

[0088] Combination According to one embodiment of the present invention, the compounds and pharmaceutical formulations thereof according to the present invention can be administered alone or in combination with co-drugs useful in the prevention and / or treatment of diseases.

[0089] According to one aspect, the compounds of the present invention can be administered in combination with at least one therapeutic molecule useful in the treatment of cancer, particularly cancer immunotherapy.

[0090] According to one aspect, the compounds of the present invention should be administered in combination with one or more of the treatments selected from radiotherapy, chemotherapy, adoptive cell therapy (e.g., chimeric antigen receptor T cells; CAR-T, or tumor-infiltrating lymphocytes; TIL), anti-cancer vaccine therapy, targeted biological therapy (e.g., tumor-specific antibodies), or immunomodulatory therapy, e.g., immune checkpoint inhibitors, bispecific T cell engagers, or nanobodies capable of binding to single or multiple drug targets.

[0091] According to one aspect, the compounds of the present invention can be administered in combination with an anti-cancer vaccine or at least one immune checkpoint inhibitor, e.g., at least one PD-1, PD-L1, or CTLA4 inhibitor, or a combination thereof.

[0092] According to a further aspect, at least one immune checkpoint inhibitor is selected from PD-1 inhibitors and CTLA4 inhibitors.

[0093] According to a further aspect, the compounds of the invention should be administered in combination with an anti-cancer vaccine, wherein the anti-cancer vaccine elicits cancer-specific immunity and the compounds of the invention should be administered before and / or after administration of the anti-cancer vaccine.

[0094] According to a further aspect, the compounds of the invention should be administered in combination with a cell therapy, for example, in combination with chimeric antigen receptor (CAR) T cells expanded ex vivo (CAR T cell therapy) or tumor infiltrating lymphocytes (TIL) (cell therapy based on bulk TIL cells). Chimeric antigen receptor (CAR) T cells are expanded ex vivo according to standard methods, which include: - Providing at least one genetically modified T cell in a T cell cell culture medium, wherein the genetically modified T cell expresses a polypeptide specific for a tumor-associated antigen on its membrane surface; - Expanding and recovering the genetically modified T cells in the culture medium.

[0095] TIL are expanded ex vivo according to standard methods, which include: - Isolating a polyclonal mixture of T cells from a tumor fragment; - Expanding these polyclonal T cells in a culture medium.

[0096] According to a further aspect, the compounds of the invention should be administered in combination with the expanded chimeric antigen receptor (CAR) T cells or TIL by the same or different routes.

[0097] The present invention encompasses administration of the compounds of the invention, wherein the compounds are administered to a subject before, simultaneously with, or after a treatment regimen or at least one co-drug. The compounds according to the invention administered simultaneously with the at least one co-drug can be administered in the same or different compositions and by the same or different routes of administration.

[0098] Subject In one embodiment, the subject in the present invention is a subject suffering from cancer.

[0099] In a further specific embodiment, the subject in the present invention is, but not limited to, a subject suffering from a solid tumor cancer selected from lung cancer (small cell and non-small cell), breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, melanoma, gastrointestinal cancer, hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer and liver cancer.

[0100] In one embodiment, the subject in the present invention is a subject presenting a tumor having an increased expression of pCREB or A2AR as described above, which is an adenosine signaling gene signature.

[0101] In a further specific embodiment, the subject in the present invention is a subject suffering from a solid tumor cancer which is a gastrointestinal cancer, particularly pancreatic cancer and colorectal cancer.

[0102] Use according to the invention In a specific embodiment, the present invention provides compounds, methods, uses and compositions useful in the treatment of cancer, particularly in immunotherapy.

[0103] According to a further specific embodiment, the methods, uses and compositions of the present invention are useful for reducing tumorigenesis and / or for enhancing 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, e.g., immune checkpoint inhibitors.

[0104] Another aspect of the present invention is a method for preventing resistance to anti-cancer immunotherapy and / or treating cancer in a subject having cancer or at risk of having resistance to anti-cancer immunotherapy, the method comprising administering to a subject in need thereof a compound according to the present invention or a pharmaceutical formulation thereof.

[0105] Another aspect of the present invention is a method for inducing or increasing an immune response to immunotherapy, particularly anti-cancer immunotherapy, the method comprising administering to a subject in need thereof an effective amount of one or more compounds of the present invention or a pharmaceutical formulation thereof in combination with an immunotherapeutic agent or in combination with radiotherapy.

[0106] In certain embodiments, the present invention provides compounds, methods, uses and compositions in the form of a combination that are useful for the treatment of solid tumor cancer, wherein at least one compound of the present invention should be administered in combination with at least one anti-cancer immunotherapeutic agent.

[0107] According to certain aspects, there is provided a method for treating a subject having cancer, the method comprising administering to a subject in need thereof an effective amount of one or more compounds of the present invention in combination with at least anti-cancer treatment, particularly an anti-cancer immunotherapeutic agent.

[0108] According to another particular embodiment, the compounds, methods and compositions of the present invention are useful in combination with cell therapy, particularly for improving the immunosuppressive tumor microenvironment and thereby enhancing the infiltration of cell therapy injected into the tumor. According to a further aspect, this combination increases the ability of T cells to kill cancer cells at the disease site.

[0109] References cited in this specification are hereby incorporated by reference in their entirety as part of the disclosure of this specification. Such modifications are intended to be within the scope of the appended claims. Although the invention has been described, the following examples are presented for illustrative purposes and are not limiting.

[0110] Synthesis of the compound according to the invention The compounds according to formula (I) can be prepared from readily available starting materials using the following general methods and procedures. It will be understood that other experimental conditions can also be used, unless otherwise specified, when typical or preferred experimental conditions (i.e., reaction temperature, time, molar amounts of reagents, solvents, etc.) are given. The optimal reaction conditions can vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art using routine optimization procedures.

[0111] General synthetic approaches for obtaining the compounds of formula (I) are represented in Schemes 1 - 5 below.

Chemical formula

[0112] Scheme 1 represents the preparation of the compounds of the present invention in a single-step procedure by substitution of cyanopyridine of formula (i) with a commercially available or synthetic nucleophile of formula (ii) (wherein X, R and R’ are as defined herein) in an aprotic solvent such as THF in the presence of a base such as trimethylamine. The synthesis of cyanopyridine of formula (i) is well described in the literature and can be 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) or Piper et al., 1986 (J. Med. Chem., 29, 1080, DOI: 10.1021 / jm00156a029), or a one-pot protocol described by, for example, Duindam et al., 1993 (Synthetic Commun., 23, 2605, DOI: 10.1080 / 00397919308012595).

[0113]

Chemical formula

[0114] Scheme 2 represents the cross-coupling of intermediates of formulas (iii) and (iv) (wherein R 2 CO 3 etc.) and a catalytic amount of a palladium source (such as palladium(II) dichloride or tetrakis(triphenylphosphine)palladium(0)) in the presence of a mineral base (such as K 1 , R 2 , R 3 and R 4 are as defined above, and R 8 and R 9 are selected from halogeno, boronic acid, or boronic acid pinacol ester groups). This cross-coupling step can be carried out under thermal conditions in a nonpolar solvent such as toluene (described in Pan et al., 2008, Catal. Commun., 9, 508 - 510 etc.), which is also called thermal activation, or under microwave irradiation in a polar solvent (such as methanol, water... etc.).

[0115]

Chem.

[0116] Scheme 3 shows a specific example of Scheme 2 for the synthesis of an intermediate of formula (iiib) by a cross-coupling reaction between a bromoaryl of formula (iiia) (wherein R 2 , R 3 and R 4 are as defined above) and commercially available bis(pinacolato)diboron of formula (v) under thermal activation of microwave irradiation described in Scheme 2.

[0117]

Chem.

[0118] Scheme 4 shows the preparation of compound (Ib) in a single-step procedure by cyclization of a cyanopyridine from a commercially available or synthesized aldehyde of formula (vi) in the presence of a cyclic amine of formula (vii) (wherein R 1 and R 2 are as defined above, and R 6 and R 7 together form an optionally substituted heterocyclic alkyl as defined herein), 2 equivalents of malononitrile (viii), a final catalyst (such as 4-(dimethylamino)pyridine (4-DMAP)) and an oxidizing agent (such as air) in a polar protic solvent (such as methanol).

[0119]

Chem.

[0120] Scheme 5 shows the preparation of an intermediate compound of formula (iiic) in a two-step manner. First, the synthesis of the intermediate of formula (vi) is carried out in a polar protic solvent (such as methanol) with 1 equivalent of malononitrile (viii) and a final catalytic amount of a cyclic amine of formula (vii) (wherein R 2is as described above, and R 6 and R 7 (which, together, form an optionally substituted heterocyclic alkyl as defined herein), and can be achieved from the reaction of a commercially available bromobenzaldehyde of formula (ix) in the presence of an oxidizing agent (such as air). The isolated intermediate (vi) can be further reacted in the presence of 1 equivalent of malononitrile (iii) and 1 equivalent of an amine of formula (vii).

[0121] This two-step approach can be further adapted to obtain gram quantities.

[0122] The compound (iiic) described above is a specific example of the compound (iiia) and can therefore be used in the synthesis of the compound (I) of the invention according to the procedure of Scheme 3 shown.

Examples

[0123] The following abbreviations have the following meanings respectively: CV (column dead volume); cyclo (cyclohexane); DCM (dichloromethane); DHP (2,4-dihydropyran); DMF (dimethylformamide); eq. (equivalent (mol%)); ESI (electrospray ionization); HRMS (high resolution mass spectrum); NMR (nuclear magnetic resonance); PBS (phosphate buffered saline); r.t. (room temperature); TFA (trifluoroacetic acid); TLC (thin layer chromatography); TOF (time of flight); UPLC (ultra performance liquid chromatography); UV (ultraviolet).

[0124] Example 1: Synthesis of the compound according to the invention The compounds of the invention were prepared according to Schemes 1-5 as follows.

[0125] General experiments: Unless otherwise specified, all products were obtained from commercial sources and used without further purification. Aprotic solvents, such as methanol, DMF or toluene, were purchased on molecular sieves sealed with AcroSeal®.

[0126] All preparative columns were run by flash chromatography on a Buchi Pure C-815 Flash system with a UV detector. The corresponding PureFlash ID cartridges (4 g, 12 g, 24 g, 40 g or 120 g, amorphous silica, 35–45 μm mesh) were purchased from Buchi, and the flow rates were set according to the preset parameters (15, 30, 32, 45 and 85 mL / min, respectively). Samples were loaded as solid deposits prepared using amorphous silica with a 40–60 μm mesh.

[0127] All reported yields are isolation yields unless otherwise specified.

[0128] 1 1H NMR spectra were recorded on a Bruker 600 MHz spectrometer equipped with a cryoprobe and calibrated to the residual protonated solvent. 13 13C NMR spectra were recorded at 125 MHz, and the solvent resonance was used as an internal standard. 1 1H NMR and 13 both 13C NMR chemical shifts are reported in parts per million from tetramethylsilane downfield.

[0129] Low-resolution mass spectra were recorded on an Advion PressionL coupled to an ESI source operating simultaneously in positive and negative ion modes. HRMS was recorded on a Xevo G2 TOF coupled to an ESI source operating in either positive or negative acquisition mode.

[0130] Small-scale one-pot cyanopyridine cyclization according to Scheme 4 In a small round-bottom flask equipped with a stirrer, the aldehyde of formula (vi) (1 eq.) and malononitrile (viii) (1 eq.) were dissolved in methanol (0.2 M) (solution A). Solution A was immediately and vigorously stirred. When the aldehyde contains a basic site (e.g., pyridine), a catalyst is not necessary. In other cases, in a small vial, a solution of the cyclic amine of formula (vii) (1.2 eq.) in MeOH (0.2 M) was prepared (solution B). One or two drops of solution B were added to solution A. After stirring at r.t. for 30 minutes, additional malononitrile (viii) (1 eq.) was added, followed by dropwise addition of the remainder of solution B. The reaction was exposed to air until completion, stirred at r.t., and completion was evaluated by TLC (cyclo:EtOAc 8:2). The medium was diluted with DCM (5×V MeOH ) and silica 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 cartridge for 0.5 - 1 mmol of starting aldehyde) using a cyclo:EtOAc gradient (95:5, 3 CV; 95:5 → 8:2, 12 CV; 8:2, 6 CV; methanol wash).

[0131] Gram-scale cyanopyridine cyclization according to Scheme 5 In a mortar or the bowl of a mechanical stirring device, the aldehyde of formula (ix) (1.05 - 1.2 eq.) and malononitrile (viii) (1 eq.) were dissolved in MeOH (2 - 1 M) (solution A). Solution A was immediately and vigorously stirred. When the aldehyde contains a basic site (e.g., pyridine), a catalyst is not necessary. In other cases, in a small vial, a solution of the cyclic amine of formula (vii) in MeOH (0.1 M) was prepared (solution B). While stirring, solution B was added dropwise to solution A up to a maximum of 5 mol% amine. A thick white paste must form in less than 3 - 5 minutes and stirring was maintained for 5 - 30 minutes before addition of cold water. The precipitate was filtered, rinsed twice with cold water and then three times with minimal cold ether. The powder was dried under reduced pressure for 16 - 24 hours to give intermediate (vi), which was used without further purification.

[0132] In a round-bottom flask, the obtained intermediate of formula (vi) (1 eq.) was suspended in MeOH (approx. 0.5 M). At 0 °C, the addition of malononitrile (viii) (1 eq.) was carried out continuously, followed by the dropwise addition of the cyclic amine of formula (vii) (1.2 eq.). The flask was closed with a guard filled with CaCl 2 beads (to allow air exchange), stirred at 0 °C for 30 minutes and then at r.t. until completion, and completion was evaluated by TLC (cyclo:EtOAc 8:2 or DCM:MeOH 9:1). The crude product was purified by flash chromatography.

[0133] Suzuki cross-coupling (thermally activated) The procedure used was adapted from the aforementioned Pan et al., 2008.

[0134] In a 25 mL round-bottom flask equipped with a magnetic stirrer, a commercially available or synthetic bromoaryl (1 eq.), a boronic acid or boronic acid pinacol ester (2 eq.), potassium carbonate (3 eq.) and palladium(II) dichloride (0.05 eq.) were successively added to toluene (0.5 M). The mixture was exposed to air and stirred at 110 °C for 24 hours. Completion was evaluated by TLC (cyclo:DCM 8:2). Palladium and salts were removed by filtration over 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 using a cyclo:DCM gradient.

[0135] Microwave-assisted Suzuki cross-coupling In a 2 - 5 mL microwave tube equipped with a magnetic stirrer, a commercially available or synthetic bromoaryl (1 eq.), a boronic acid or boronic acid pinacol ester (1.2 - 3 eq.) and potassium carbonate (3 eq.) were added to THF:H 2It was dissolved in a mixture of O₂:1 (0.05 M). The solution was bubbled with argon for 10 minutes, and then tetrakis(triphenylphosphine)palladium(0) (0.05 eq.) was added. The tube was sealed and heated at 80 °C or 100 °C for 15 - 30 minutes under microwave irradiation. The medium was diluted with water, and the crude product was extracted with EtOAc×3. The organic phases were combined, washed with brine, dried over MgSO 4 and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (4 g cartridge for starting bromoaryl less than 0.5 mmol) using a cyclo:MixA [toluene:acetone 8:2 mixture] gradient (5:5, 6 CV; 5:5 → 0:10, 6 CV; 0:10, 6 CV; methanol wash).

[0136] Intermediate of formula (i) 4-([1,1'-Biphenyl]-3-yl)-2-amino-6-chloropyridine-3,5-dicarbonitrile (wherein R 1 is phenyl, R 2 is hydrogen, and R 3 is cyano)

Chemical formula

[0137] The intermediate was prepared starting from 3-(trimethoxymethyl)-1,1'-biphenyl (1.29 g, 5.0 mmol, 1 eq.) according to the two-step protocol described by Murray et al., 1995 (Tetrahedron, 1995, 51, 635, DOI: 10.1016 / 0040-4020(94)00922-H) and isolated as a crude powder (650 mg, 2.0 mmol, isolated yield 39%). 1 H NMR (600 MHz, (CD 3 )) 2SO) δ 7.91 (d, J = 1.8 Hz, 1H), 7.90 (dt, J = 7.7, 1.5 Hz, 1H), 7.75 - 7.71 (m, 2H), 7.68 (t, J = 7.7 Hz, 1H), 7.59 (dt, J = 7.7, 1.5 Hz, 1H), 7.52 (t, J = 7.7 Hz, 2H), 7.45 - 7.39 (m, 1H). 13 C NMR (151 MHz, (CD 3 ) 2 SO) δ 160.3, 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 - ) C 19 H 11 N 4 Cl for calculated value: [M - H + m / z = 329.1, found m / z = 329.3.

[0138] Intermediate of formula (vi) 4’-Methyl-[1,1’-biphenyl]-3-carbaldehyde (wherein, R 1 is 4 - methylphenyl, R 2 is hydrogen)

Chemical formula

[0139] 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, isolated yield 58%). 1 H NMR (600 MHz, CDCl 3 ) δ 10.09 (s, 1H), 8.09 (t, J = 1.8 Hz, 1H), 7.86 - 7.83 (m, 2H), 7.60 (t, J = 7.6 Hz, 1H), 7.56 - 7.51 (m, 2H), 7.32 - 7.27 (m, 2H), 2.42 (s, 3H). 13 C NMR (151 MHz, CDCl 3) δ 192.6, 142.3, 138.1, 136.9, 133.0, 129.9, 129.6, 128.5, 128.1, 127.1, 21.3.

[0140] 2’-Fluoro-[1,1’-biphenyl]-3-carbaldehyde (wherein, R 1 is 2-fluorophenyl and R 2 is hydrogen) [Chemical formula]

[0141] 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, isolated yield 68%). 1 H NMR (600 MHz, CDCl 3 ) δ 10.09 (s, 1H), 8.06 (q, J = 1.9 Hz, 1H), 7.90 (dt, J = 7.7, 1.4 Hz, 1H), 7.83 (dtd, J = 7.7, 1.7, 1.2 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.48 (td, J = 7.7, 1.8 Hz, 1H), 7.38 (dddd, J = 8.3, 7.4, 5.0, 1.8 Hz, 1H), 7.25 (td, J = 7.5, 1.2 Hz, 1H), 7.19 (ddd, J = 10.8, 8.3, 1.2 Hz, 1H). 13 C NMR (151 MHz, CDCl 3 ) δ 192.3 (d, J = 1.5 Hz), 160.7, 159.1, 136.9 (d, J = 22.7 Hz), 135.1 (d, J = 3.3 Hz), 130.8 (d, J = 3.2 Hz), 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).

[0142] 3’-Fluoro-[1,1’-biphenyl]-3-carbaldehyde (wherein, R 1 is 3-fluorophenyl and R2 is hydrogen)

Chem.

[0143] 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, isolated yield 77%). 1 H NMR (600 MHz, CDCl 3 ) δ 10.10 (s, 1H), 8.11 - 8.06 (m, 1H), 7.89 (dt, J = 7.6, 1.4 Hz, 1H), 7.84 (ddd, J = 7.7, 2.0, 1.2 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.46 - 7.40 (m, 2H), 7.33 (ddd, J = 9.9, 2.6, 1.7 Hz, 1H), 7.13 - 7.08 (m, 1H). 13 C NMR (151 MHz, CDCl 3 ) δ 192.2 (d, J = 1.8 Hz), 163.4 (d, J = 246.2 Hz), 142.1 (d, J = 7.7 Hz), 141.1 (d, J = 2.3 Hz), 137.2, 133.1, 130.7 (d, J = 8.4 Hz), 129.8, 129.4, 128.2, 123.0 (d, J = 3.0 Hz), 115.0 (d, J = 21.1 Hz), 114.3 (d, J = 22.2 Hz).

[0144] 4’-Fluoro-[1,1’-biphenyl]-3-carbaldehyde (wherein R 1 is 4-fluorophenyl and R 2 is hydrogen)

Chem.

[0145] 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, isolated yield 58%). 1 H NMR (600 MHz, CDCl 3 ) δ 10.09 (s, 1H), 8.06 (t, J = 1.8 Hz, 1H), 7.86 (dt, J = 7.6, 1.4 Hz, 1H), 7.82 (ddd, J = 7.7, 2.0, 1.2 Hz, 1H), 7.63 - 7.57 (m, 3H), 7.19 - 7.14 (m, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 192.4 (d, J = 2.0 Hz), 163.0 (d, J = 247.7 Hz), 141.4, 137.1, 136.0 (d, J = 3.2 Hz), 133.0, 129.7, 129.0, 128.9, 128.0, 116.1 (d, J = 21.7 Hz).

[0146] 3-(2-(Tetrahydro-2H-pyran-2-yl)-2H-tetrazol-5-yl)benzaldehyde (wherein R 1 is 2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazol-5-yl and R 2 is hydrogen)

Chemical formula

[0147] The intermediate 3-(tetrazol-5-yl)benzaldehyde can be prepared according to the literature (Bianchini et al., 2021, Med. Chem., 64, 16820-16837). Next, 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-(tetrazol-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. After the completion of the reaction was evaluated by TLC (Rf = 0.57 in DCM:MeOH 98:2), it was stopped by the addition of 10% Na 2 CO 3 (10 mL) and water (20 mL). The crude product was extracted with EtOAc (3 × 15 mL), the organic phases were combined, washed with brine (20 mL), dried over MgSO 4 and the solvent was removed under reduced pressure. Purification was performed by flash chromatography on a 12 g silica cartridge using a cyclo:DCM gradient (8:2, 3 CV; 8:2 → 0:10, 6 CV; methanol wash) and isolated as an oil (66 mg, 0.26 mmol, isolated yield 26%). 1 H NMR (600 MHz, CDCl 3 ) δ 10.11 (s, 1H), 8.70 (t, J = 1.8 Hz, 1H), 8.47 (dt, J = 7.7, 1.5 Hz, 1H), 8.01 (dt, J = 7.7, 1.4 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 6.10 (dd, J = 7.6, 2.9 Hz, 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). 13 C NMR (151 MHz, CDCl 3 ) δ 191.81, 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 + ) C13 H 14 N 4 O 2 Calculated value for: [M+Cl - m / z = 293.1, measured value m / z = 293.1.

[0148] Intermediate of formula (iv) 3-Bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (In the formula, R 1 is 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole, and R 9 is bromo)

[0149]

Chemical formula

[0150] 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). After flowing argon through the flask, 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. Completion was evaluated by TLC and revealed with basic KMnO 4 (cyclo: EtOAc 8:2, Rf = 0.18). The reaction was quenched with 0.5 M aqueous NaOH (20 mL), and the crude intermediate was extracted with EtOAc (3 × 15 mL). The organic phases were combined, washed with brine (20 mL), and MgSO 4It was dried 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 cartridge using a cyclo:EtOAc gradient (9:1, 3 CV; 9:1 → 8:2, 3 CV; 8:2, 3 CV; methanol wash) and isolated as a translucent oil (421 mg, 2.8 mmol, isolated yield 92%). The 3-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole intermediate (va) was prepared from the 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) and isolated as a pale yellow oil (460 mg, 2.0 mmol, isolated yield 74%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.50 (d, J = 2.4 Hz, 1H), 6.31 (d, J = 2.5 Hz, 1H), 5.32 (dd, J = 9.3, 2.9 Hz, 1H), 4.07 - 4.03 (m, 1H), 3.68 (td, J = 11.1, 2.8 Hz, 1H), 2.11 - 2.00 (m, 2H), 1.70 - 1.65 (m, 3H), 1.63 - 1.58 (m, 1H). 13 C NMR (151 MHz, CDCl 3 ) δ 129.8, 126.5, 109.1, 87.9, 67.9, 30.5, 25.0, 22.3.

[0151] Intermediate of formula (iiia) 2-Amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (wherein R 2 is hydrogen, R 3 is cyano, and R 4 is piperidine)

Chemical Structure

[0152] 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 and vigorously stirred. 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 hour. Additional malononitrile (viii) (330 mg, 1 eq.) dissolved in MeOH (5 mL) was added and the reaction was exposed to air and stirred at r.t. until completion. Completion was evaluated by TLC (Rf = 0.16 cyclo:EtOAc 8:2). The medium was diluted with DCM (20 mL), silica was added directly on top of the mixture, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on a 24 g cartridge using a cyclo:EtOAc gradient (9:1, 3 CV; 9:1 → 8:2, 6 CV; 8:2, 8 CV; methanol wash). 1 H NMR (600 MHz, CDCl 3 ) δ 7.65 (dt, J = 7.3, 1.9 Hz, 1H), 7.61 (t, J = 1.8 Hz, 1H), 7.43 - 7.36 (m, 2H), 5.50 (s, 2H), 3.84 - 3.79 (m, 4H), 1.76 - 1.68 (m, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 160.9, 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 + ) C 18 H 16 N 5 Calculated for + C + H 18 N 16 Br [M+H 5 m / z = 382.1 and 384.1, found m / z = 382.1 and 384.1. HRMS (ESI + ) Calculated for

[0153] 2-Amino-4-(3-bromo-2-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (wherein, R 2 is 2-fluoro, R 3 is cyano, R 4 is piperidine) [Chemical Formula]

[0154] 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 r.t. for 30 minutes, the remainder of Solution B was added slowly. The reaction was exposed to air until completion and stirred at r.t. 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 MgSO 4 and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on a 12 g silica cartridge using a cyclo:DCM gradient (8:2, 4 CV; 8:2 → 4:6, 8 CV; 4:6, 4 CV; methanol wash) and isolated as a white powder (63 mg, 0.16 mmol, isolated yield 10%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.60 (ddd, J = 8.9, 4.5, 2.5 Hz, 1H), 7.49 (dd, J = 6.3, 2.5 Hz, 1H), 7.13 (t, J = 9.0 Hz, 1H), 5.39 (s, 2H), 3.84 - 3.80 (m, 4H), 1.73 - 1.68 (m, 6H). 13 C NMR (151 MHz, CDCl 3)δ 160.2, 159.1, 158.2 (d, J = 250.9 Hz), 155.2, 135.4 (d, J = 8.4 Hz), 133.1 (d, J = 2.1 Hz), 124.8 (d, J = 16.8 Hz), 118.4 (d, J = 23.0 Hz), 117.2 (d, J = 3.6 Hz), 116.9, 115.7, 84.3, 82.3, 49.2, 26.1, 24.5. MS (ESI + ) C 18 H 15 N 5 Calculated values for FBr: [M + H + m / z = 400.1 and 402.1, found m / z = 400.4 and 402.3.

[0155] 2-Amino-4-(3-bromo-2-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (wherein R 2 is 2-fluoro, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0156] 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). 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 r.t. for 30 minutes, the remainder of solution B was added slowly. The reaction was exposed to air until completion and stirred at r.t. 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, MgSO 4It was dried and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on a 12 g silica cartridge using a cyclo:DCM gradient (8:2, 4 CV; 8:2 → 4:6, 8 CV; 4:6, 4 CV; methanol wash) and isolated as a white powder (63 mg, 0.16 mmol, isolated yield 10%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.70 (ddd, J = 8.1, 6.4, 1.6 Hz, 1H), 7.32 (ddd, J = 7.9, 6.2, 1.6 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 5.38 (s, 2H), 3.84 - 3.78 (m, 4H), 1.76 - 1.66 (m, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 160.2, 159.1, 155.6, 155.5 (d, J = 250.8 Hz), 135.8, 129.5 (d, J = 2.1 Hz), 125.6 (d, J = 4.2 Hz), 124.2 (d, J = 16.2 Hz), 116.8, 115.7, 110.3 (d, J = 20.9 Hz), 84.2, 82.2, 49.1, 26.0, 24.4. MS (ESI + ) C 18 H 15 N 5 FBr for calculated value: [M + H + m / z = 400.1 and 402.1, found m / z = 400.3 and 402.3.

[0157] 2-Amino-4-(3-chloro-5-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (wherein R 2 is 5 - fluoro, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0158] 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 and vigorously stirred and cooled to 0 °C. Piperidine (0.24 mL, 1.2 eq.) in MeOH (1 mL) was slowly added. The reaction was exposed to air until completion and stirred at r.t. The medium was diluted with brine (20 mL), and the crude product was extracted with AcOEt (3 × 15 mL). The organic phases were combined and dried over MgSO 4 and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on a 24 g silica cartridge using a cyclo:EtOAc isocratic gradient (85:15, 12 CV; methanol wash) and isolated as a white powder (136 mg, 0.38 mmol, isolated yield 19%). 1 1H NMR (600 MHz, CDCl 3 ) δ 7.26 - 7.22 (m, 2H), 7.09 (dt, J = 8.1, 1.7 Hz, 1H), 5.51 (s, 2H), 3.84 - 3.80 (m, 4H), 1.73 - 1.67 (m, 6H). 13 13C NMR (151 MHz, CDCl 3 ) δ 163.4, 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 + ) C 18 H 15 N 5 FCl for calculated: [M + H + m / z = 356.1, found m / z = 356.4.

[0159] Intermediate of formula (iiib) (3-(2-Amino-3,5-dicyano-6-(piperidin-1-yl)pyridin-4-yl)phenyl)boronic acid pinacol ester (wherein R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chem.

[0160] 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(pinacolato)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 the medium was heated at 110 °C for 16 h. Completion was evaluated by TLC (Rf = 0.06 in cyclo:DCM 4:6). The palladium and salts were removed by filtration over a Celite pad, washed with EtOAc (3 × 5 mL), and the solvent was removed under reduced pressure. The product was isolated by flash chromatography on a 12 g cartridge using a cyclo:DCM gradient (5:5, 6 CV; 5:5 → 0:10, 6 CV; 0:10, 9 CV; methanol wash) and isolated as an off-white powder (213 mg, 0.5 mmol, isolated yield 50%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.94 (dt, J = 7.3, 1.3 Hz, 1H), 7.87 (dt, J = 1.9, 0.8 Hz, 1H), 7.54 (dt, J = 7.7, 1.7 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 5.33 (s, 2H), 3.82 - 3.77 (m, 2H), 1.72 - 1.66 (m, 5H), 1.35 (s, 12H). 13 C NMR (151 MHz, CDCl 3 ) δ 162.9, 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 N 5 O2 B: [M+H + m / z = 430.2, measured m / z = 430.4.

[0161] Compound of formula (I) 4-([1,1’-Biphenyl]-3-yl)-2-amino-6-(azetidin-1-yl)pyridine-3,5-dicarbonitrile (1) (In the formula, R 1 is phenyl, R 2 is hydrogen, R 3 is cyano, R 4 is azetidine)

Chemical formula

[0162] The product was prepared from commercially available [1,1'-biphenyl]-3-carbaldehyde (55 mg, 0.30 mmol, 1 eq.) (intermediate of formula (vi) (wherein R 1 is phenyl, R 2 is hydrogen)) and azetidine (0.024 mL, 1.2 eq.) (cyclic amine of formula (vii)) according to the general procedure under Scheme 4 and isolated as an off-white powder (27 mg, 0.08 mmol, isolated yield 26%). 1 1H NMR (600 MHz, CDCl 3 ) δ 7.71 (ddd, J = 7.7, 1.8, 1.1 Hz, 1H), 7.68 (t, J = 1.8 Hz, 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, 6.0 Hz, 2H). 13 13C NMR (151 MHz, CDCl 3 ) δ 161.0, 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 + ) C 22 H 17 N 5Calculated value for: [M+H + m / z = 352.2, measured m / z = 352.0. HRMS (ESI + ) C 22 H 17 N 5 Calculated value for: [M+H + m / z = 352.1557, measured m / z = 352.1575.

[0163] 4-([1,1’-Biphenyl]-3-yl)-2-amino-6-(pyrrolidin-1-yl)pyridine-3,5-dicarbonitrile (2) (In the formula, R 1 is phenyl, R 2 is hydrogen, R 3 is cyano, R 4 is pyrrolidine)

Chemical formula

[0164] The product was prepared according to the general procedure under Scheme 4 starting from commercially available [1,1'-biphenyl]-3-carbaldehyde (intermediate of formula (vi) (in the formula, R 1 is phenyl, R 2 is hydrogen)) (55 mg, 0.30 mmol, 1 eq.) and pyrrolidine (0.030 mL, 1.2 eq.) (cyclic amine of formula (vii)), and isolated as an off-white powder (19 mg, 0.05 mmol, isolated yield 17%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.71 (dt, J = 7.8, 1.5 Hz, 1H), 7.69 (t, J = 1.8 Hz, 1H), 7.65 - 7.62 (m, 2H), 7.58 (t, J = 7.6 Hz, 1H), 7.48 - 7.44 (m, 3H), 7.36 (t, J = 7.4 Hz, 1H), 5.42 (s, 2H), 3.87 - 3.82 (m, 4H), 2.03 - 1.95 (m, 4H). 13 C NMR (151 MHz, CDCl 3) δ 162.2, 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 + ) C 23 H 19 N 5 Calculated values for: [M + H + m / z = 366.2, found m / z = 366.0. HRMS(ESI + ) C 23 H 19 N 5 Calculated values for: [M + H + m / z = 366.1714, found m / z = 366.1706.

[0165] 4-([1,1’-Biphenyl]-3-yl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (3) (wherein R 1 is phenyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0166] The product was prepared starting from commercially available [1,1'-biphenyl]-3-carbaldehyde (intermediate of formula (vi) wherein R 1 is phenyl, R 2 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, isolated yield 7%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.73 - 7.70 (m, 2H), 7.66 - 7.62 (m, 2H), 7.58 (t, J = 7.7 Hz, 1H), 7.49 (dt, J = 7.7, 1.5 Hz, 1H), 7.45 (t, J = 7.7 Hz, 2H), 7.37 (tt, J = 7.4, 1.3 Hz, 1H), 5.49 (s, 2H), 3.86 - 3.80 (m, 4H), 1.73 - 1.70 (m, 6H).13 13C NMR (151 MHz, CDCl 3 ) δ 162.4, 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 + ) C 24 H 21 N 5 calculated for: [M + H + m / z = 380.2, found m / z = 380.1. HRMS (ESI + ) C 24 H 21 N 5 calculated for: [M + H + m / z = 380.1870, found m / z = 380.1859.

[0167] 2-Amino-4-(2’-fluoro-[1,1’-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (4) (wherein R 1 is 2-fluorophenyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0168] The product was prepared starting from 2'-fluoro-[1,1'-biphenyl]-3-carbaldehyde (intermediate of formula (vib)) (100 mg, 0.5 mmol, 1 eq.) according to Sarkar et al., 2014, and isolated as a white powder (45 mg, 0.11 mmol, isolated yield 23%). 1 1H NMR (600 MHz, CDCl 3 ) δ 7.72 - 7.67 (m, 2H), 7.59 (td, J = 7.6, 0.7 Hz, 1H), 7.51 (dt, J = 7.7, 1.5 Hz, 1H), 7.41 (td, J = 4.4, 3.8, 2.8 Hz, 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 (151 MHz, CDCl 3 ) δ 163.3 (d, J = 246.0 Hz), 162.1, 161.3, 159.6, 142.8 (d, J = 7.5 Hz), 140.7 (d, J = 2.1 Hz), 135.5, 130.5 (d, J = 8.4 Hz), 129.5 (d, J = 26.6 Hz), 128.2, 127.9, 123.3, 117.9, 116.7, 114.6 (d, J = 21.0 Hz), 114.4 (d, J = 22.0 Hz), 83.7, 81.8, 49.4, 26.2, 24.6. MS (ESI + ) C 24 H 20 N 5 Calculated for C + H + N 24 F: [M + H 20 m / z = 398.2, found m / z = 398.8. HRMS (ESI 5 Calculated for C + H

[0169] 2-Amino-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (5) (wherein R 1 is 3-fluorophenyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0170] The product was prepared starting from 3'-fluoro-[1,1'-biphenyl]-3-carbaldehyde (intermediate of formula (vic)) (100 mg, 0.5 mmol, 1 eq.) according to Sarkar et al., 2014, and isolated as a white powder (34 mg, 0.08 mmol, isolated yield 17%). 1 H NMR (600 MHz, CDCl 3) δ 7.71 (dq, J = 7.7, 1.6 Hz, 1H), 7.68 (q, J = 1.6 Hz, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.55 - 7.49 (m, 2H), 7.37 - 7.30 (m, 1H), 7.23 (td, J = 7.5, 1.2 Hz, 1H), 7.17 (ddd, J = 10.9, 8.2, 1.2 Hz, 1H), 5.37 (s, 2H), 3.83 - 3.79 (m, 4H), 1.71 (t, J = 3.1 Hz, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 162.0, 161.4, 159.9 (d, J = 248.7 Hz), 159.6, 136.5, 135.1, 131.3 (d, J = 3.3 Hz), 131.1 (d, J = 3.2 Hz), 129.59 (d, J = 1.1 Hz), 129.55 (d, J = 4.0 Hz), 129.1, 128.1, 124.7 (d, J = 3.6 Hz), 117.8, 116.7, 116.3 (d, J = 22.5 Hz), 83.8, 81.8, 49.4, 26.1, 24.6. MS (ESI + ) C 24 H 20 N 5 Calculated for C + H + N 24 F: [M + H 20 m / z = 398.2, found m / z = 398.8. HRMS (ESI 5 Calculated for C + H

[0171] 2-Amino-4-(4'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (6) (wherein, R 1 is 4-fluorophenyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0172] The product was prepared starting from 4'-fluoro-[1,1'-biphenyl]-3-carbaldehyde (intermediate of formula (vid)) (100 mg, 0.5 mmol, 1 eq.) according to Sarkar et al., 2014, and isolated as a white powder (34 mg, 0.08 mmol, isolated yield 17%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.68 - 7.64 (m, 2H), 7.61 - 7.55 (m, 3H), 7.48 (dt, J = 7.7, 1.5 Hz, 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 (151 MHz, CDCl 3 ) δ 162.9 (d, J = 246.7 Hz), 162.2, 161.3, 159.6, 141.0, 136.8 (d, J = 3.4 Hz), 135.4, 129.5, 129.3, 129.2 (d, J = 8.2 Hz), 127.8 (d, J = 19.2 Hz), 117.9, 116.8, 115.9 (d, J = 21.5 Hz), 83.7, 81.8, 49.4, 26.2, 24.6. MS (ESI + ) C 24 H 20 N 5 F calculated for: [M + H + m / z = 398.2, found m / z = 398.8. HRMS (ESI + ) C 24 H 20 N 5 F calculated for: [M + Na + m / z = 420.1595, found m / z = 420.1594.

[0173] 2-Amino-4-(2'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (7) (wherein R 1 is 2-methylphenyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical Structure

[0174] The product was prepared as described above by thermal activation starting from 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate of formula (iiiaa)) (60 mg, 0.16 mmol, 1 eq.) and 2-methylphenylboronic acid (intermediate of formula (iv) (wherein R 1 is 2-methylphenyl and R 2 is boronic acid)) (63 mg, 3 eq.) and isolated as a white powder (30 mg, 0.08 mmol, isolated yield 49%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.55 (t, J = 7.7 Hz, 1H), 7.47 (ddt, J = 7.4, 5.8, 1.4 Hz, 2H), 7.43 (t, J = 1.8 Hz, 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 (151 MHz, CDCl 3 ) δ 162.3, 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 for C 24 H 21 N 5 : [M + H + m / z = 394.2, found m / z = 394.9. HRMS (ESI + ) Calculated for C 24 H 21 N 5 : [M + Na + m / z = 416.1846, found m / z = 416.1857.

[0175] 2-Amino-4-(3'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (8) (wherein R 1 is 3-methylphenyl, R 2 is hydrogen, R 3 is cyano, and R 4 is piperidine) [Chemical formula]

[0176] The product was prepared as described above by thermal activation starting from 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate of formula (iiiaa)) (60 mg, 0.16 mmol, 1 eq.) and 3-methylphenylboronic acid (intermediate of formula (iv) (wherein R 1 is 3-methylphenyl and R 2 is boronic acid)) (63 mg, 3 eq.) and isolated as a white powder (47 mg, 0.12 mmol, isolated yield 76%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.70 (ddd, J = 7.7, 1.9, 1.2 Hz, 1H), 7.69 (t, J = 1.8 Hz, 1H), 7.57 (t, J = 7.7 Hz, 1H), 7.47 - 7.46 (m, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H), 7.18 (d, J = 7.2 Hz, 0H), 5.36 (s, 2H), 3.82 (dd, J = 5.9, 3.1 Hz, 4H), 2.43 (s, 3H), 1.72 (q, J = 1.5 Hz, 4H). 13 C NMR (151 MHz, CDCl 3 ) δ 162.4, 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 for C 24 H 21 N 5 : [M + H + m / z = 394.2, found m / z = 394.9. HRMS (ESI + ) Calculated for C 24 H 21 N 5 : [M + Na +m / z = 416.1846, measured m / z = 416.1857.

[0177] 2-Amino-4-(4'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (9) (In the formula, R 1 is 4-methylphenyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0178] The product was prepared starting from 4'-methyl-[1,1'-biphenyl]-3-carbaldehyde (intermediate of formula (via)) (98 mg, 0.5 mmol, 1 eq.) according to Sarkar et al., 2014, and isolated as a white powder (56 mg, 0.14 mmol, isolated yield 28%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.71 - 7.67 (m, 2H), 7.56 (t, J = 7.6 Hz, 1H), 7.53 (d, J = 7.9 Hz, 2H), 7.45 (dd, J = 7.7, 1.5 Hz, 1H), 7.26 (d, J = 8.1 Hz, 2H), 5.37 (s, 2H), 3.84 - 3.79 (m, 4H), 1.74 - 1.69 (m, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 162.4, 161.4, 159.6, 141.9, 137.8, 137.6, 135.2, 129.7, 129.31, 129.28, 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 for C 24 H 21 N 5 : [M + H + m / z = 394.2, measured m / z = 394.8. HRMS (ESI + ) Calculated value for C 24 H 21 N 5 : [M + Na +m / z = 416.1846, measured m / z = 416.1857.

[0179] 2-Amino-6-(piperidin-1-yl)-4-(3-(pyridin-2-yl)phenyl)pyridine-3,5-dicarbonitrile (10) (In the formula, R 1 is 2-pyridinyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0180] The product was prepared as described above using microwave-assisted Suzuki cross-coupling at 80 °C for 15 minutes starting from (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 2-bromopyridine (intermediate of formula (iv) (wherein R 1 is 2-pyridinyl, R 2 is bromo)) (28 mg, 1.2 eq.) and isolated as a white powder (17 mg, 0.04 mmol, isolated yield 30%). 1 1H NMR (600 MHz, CDCl 3 ) δ 8.92 (s, 1H), 8.65 (s, 1H), 7.99 (dt, J = 8.0, 1.8 Hz, 1H), 7.75 - 7.68 (m, 2H), 7.64 (t, J = 7.7 Hz, 1H), 7.56 (dt, J = 7.7, 1.4 Hz, 1H), 7.44 (t, J = 5.9 Hz, 1H), 5.40 (s, 2H), 3.86 - 3.80 (m, 4H), 1.76 - 1.70 (m, 6H). 13 13C NMR (151 MHz, CDCl 3 ) δ 162.0, 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 + ) C 23 H 20 N 6Calculated value for: [M+H + m / z = 381.2, measured m / z = 381.0. HRMS (ESI + ) Calculated value for C23H20N6: [M+H+] m / z = 381.1823, measured m / z = 381.1803.

[0181] 2-Amino-6-(piperidin-1-yl)-4-(3-(pyridin-3-yl)phenyl)pyridine-3,5-dicarbonitrile (11) (In the formula, R 1 is 3-pyridinyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0182] The product was prepared as described above starting from 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate of formula (iiiaa)) (58 mg, 0.15 mmol, 1 eq.) and 3-pyridineboronic acid (intermediate of formula (iv) (wherein R 1 is 3-pyridinyl and R 2 is boronic acid)) (55 mg, 3 eq.) using microwave-assisted Suzuki cross-coupling at 100 °C for 30 minutes and isolated as a white powder (79 mg, 0.15 mmol, quantitative isolation yield). 1 1H NMR (600 MHz, CDCl 3 ) δ 8.92 (s, 1H), 8.65 (s, 1H), 7.99 (dt, J = 8.0, 1.8 Hz, 1H), 7.75 - 7.68 (m, 2H), 7.64 (t, J = 7.7 Hz, 1H), 7.56 (dt, J = 7.7, 1.4 Hz, 1H), 7.44 (t, J = 5.9 Hz, 1H), 5.40 (s, 2H), 3.86 - 3.80 (m, 4H), 1.76 - 1.70 (m, 6H). 13 13C NMR (151 MHz, CDCl 3) δ 161.9, 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 + ) C 23 H 20 N 6 Calculated values for: [M + H + m / z = 381.2, found m / z = 381.1. HRMS (ESI + ) C 23 H 20 N 6 Calculated values for: [M + H + m / z = 381.1823, found m / z = 381.1836.

[0183] 2-Amino-6-(piperidin-1-yl)-4-(3-(pyridin-4-yl)phenyl)pyridine-3,5-dicarbonitrile (12) (wherein R 1 is 4-pyridinyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical Structure

[0184] The product was prepared as described above using microwave-assisted Suzuki cross-coupling at 80 °C for 15 minutes starting from 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate of formula (iiiaa)) (58 mg, 0.15 mmol, 1 eq.) and 4-pyridineboronic acid (intermediate of formula (iv) (wherein R 1 is 4-pyridinyl, R 2 is boronic acid)) (55 mg, 3 eq.) and isolated as a white powder (5 mg, 0.013 mmol, isolated yield 9%). 1 H NMR (600 MHz, CDCl 3)δ 8.71 - 8.67 (m, 2H), 7.79 - 7.74 (m, 2H), 7.65 (t, J = 8.1 Hz, 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 (151 MHz, CDCl 3 )δ 161.7, 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 + )C 23 H 20 N 6 for the calculated value of: [M + H + m / z = 381.2, found m / z = 381.1. HRMS (ESI + )C 23 H 20 N 6 for the calculated value of: [M + H + m / z = 381.1823, found m / z = 381.1836.

[0185] 2-Amino-6-(piperidin-1-yl)-4-(3-(thiophen-2-yl)phenyl)pyridine-3,5-dicarbonitrile (13) (wherein, R 1 is 2 - thiophenyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0186] The product was treated with 2 - amino - 4 - (3 - bromophenyl) - 6 - (piperidin - 1 - yl) pyridine - 3,5 - dicarbonitrile (intermediate of formula (iiiaa)) (58 mg, 0.16 mmol, 1 eq.) and 2 - thiopheneboronic acid (intermediate of formula (iv) (wherein R 1 is 2 - thiophenyl, R 2Starting from boronic acid ((23 mg, 3 eq.)), it was prepared as described above using microwave-assisted Suzuki cross-coupling at 100 °C for 30 minutes and isolated as a white powder (58 mg, 0.16 mmol, quantitative isolation yield). 1 H NMR (600 MHz, CDCl 3 ) δ 7.74 (ddd, J = 7.8, 1.9, 1.1 Hz, 1H), 7.71 (t, J = 1.8 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.39 (ddd, J = 7.7, 1.8, 1.1 Hz, 1H), 7.37 (dd, J = 3.6, 1.2 Hz, 1H), 7.31 (dd, J = 5.1, 1.2 Hz, 1H), 7.09 (dd, J = 5.1, 3.6 Hz, 1H), 5.44 (s, 2H), 3.85 - 3.80 (m, 4H), 1.75 - 1.69 (m, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 162.0, 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 + ) C 22 H 19 N 5 Calculated for C + m / z = 386.1, found m / z = 386.1. HRMS (ESI + ) C 22 H 19 N 5 Calculated for C + m / z = 386.1435, found m / z = 386.1438.

[0187] 2-Amino-6-(piperidin-1-yl)-4-(3-(thiophen-3-yl)phenyl)pyridine-3,5-dicarbonitrile (14) (wherein R 1 is 3 - thiophenyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0188] The product was prepared as described above using microwave-assisted Suzuki cross-coupling at 80 °C for 15 minutes starting from 2-amino-4-(3-bromophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate of formula (iiiaa)) (58 mg, 0.16 mmol, 1 eq.) and 3-thiopheneboronic acid (intermediate of formula (iv) (wherein R 1 is 3-thienyl and R 2 is boronic acid)) (23 mg, 1.2 eq.), and isolated as a white powder (42 mg, 0.11 mmol, isolated yield 73%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.66 - 7.62 (m, 2H), 7.46 (t, J = 7.7 Hz, 1H), 7.44 (dd, J = 2.9, 1.4 Hz, 1H), 7.36 - 7.33 (m, 2H), 7.32 (dd, J = 5.0, 2.9 Hz, 1H), 5.33 (s, 2H), 3.76 - 3.72 (m, 4H), 1.69 - 1.62 (m, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 162.2, 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 + ) C 22 H 19 N 5 Calculated for S: [M + Na + m / z = 408.1, found m / z = 408.0. HRMS (ESI + ) C 22 H 19 N 5 Calculated for S: [M + H + m / z = 386.1435, found m / z = 386.1438.

[0189] 4-(3-(1H-Pyrazol-5-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (15) (wherein R 1 is 1H-pyrazol-5-yl, R 2 is hydrogen, R3 is cyano, and R 4 is piperidine)

Chemical formula

[0190] The Suzuki cross-coupling step was carried out using microwave-assisted Suzuki cross-coupling starting from (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.). Deprotection of the pyrazole was carried out in a dry 10 mL round-bottom flask under a stream of argon. The intermediate (1 eq.) was dissolved in a 1:1 DCM:MeOH mix (3 mL). TsOH (39 mg, 1.5 eq.) was added and the solution was stirred at r.t. for 20 h. Completion was evaluated by TLC (Rf = 0.18 in MixA). Two purifications were carried out by flash chromatography on a pre-packed 4 g silica cartridge using a MixA 3 :MeOH isocratic gradient (9:1, 12 CV), then a cyclo:DCM gradient (95:5, 3 CV; 95:5 → 0:10, 18 CV; methanol wash) (42 mg, 0.11 mmol, overall isolated yield 76%). 1 1H NMR (600 MHz, CDCl 3 ) δ 7.88 (dt, J = 7.8, 1.4 Hz, 1H), 7.86 (t, J = 1.8 Hz, 1H), 7.63 (s, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.44 (dt, J = 7.8, 1.3 Hz, 1H), 6.65 (s, 1H), 5.33 (s, 2H), 3.77 - 3.73 (m, 4H), 1.67 - 1.62 (m, 6H). 13 13C NMR (151 MHz, CDCl 3) δ 161.8, 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 + ) C 21 H 19 N 7 Calculated values for: [M + H + m / z = 370.2, found m / z = 370.3. HRMS (ESI + ) C 21 H 19 N 7 Calculated values for: [M + H + m / z = 370.1775, found m / z = 370.1779.

[0191] 4-(3-(1H-Tetrazol-5-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (16) (wherein R 1 is 1H - tetrazol - 5 - yl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0192] The product was prepared starting from 3 - (2 - (tetrahydro - 2H - pyran - 2 - yl) - 2H - tetrazol - 5 - yl) benzaldehyde (intermediate of formula (vie)) (60 mg, 0.23 mmol, 1 eq.) according to the general procedure under Scheme 4 and purified by flash chromatography on a pre - packed 4 g silica cartridge using a cyclo:DCM gradient (95:5, 3 CV; 95:5 → 0:10, 18 CV; methanol wash). Deprotection of the tetrazole was carried out at 80 °C for 16 h in 98:2 EtOH:H 2 O mix (1 mL) in the presence of Dowex 50WX8 H + (1 g of resin for 100 mg of compound). The resin was filtered off, washed with warm EtOH (3 × 2 mL), and the product was isolated as a white powder (10 mg, 0.03 mmol, overall isolated yield 11%). 11H NMR (600 MHz, (CD 3 )) 2 SO) δ 10.20 (s, 1H), 8.20 (dt, J = 7.8, 1.5 Hz, 1H), 8.15 (t, J = 1.8 Hz, 1H), 7.78 (t, J = 7.7 Hz, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.50 (s, 2H), 3.77 - 3.73 (m, 4H), 1.68 - 1.58 (m, 6H). MS (ESI - ) C 19 H 17 N 9 for the calculated value of: [M - H + m / z = 370.2, found m / z = 370.0. HRMS (ESI - ) C 19 H 17 N 9 for the calculated value of: [M - H + m / z = 370.1524, found m / z = 370.1500.

[0193] 4-([1,1'-Biphenyl]-3-yl)-2-amino-6-(diethylamino)pyridine-3,5-dicarbonitrile (17) (wherein, R 1 is phenyl, R 2 is hydrogen, R 3 is cyano, R 4 is diethylamino)

Chemical formula

[0194] The product was prepared according to the general procedure based on Scheme 1 starting from 4-([1,1'-biphenyl]-3-yl)-2-amino-6-chloropyridine-3,5-dicarbonitrile (50 mg, 0.15 mmol) (the intermediate of formula (i) (wherein, R 1 is phenyl, R 2 is hydrogen, R 3 is cyano)) and diethylamine (0.10 mL, 6 eq.) (the nucleophile of formula (ii)), and isolated as a white amorphous powder (22 mg, 0.06 mmol, isolated yield 40%). 1 1H NMR (600 MHz, CDCl 3) δ 7.71 (dt, J = 7.8, 1.4 Hz, 1H), 7.68 (t, J = 1.8 Hz, 1H), 7.66 - 7.61 (m, 2H), 7.58 (t, J = 7.7 Hz, 1H), 7.48 - 7.42 (m, 3H), 7.36 (dd, J = 7.4, 1.4 Hz, 1H), 5.50 (s, 2H), 3.77 (q, J = 7.0 Hz, 4H), 1.32 (t, J = 7.0 Hz, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 162.9, 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 + ) C 23 H 21 N 5 for the calculated value of: [M + H + m / z = 368.2, found m / z = 368.4. HRMS (ESI + ) C 23 H 21 N 5 for the calculated value of: [M + H + m / z = 368.1870, found m / z = 368.1839.

[0195] 4-([1,1'-Biphenyl]-3-yl)-2-amino-6-morpholinopyridine-3,5-dicarbonitrile (18) (wherein, R 1 is phenyl, R 2 is hydrogen, R 3 is cyano, R 4 is morpholine)

Chemical formula

[0196] The product was 4-([1,1'-biphenyl]-3-yl)-2-amino-6-chloropyridine-3,5-dicarbonitrile (50 mg, 0.15 mmol) (the intermediate of formula (i) (wherein, R 1 is phenyl, R 2 is hydrogen, R 3Starting from cyanide)) and morpholine (0.10 mL, 8 eq.) (the nucleophile of formula (ii)), it was prepared according to the general procedure based on Scheme 1 and isolated as a white amorphous powder (12 mg, 0.03 mmol, isolated yield 21%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.73 (dt, J = 7.8, 1.4 Hz, 1H), 7.70 (t, J = 1.8 Hz, 1H), 7.65 - 7.61 (m, 2H), 7.59 (t, J = 7.7 Hz, 1H), 7.50 - 7.43 (m, 3H), 7.37 (dd, J = 7.4, 1.2 Hz, 1H), 5.51 (s, 2H), 3.88 (dd, J = 5.7, 3.6 Hz, 4H), 3.82 (dd, J = 5.6, 3.7 Hz, 4H). 13 C NMR (151 MHz, CDCl 3 ) δ 162.4, 161.5, 159.5, 142.1, 140.5, 134.9, 129.7, 129.5, 129.0, 127.88, 127.85, 127.6, 127.5, 117.5, 116.3, 84.3, 83.0, 66.8, 48.5. MS (ESI + ) C 23 H 19 N 5 O calculated value for: [M + H + m / z = 382.2, measured value m / z = 328.3. HRMS (ESI + ) C 23 H 19 N 5 O calculated value for: [M + H + m / z = 382.1663, measured value m / z = 328.1636.

[0197] 4-([1,1'-Biphenyl]-3-yl)-2-amino-6-(4-methylpiperazin-1-yl)pyridine-3,5-dicarbonitrile (19) (wherein R 1 is phenyl, R 2 is hydrogen, R 3 is cyano, R 4 is N - methylpiperazine)

Chemical Structure

[0198] The product was prepared according to the general procedure based on 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 R 1 is phenyl, R 2 is hydrogen, R 3 is cyano)) and N-methylpiperazine (0.10 mL, 6 eq.) (nucleophile of formula (ii)), and isolated as a white amorphous powder (13 mg, 0.03 mmol, isolated yield 22%). 1 H NMR (600 MHz, CD 3 OD) δ 7.79 (ddd, J = 7.8, 1.9, 1.1 Hz, 1H), 7.76 (t, J = 1.8 Hz, 1H), 7.70 - 7.65 (m, 2H), 7.62 (t, J = 7.7 Hz, 1H), 7.51 (ddd, J = 7.7, 1.8, 1.1 Hz, 1H), 7.49 - 7.43 (m, 2H), 7.38 (dd, J = 7.4, 1.1 Hz, 1H), 3.89 (t, J = 5.1 Hz, 4H), 2.71 (t, J = 4.9 Hz, 4H), 2.45 (s, 3H). 13 C NMR (151 MHz, CD 3 OD) δ 163.6, 163.3, 161.9, 143.1, 141.7, 137.0, 130.3, 130.0, 128.8, 128.78, 128.76, 128.3, 118.9, 117.1, 84.2, 83.5, 55.5, 48.2, 45.6. MS (ESI + ) C 24 H 22 N 6 Calculated for: [M + H + m / z = 395.2, found m / z = 395.5. HRMS (ESI + ) C 24 H 22 N 6 Calculated for: [M + H + m / z = 395.1979, found m / z = 395.1955.

[0199] 4-([1,1'-Biphenyl]-3-yl)-2-amino-6-(benzylthio)pyridine-3,5-dicarbonitrile (20) (wherein R 1 is phenyl, R 2 is hydrogen, R3 is cyano, and R 4 is benzyl mercaptan) [Chemical formula]

[0200] The product was prepared according to the general procedure based on 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 R 1 is phenyl, and R 2 is hydrogen, and R 3 is cyano)), benzyl mercaptan (0.03 mL, 1.5 eq.) (nucleophile of formula (ii)) and triethylamine (0.04 mL, 2 eq.), and isolated as a white amorphous powder (19 mg, 0.04 mmol, isolated yield 30%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.75 (dt, J = 7.9, 1.4 Hz, 1H), 7.72 (t, J = 1.8 Hz, 1H), 7.64 - 7.58 (m, 3H), 7.50 (dt, J = 7.8, 1.3 Hz, 1H), 7.46 (t, J = 7.7 Hz, 2H), 7.43 - 7.40 (m, 2H), 7.40 - 7.36 (m, 1H), 7.34 (dd, J = 8.3, 6.6 Hz, 2H), 7.32 - 7.27 (m, 1H), 5.70 (s, 2H), 4.48 (s, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 168.9, 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 - ) C 26 H 18 N 4 Calculated for C + [M - H + m / z = 417.1, found m / z = 416.8. HRMS (ESI + ) C26 H 18 N 4 Calculated value for S: [M+Na + m / z = 441.1145, measured value m / z = 441.1118.

[0201] 4-([1,1'-Biphenyl]-3-yl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile (21) (wherein R 1 is phenyl, R 2 is hydrogen, R 3 is cyano, R 4 is methoxy)

Chemical formula

[0202] The product was prepared 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 R 1 is phenyl, R 2 is hydrogen, R 3 is cyano)), methanol (1 mL) (nucleophile of formula (ii)) and 1.8 M lithium diisopropylamine in THF (0.19 mL, 3 eq.) according to a general procedure based on Scheme 1 and isolated as a white amorphous powder (22 mg, 0.07 mmol, isolated yield 67%). 1 H NMR (600 MHz, (CD 3 ) 2 SO) δ 8.09 (broad s, 2H), 7.89 - 7.85 (m, 1H), 7.84 (d, J = 1.9 Hz, 1H), 7.76 - 7.71 (m, 2H), 7.67 (t, J = 7.7 Hz, 1H), 7.55 - 7.50 (m, 3H), 7.44 - 7.40 (m, 1H), 4.00 (s, 3H). 13 C NMR (151 MHz, (CD 3 ) 2 SO) δ 165.8, 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+ )C 20 H 14 N 4 Calculated values for C, H, N, O: [M+Na + m / z = 349.1, found m / z = 349.0.

[0203] 4-([1,1'-Biphenyl]-3-yl)-2-amino-6-ethoxypyridine-3,5-dicarbonitrile (22) (In the formula, R 1 is phenyl, R 2 is hydrogen, R 3 is cyano, R 4 is ethoxy) [Chemical formula]

[0204] The product was prepared according to the general procedure based on 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) in which R 1 is phenyl, R 2 is hydrogen, R 3 is cyano), ethanol (1 mL) (nucleophile of formula (ii)) and 1.8 M lithium diisopropylamine in THF (0.13 mL, 3 eq.), and isolated as a white amorphous powder (19 mg, 0.055 mmol, isolated yield 80%). 1 H NMR (600 MHz, (CD 3 ) 2 SO) δ 7.97 (broad s, 2H), 7.86 (ddd, J = 7.8, 1.9, 1.1 Hz, 1H), 7.84 (t, J = 1.8 Hz, 1H), 7.75 - 7.70 (m, 2H), 7.66 (t, J = 7.7 Hz, 1H), 7.55 - 7.48 (m, 3H), 7.45 - 7.39 (m, 1H), 4.46 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H). 13 C NMR (151 MHz, (CD 3 ) 2SO) δ 165.4, 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 + ) C 21 H 16 N 4 Calculated values for O: [M + Na + m / z = 363.1, found m / z = 363.0.

[0205] 4-([1,1'-Biphenyl]-3-yl)-2-amino-6-(2-hydroxyethoxy)pyridine-3,5-dicarbonitrile (23) (wherein R 1 is phenyl, R 2 is hydrogen, R 3 is cyano, R 4 is ethoxy,1 - ol)

Chemical formula

[0206] The product was prepared 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 R 1 is phenyl, R 2 is hydrogen, R 3 is cyano), methanol (1 mL) (nucleophile of formula (ii)) and 1.8 M lithium diisopropylamine in THF (0.19 mL, 3 eq.) according to a general procedure based on Scheme 1 and isolated as a white amorphous powder (11 mg, 0.03 mmol, isolated yield 31%). 1 H NMR (600 MHz, (CD 3 ) 2SO) δ 7.97 (broad s, 2H), 7.87 (dt, J = 7.8, 1.5 Hz, 1H), 7.84 (t, J = 1.8 Hz, 1H), 7.76 - 7.72 (m, 2H), 7.66 (t, J = 7.7 Hz, 1H), 7.55 - 7.48 (m, 3H), 7.46 - 7.39 (m, 1H), 4.91 (t, J = 5.3 Hz, 1H), 4.43 (t, J = 5.1 Hz, 2H), 3.74 (q, J = 5.2 Hz, 2H). 13 C NMR (151 MHz, (CD 3 ) 2 SO) δ 165.6, 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 + ) C 21 H 16 N 4 O 2 for the calculated values of: [M + Na + m / z = 379.1, found m / z = 379.0.

[0207] 2-Amino-4-(3-(3-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (24) (wherein, R 1 is 3 - fluoro - 2 - pyridinyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical Structure

[0208] The product was treated with (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 - 3 - fluoropyridine (intermediate of formula (iv) (wherein R 1 is 3 - fluoro - 2 - pyridinyl, R 9Starting from bromo ((53 mg, 1.2 eq.)), it was prepared as described above using microwave-assisted Suzuki cross-coupling at 80 °C for 15 minutes and isolated as a white powder (66 mg, 0.16 mmol, isolated yield 72%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.54 (dt, J = 4.6, 1.6 Hz, 1H), 8.19 (dd, J = 7.8, 1.5 Hz, 1H), 8.17 (d, J = 1.5 Hz, 1H), 7.64 (t, J = 7.7 Hz, 1H), 7.57 (dt, J = 7.7, 1.4 Hz, 1H), 7.53 (ddd, J = 11.0, 8.2, 1.4 Hz, 1H), 7.31 (p, J = 4.2 Hz, 1H), 5.44 (s, 2H), 3.84 - 3.79 (m, 4H), 1.73 - 1.70 (m, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 162.1, 161.1, 159.5, 157.9 (d, J = 261.4 Hz), 145.5 (d, J = 5.2 Hz), 145.0 (d, J = 10.3 Hz), 135.8 (d, J = 5.6 Hz), 135.2, 131.0 (d, J = 6.6 Hz), 129.7, 129.3 (d, J = 6.1 Hz), 129.2, 124.7 (d, J = 20.6 Hz), 124.1 (d, J = 4.1 Hz), 117.6, 116.5, 83.9, 82.0, 49.5, 26.2, 24.5. MS (ESI + ) C 23 H 19 N 6 Calculated for C + H + N 23 F: [M + H 19 N 6 F: [M + H + m / z = 399.2, found m / z = 399.1. HRMS (ESI

[0209] 2-Amino-4-(3-(6-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (25) (wherein R 1 is 6-fluoro-2-pyridinyl, R 2 is hydrogen, R 3 is cyano, R 4is piperidine) [Chemical formula]

[0210] The product was prepared as described above using microwave-assisted Suzuki cross-coupling at 80 °C for 15 minutes 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-6-fluoropyridine (intermediate of formula (iv) (wherein R 1 is 6-fluoro-2-pyridinyl and R 9 is bromo)) (53 mg, 1.2 eq.) and isolated as a white powder (35 mg, 0.09 mmol, isolated yield 38%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.17 - 8.12 (m, 2H), 7.86 (q, J = 8.0 Hz, 1H), 7.68 (dd, J = 7.4, 2.2 Hz, 1H), 7.64 - 7.61 (m, 1H), 7.55 (dt, J = 7.6, 1.5 Hz, 1H), 6.90 (dd, J = 8.1, 3.0 Hz, 1H), 5.47 (s, 2H), 3.85 - 3.80 (m, 4H), 1.75 - 1.69 (m, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 163.6 (d, J = 238.9 Hz), 162.1, 160.9, 159.34, 155.4 (d, J = 13.4 Hz), 141.9 (d, J = 7.7 Hz), 138.3, 135.5, 129.8, 129.5, 129.0, 127.6, 117.8 (d, J = 4.0 Hz), 117.6, 116.5, 108.4 (d, J = 37.5 Hz), 83.8, 81.9, 49.5, 26.2, 24.5. MS (ESI + ) C 23 H 19 N 6 Calculated for [M + H + m / z = 399.2, found m / z = 399.2. HRMS (ESI + ) C 23H 19 N 6 Calculated value for F: [M+H + m / z = 399.1728, measured value m / z = 399.1701.

[0211] 2-Amino-4-(3-(4-fluoropyridin-2-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (26) (In the formula, R 1 is 4-fluoro-2-pyridinyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0212] The product was prepared as described above using microwave-assisted Suzuki cross-coupling at 80 °C for 15 minutes 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 R 1 is 4-fluoro-2-pyridinyl, R 9 is bromo)) (53 mg, 1.2 eq.) and isolated as a white powder (35 mg, 0.09 mmol, isolated yield 38%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.70 (dd, J = 8.5, 5.6 Hz, 1H), 8.17 (d, J = 7.8 Hz, 1H), 8.14 (s, 1H), 7.66 (t, J = 7.7 Hz, 1H), 7.60 (dd, J = 7.7, 1.6 Hz, 1H), 7.54 (dd, J = 10.1, 2.4 Hz, 1H), 7.05 (ddd, J = 6.5, 5.1, 2.6 Hz, 1H), 5.39 (s, 2H), 3.84 - 3.80 (m, 4H), 1.74 - 1.70 (m, 6H). 13 C NMR (151 MHz, CDCl 3)δ 169.9 (d, J = 264.1 Hz), 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 = 17.0 Hz), 109.0 (d, J = 17.8 Hz), 83.7, 81.8, 49.4, 26.2, 24.6. MS (ESI + ) C 23 H 19 N 6 Calculated for: [M + H + m / z = 399.2, found m / z = 399.2. HRMS (ESI + ) C 23 H 19 N 6 Calculated for: [M + H + m / z = 399.1728, found m / z = 399.1701.

[0213] 2-Amino-4-(3-(5-fluoropyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (27) (wherein, R 1 is 5-fluoro-3-pyridinyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical Structure

[0214] The product was prepared as described above 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, R 1 is 5-fluoro-3-pyridinyl, R 9 is bromo)) (53 mg, 1.2 eq.) and isolated as a white powder (76 mg, 0.19 mmol, isolated yield 83%). 1 1H NMR (600 MHz, CDCl 3)δ 8.74 (d, J = 1.7 Hz, 1H), 8.51 (d, J = 2.2 Hz, 1H), 7.72 (ddt, J = 19.1, 9.9, 1.6 Hz, 3H), 7.66 (t, J = 7.3 Hz, 1H), 7.60 (dd, J = 7.7, 1.5 Hz, 1H), 5.43 (s, 2H), 3.85 - 3.78 (m, 4H), 1.74 - 1.70 (m, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 161.5, 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 + ) C 23 H 19 N 6 Calculated for C + H + N 23 F: [M + H 19 m / z = 399.2, found m / z = 399.2. HRMS (ESI 6 Calculated for C + H

[0215] 2-Amino-4-(3-(2-fluoropyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (28) (wherein R 1 is 2 - fluoro - 3 - pyridinyl, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0216] The product was prepared from 2 - amino - 4 - (3 - bromophenyl) - 6 - (piperidin - 1 - yl) pyridine - 3,5 - dicarbonitrile (intermediate of formula (iiiaa)) (100 mg, 0.23 mmol, 1 eq.) and 2 - fluoro - 3 - pyridineboronic acid (intermediate of formula (iv) (wherein R 1 is 2 - fluoro - 3 - pyridinyl, R 9Starting from boronic acid (109 mg, 3 eq.), it was prepared as described above using microwave-assisted Suzuki cross-coupling at 100 °C for 30 minutes and isolated as a white powder (12 mg, 0.03 mmol, isolated yield 12%). 1 H NMR (600 MHz, (CD 3 ) 2 SO) δ 8.29 (d, J = 4.6 Hz, 0H), 8.16 (ddd, J = 10.3, 7.4, 1.9 Hz, 1H), 7.81 (dq, J = 7.7, 1.6 Hz, 1H), 7.78 (s, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.60 (dt, J = 7.7, 1.4 Hz, 1H), 7.52 (ddd, J = 6.9, 4.8, 1.7 Hz, 1H), 7.46 (s, 3H), 3.76 - 3.71 (m, 4H), 1.71 - 1.55 (m, 6H). 13 C NMR (151 MHz, (CD 3 ) 2 SO) δ 161.1, 160.7, 159.8, 159.6 (d, J = 237.7 Hz), 146.9 (d, J = 14.6 Hz), 141.6 (d, J = 4.2 Hz), 135.8, 133.6 (d, J = 5.1 Hz), 130.4 (d, J = 3.1 Hz), 129.1, 129.0 (d, J = 24.0 Hz), 122.8 (d, J = 4.2 Hz), 122.2 (d, J = 28.2 Hz), 117.8, 116.3, 81.5, 80.8, 48.5, 25.6, 23.9. MS (ESI + ) C 23 H 19 N 6 F for calculated value: [M + H + m / z = 399.2, found m / z = 399.5. HRMS (ESI + ) C 23 H 19 N 6 F for calculated value: [M + H + m / z = 399.1728, found m / z = 399.1707.

[0217] 2-Amino-4-(2-fluoro-5-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (29) (wherein R 1 is pyridinyl, R 2 is 6 - fluoro, R 3is cyano, R 4 is piperidine) [Chemical formula]

[0218] The product was prepared as described above starting from 2-amino-4-(5-bromo-2-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate of formula (iiiab)) (58 mg, 0.15 mmol, 1 eq.) and 3-pyridineboronic acid (intermediate of formula (iv) (wherein R 1 is 3-pyridinyl and R 9 is boronic acid)) (55 mg, 3 eq.) using microwave-assisted Suzuki cross-coupling at 100 °C for 30 minutes and isolated as a white powder (53 mg, 0.13 mmol, isolated yield 89%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.89 (d, J = 2.3 Hz, 1H), 8.66 (dd, J = 5.1, 1.5 Hz, 1H), 8.09 (dd, J = 8.0, 1.9 Hz, 1H), 7.71 (ddd, J = 8.6, 4.6, 2.4 Hz, 1H), 7.61 (dd, J = 6.5, 2.4 Hz, 1H), 7.57 (dd, J = 8.0, 5.0 Hz, 1H), 7.39 (t, J = 8.9 Hz, 1H), 5.41 (s, 2H), 3.86 - 3.81 (m, 4H), 1.76 - 1.67 (m, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 160.4, 159.6 (d, J = 253.6 Hz), 159.3, 155.8, 145.9 (d, J = 82.0 Hz), 137.3, 136.5, 133.3, 131.3 (d, J = 8.4 Hz), 129.8 (d, J = 3.1 Hz), 124.8, 124.0 (d, J = 15.7 Hz), 117.8 (d, J = 22.5 Hz), 117.2, 116.1, 84.3, 82.4, 49.2, 26.2, 24.5. MS (ESI + ) C 23 H 19 N 6 Calculated for C +m / z = 399.2, measured value m / z = 399.4. HRMS (ESI + ) C 23 H 19 N 6 Calculated value for F: [M + H + m / z = 399.1728, measured value m / z = 399.1701.

[0219] 2-Amino-4-(2-fluoro-3-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (30) (In the formula, R 1 is pyridinyl, R 2 is 2-fluoro, R 3 is cyano, R 4 is piperidine)

Chemical Structure

[0220] The product was prepared as described above using microwave-assisted Suzuki cross-coupling at 100 °C for 30 minutes starting from 2-amino-4-(3-bromo-2-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate of formula (iiiac)) (58 mg, 0.15 mmol, 1 eq.) and 3-pyridineboronic acid (intermediate of formula (iv) (wherein R 1 is 3-pyridinyl and R 9 is boronic acid)) (55 mg, 3 eq.) and isolated as a paste (15 mg, 0.04 mmol, isolated yield 25%). 1 1H NMR (600 MHz, CDCl 3 ) δ 8.96 - 8.94 (m, 1H), 8.77 - 8.73 (m, 1H), 8.41 (d, J = 8.0 Hz, 1H), 7.83 (dd, J = 8.0, 5.3 Hz, 1H), 7.63 (td, J = 7.4, 1.8 Hz, 1H), 7.55 (ddd, J = 8.0, 6.4, 1.7 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 5.43 (s, 2H), 3.86 - 3.80 (m, 4H), 1.77 - 1.68 (m, 6H). 13 13C NMR (151 MHz, CDCl 3) δ 160.1, 159.1, 156.1 (d, J = 253.4 Hz), 155.6, 143.5, 142.6, 133.9, 132.41 (d, J = 2.6 Hz), 132.37, 125.8 (d, J = 4.7 Hz), 125.7, 124.4, 124.3, 117.1, 115.9, 84.1, 82.1, 49.1, 26.0, 24.4. MS (ESI + ) C 23 H 19 N 6 Calculated for F: [M + H + m / z = 399.2, found m / z = 399.4. HRMS (ESI + ) C 23 H 19 N 6 Calculated for F: [M + H + m / z = 399.1728, found m / z = 399.1701.

[0221] 2-Amino-4-(3-fluoro-5-(pyridin-3-yl)phenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (31) (wherein R 1 is pyridinyl, R 2 is 5-fluoro, R 3 is cyano, R 4 is piperidine)

Chemical Structure

[0222] The product was prepared as described above using microwave-assisted Suzuki cross-coupling at 100 °C for 30 minutes starting from 2-amino-4-(3-chloro-5-fluorophenyl)-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (intermediate of formula (iiiad)) (53 mg, 0.15 mmol, 1 eq.) and 3-pyridineboronic acid (intermediate of formula (iv) (wherein R 1 is 3-pyridinyl, R 9 is boronic acid)) (55 mg, 3 eq.) and isolated as a white powder (56 mg, 0.14 mmol, isolated yield 96%). 1 1H NMR (600 MHz, CDCl 3) δ 8.93 (s, 1H), 8.70 (s, 1H), 8.15 (dt, J = 8.0, 1.8 Hz, 1H), 7.65 - 7.59 (m, 1H), 7.49 (d, J = 1.6 Hz, 1H), 7.44 (dt, J = 8.9, 1.9 Hz, 1H), 7.31 (dt, J = 8.5, 1.9 Hz, 1H), 5.45 (s, 2H), 3.86 - 3.81 (m, 4H), 1.76 - 1.68 (m, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 163.2 (d, J = 250.3 Hz), 160.8, 160.1, 159.4, 146.3, 145.4, 138.0 (d, J = 8.4 Hz), 137.8, 125.1, 123.8 (d, J = 3.1 Hz), 117.5, 116.7 (d, J = 23.0 Hz), 116.32 (d, J = 22.5 Hz), 116.27, 83.2, 81.5, 49.3, 26.2, 24.5. MS (ESI + ) C 23 H 19 N 6 Calculated for C + H + N 23 F: [M + H 19 m / z = 399.2, found m / z = 399.3. HRMS (ESI 6 Calculated for C + H

[0223] 4-(3-(1,3,4-Oxadiazol-2-yl)phenyl)-2-amino-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile (32) (wherein, R 1 is 1,3,4 - oxadiazole, R 2 is hydrogen, R 3 is cyano, R 4 is piperidine)

Chemical formula

[0224] In a 25 mL 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 (54 mg, 0.15 mmol, 1 eq.) was suspended in anhydrous methanol (1.5 mL). Hydrazine monohydrate (37 mg, 5 eq.) was added to the solution, and then the vessel was purged with argon and sealed. The reaction was heated at 80 °C for 16 h. The solvent was removed under reduced pressure, and the crude product was rinsed with DCM:MeOH 7:3 (10 mL) and dried under reduced pressure. In a 25 mL round-bottom flask equipped with a magnetic stirrer and a condenser, the intermediate was suspended in excess triethyl orthoformate (1 mL). Argon was passed through the vessel and heated at 100 °C for 20 h. The reaction was quenched by the addition of water (20 mL), and the crude product was extracted with EtOAc (3 × 15 mL). The organic phases were combined, washed with brine, dried over MgSO 4 and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on a silica cartridge with a cyclo:EtOAc gradient (8:2, 3 CV; 8:2 → 6:4, 12 CV; 6:4, 6 CV) on a 12 g silica cartridge solid deposit and isolated as a white powder (32 mg, 0.09 mmol, overall isolated yield 58%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.50 (s, 1H), 8.26 (dt, J = 7.6, 1.6 Hz, 1H), 8.21 (t, J = 1.8 Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H), 7.67 (dt, J = 7.8, 1.6 Hz, 1H), 5.66 (s, 2H), 3.87 - 3.82 (m, 4H), 1.76 - 1.70 (m, 6H). 13 C NMR (151 MHz, CDCl 3 ) δ 164.1, 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 + ) C 20 H 17 N 7 O calculated for: [M + H+ m / z = 372.2, measured m / z = 372.4. HRMS (ESI + ) C 20 H 17 N 7 Calculated values for O: [M + H + m / z = 372.1568, measured m / z = 372.1565.

[0225] Example 2: Effect of the compounds of the present invention in CHO cells The efficacy of the compounds of the present invention was tested using the adenosine receptor-expressing cells described below.

[0226] a) Characterization of the potency using adenosine receptor-expressing cells The efficacy of the A2AR antagonists according to the present invention was characterized using Chinese hamster ovary (CHO) cells expressing recombinant A2AR and containing the in-house cAMP-based biosensor described below. The biosensor contains a cAMP-binding domain that enables the measurement of Gs and Gi coupled receptor activation. Cells were treated with the indicated concentrations of the A2AR antagonist in 384-well plates, followed by the addition of adenosine, an A2AR agonist. cAMP levels were measured in a luminescence / fluorescence plate reader.

[0227] CHO cells expressing A2AR and the biosensor were seeded in black 384-well plates (Nunc) and grown overnight at 37 °C and 5% CO 2 . They were pre-incubated for 6 minutes with various concentrations of the compounds of the present invention up to 30 μM, followed by the addition of 400 nM adenosine (Sigma), the corresponding EC 80 of the A2AR agonist. The cAMP biosensor enabled real-time monitoring of the signal using FDSS / μCELL (Hamamatsu). The assay was performed in 1× Hank's balanced salt solution (HBSS). The total volume of the reaction was 80 μl (45 μl of cells, 15 μl of antagonist and 20 μl of agonist). Data analysis was performed using GraphPad Prism.

[0228] As reported in Table 1 below, the compounds of the present invention can antagonize the production of cAMP in cell lines overexpressing A2AR in the absence of other adenosine receptors, where the activity represents the ability of a compound to antagonize the production of cAMP in response to stimulation of A2AR-expressing CHO cells with adenosine, an A2AR agonist, as described in the cAMP assay method above. Thus, the compounds can be classified as A2AR antagonists.

[0229] [Table 1] The activity scale represents the IC50 range of: +: > 1000 nM, ++: 200 - 1000 nM, +++: < 200 nM.

[0230] b) Verification of the mechanism of action of small molecule negative allosteric molecules using the "shift assay" The allosteric molecular mechanism of A2AR inhibition mediated by the compounds of the present invention was demonstrated using a "shift assay" using an A2AR-transfected cell line. These assays contain a BRET-based biosensor and use CHO cells expressing recombinant A2AR. The "shift assay" was performed in 384-well plates using the addition of the indicated concentrations of an A2AR antagonist followed by the addition of a concentration-response curve (CRC) of adenosine, an A2AR agonist. cAMP levels were measured in a luminescence / fluorescence plate reader. Briefly, CHO cells expressing A2AR and the biosensor were seeded in black 384-well plates and incubated at 37 °C and 5% CO 2They were grown overnight and pre-incubated for 6 minutes with various concentrations of the compounds of the invention up to 30 μM, after which adenosine was added at increasing concentrations up to 1 mM (CRC). The cAMP biosensor readings and assay volumes were as described in the previous section. Data analysis was performed using GraphPad Prism. Schild regression plots were calculated for the compounds by using shifted assay data. Log (dose ratio -1) was plotted on the y-axis against the log of the antagonist concentration on the x-axis.

[0231] Negative allosteric modulation of A2AR can be identified using a "shift assay". The results are shown in Figure 1A, where it can be observed that the concentration-response curves (CRCs) of adenosine begin to overlap with each other as the compound concentration increases (from small white circles to large white circles). The conversion of this data to a Schild regression plot (Figure 1B) confirms this classification of the compounds of the invention as NAMs. This is because the allosteric modulator causes a plateau in the dose ratio (DR) observed at the upper right of the Schild plot. In contrast, orthosteric compounds are characterized by a complete linear DR profile in the Schild regression plot (Kenakin, 2017, Curr Protoc Pharmacol doi:10.1002 / cpph.18).

[0232] Example 3: Effect of the compounds of the present invention on the restoration of immune response in healthy human peripheral blood immune cells Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood of healthy human blood donors using density centrifugation. The cells were cryopreserved and thawed and allowed to rest for several hours in complete cell culture medium containing human albumin and antibiotics (penicillin and streptomycin; 100 units or mg per 1 ml respectively). The cells were incubated in 96-well plates with the indicated concentrations of the A2AR antagonist of the present invention, followed by the addition of 5 μM of the adenosine analog adenosine-5'-N-ethyluronamide (NECA) (https: / / doi.org / 10.1371 / journal.pcbi.1007818), a potent A2AR agonist. DMSO was used as a storage diluent for the concentrated A2AR antagonist. NECA and DMSO controls (% v / v) were also tested in parallel with the therapeutic agent candidates. After the indicated incubation time with NECA, the cells were stained to determine their intracellular levels of pCREB as described below, or stained for intracellular cytokines after activation overnight with Dynabeads.

[0233] a) Evaluation of intracellular pCREB using flow cytometry The ability of the compounds of the present invention to restore normal pCREB levels in human-derived immune cells was tested as follows.

[0234] PBMCs were cultured with an A2AR antagonist in the presence of NECA and then 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 supplied by Life Technologies), and CD3 (BD Biosciences). Stained cells were acquired using a Fortessa flow cytometer (BD). Gating of the desired cell subsets was performed using FSC-A versus FSC-H to remove doublet cells, and cells with a lymphocyte-like morphology were identified using the FSC versus SSC parameters, followed by CD3 + T cells and CD4 + or CD8 + sub-gating. The fold change in mean fluorescence intensity (MFI) of intracellular pCREB in gated CD4 or CD8 + T cells was calculated using the NECA-only condition and divided by that of NECA- and A2AR antagonist-cultured cells from the same donor. The total activity of the test compound was calculated using the mean of at least three donors.

[0235] The compounds of the present invention were tested using healthy human peripheral blood mononuclear cells under physiological conditions and in the presence of human albumin to increase the physiological relevance of the assay. The compounds of the present invention were also compared to control molecules that have previously been reported to antagonize A2AR (orthosteric antagonists) and have been tested in human Phase I or II clinical trials. These include CPI-444, 7-(5-methylfuran-2-yl)-3-[[6-[[(3S)-oxolan-3-yl]oxymethyl]pyridin-2-yl]methyl]triazolo[4,5-d]pyrimidin-5-amine (siforadenant), 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-hexatricyclo[7.3.0.0 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-triazin-3-amine (imaladenant), and AB928, 3-[2-amino-6-[1-[[6-(2-hydroxypropan-2-yl)pyridin-2-yl]methyl]triazol-4-yl]pyrimidin-4-yl]-2-methylbenzonitrile (etomadenant), and two other known orthosteric compounds. Table 2 below represents the ability of the compounds to restore pCREB to normal physiological levels (immunocompetence) in gated human CD4 + cells, and Table 3 represents the ability of the compounds to restore pCREB to normal physiological levels (immunocompetence) in gated human CD8 + cells as described above. The activity scale presented is the average for at least three donors compared to a control of NECA only.

[0236] [Table 2]

[0237]

Table 3

[0238] As can be seen in Tables 2 and 3, the compounds of the present invention were able to restore normal physiological levels of pCREB in both CD4 and CD8 + T cells under conditions mimicking high adenosine concentrations. pCREB is a signaling element proximal (downstream) to cAMP in A2AR signaling and represents a surrogate marker for immunosuppression.

[0239] b) Evaluation of intracellular cytokines using flow cytometry The ability of the compounds of the present invention to restore anti-tumor cytokine responses in human-derived immune cells was tested as described below.

[0240] In addition, PBMCs cultured with an A2AR antagonist and NECA were stimulated overnight with 1 μl of anti-CD3 and anti-CD28 Dynabeads (Life Technologies) to activate T cells in the presence of GolgiStop (Life Technologies) to prevent the secretion of any newly induced cytokines. The cells were then stained with a fixable live-dead stain (LD Blue, Life Technologies), a T cell-specific Ab described for the pCREB stain, followed by fixation and permeabilization using a commercial kit (ThermoFisher) containing paraformaldehyde and a detergent-based buffer. Intracellular staining for 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 the permeabilization buffer (ThermoFisher). The cells were then washed, acquired, and gated as described for the pCREB assay with the following modifications: dead cells were excluded using the live-dead marker, and then the gated T cell subset was analyzed for the percentage of cytokine-positive cells, which was the total CD4 + or CD8 + subsets of T cells. The percentage of cytokine-positive cells in the Dynabead-only control samples was used as a 100% normal (immunocompetent) immune response standardized within each donor, while the stimulation conditions with Dynabeads and NECA represented an immunosuppressive control. The restoration of the cytokine response in the presence of the A2AR antagonist was calculated as follows using the average of at least two donors: 100 × (1 - (100 - % cytokine-positive cells in the presence of the A2AR antagonist) / (100 - % cytokine-positive cells in the immunocompetent control)).

[0241] The compound was tested in a translation assay involving the activation of healthy donor human T cells in vitro in the presence of the immunosuppressive concentration of the A2AR agonist NECA as described above. Activated human blood CD4 +Normal cytokine production and activated human blood CD8 in T cells + The ability of compounds to restore normal cytokine production in T cells is represented in Tables 4 and 5, respectively. Clinical-stage antagonist compounds are listed in Table 2.

[0242]

Table 4

[0243]

Table 5

[0244] Example 4: In vivo effects of the compounds of the present invention in a mouse tumor model To demonstrate the in vivo therapeutic activity of the A2AR antagonist, mouse xenograft MC38 or CT26 (colorectal cancer) tumor models were used. CT26 (FenicsBio) and MC38 (Creative-Biogene) mouse colon tumor cells expressing a bioluminescent luciferase construct (firefly luciferase under the control of the CMV promoter) were verified to be mycoplasma-free. Cells were cultured as recommended by the manufacturer and injected subcutaneously into immunocompetent mice (Balb / c for CT26 and C57BL / 6 for MC38) (200,000 - 1,000,000 cells, for example, between 250,000 - 500,000 tumor cells as shown). Tumor growth was monitored until the establishment of a sufficiently defined intermediate-sized tumor. Then, the mice were randomized and treated either by direct intratumoral injection or via systemic administration. Seven to fourteen days later, the mice were randomized into groups with an average tumor size of 50 mm 2 . Mice were treated at least once a day with the indicated route and compound dose. For combination therapy studies, mice were additionally treated intraperitoneally with an existing approved immunotherapy (in vivo grade anti-PD-1 or anti-CTLA ICI mAb, purchased from commercial suppliers) at the indicated doses and frequencies. Mice were monitored daily for adverse events, and tumor volume was measured by caliper or by whole-body mouse imaging using an injection of luciferin D followed by an IVIS Spectrum (Perkin Elmer) instrument. The compound of the invention of structure (Ib) mediated a significant decrease in tumor growth when compared to vehicle control treatment conducted under similar conditions.

Claims

1. Compound of formula (I): 【Chemistry 1】 [wherein, R １ is selected from one or more halogens, cyano, hydroxy or C １ -C ６ aryl which may be optionally substituted by alkyl, and one or more halogens, cyano, hydroxy or C １ -C ６ heteroaryl which may be optionally substituted by alkyl; R ２ is selected from H and halogen; R ３ is selected from H, CN and optionally substituted C １ -C ６ alkyl; R ４ is selected from XR(R') group, optionally substituted aryl, and optionally substituted heteroaryl (wherein, X is selected from O, S, N, and R and R' are independently optionally substituted C １ -C ６ alkyl, or XR R' together forms optionally substituted heterocyclic alkyl)], or a pharmaceutically acceptable salt thereof, a pharmaceutical composition for use in the treatment of cancer.

2. The compound is a cyanopyridine of formula (Ia): 【Chemistry 2】 [In the formula, R １ and R ２ is as defined in claim 1, where X is selected from O and S, and R ５ C may be replaced depending on the circumstances. １ -C ６ The pharmaceutical composition according to claim 1, wherein it is alkyl.

3. The compound is a cyanopyridine of formula (Ib): 【Transformation 3】 [In the formula, R １ and R ２ This is as defined in claim 1, and R ６ and R ７ C may be substituted independently and depending on the circumstances. １ -C ６ The pharmaceutical composition according to claim 1, wherein it is alkyl.

4. Compound of formula (I): 【Chemistry 4】 [In the formula, R1 to R4 are as defined in claim 1.] However, the compound of formula (I) is one of the following compounds: 4-(3-(1H-pyrrole-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-pyrrole-1-yl)phenyl)pyridine-3,5-dicarbonitrile (RN: 391664-25-8); 4-(3-(1H-pyrrole-1-yl)phenyl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile (RN: 391664-24-7); 4-(3-(1H-imidazole- 1-yl)phenyl)-2-amino-6-methoxypyridine-3,5-dicarbonitrile; 4-(3-(1H-imidazole-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: 1646173-50-3); 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);A compound other than 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), or a pharmaceutically acceptable salt thereof.

5. R １ However, it contains one or more halogens, cyano, hydroxy, or C １ -C ６ The pharmaceutical composition according to claim 1, wherein the aryl is optionally substituted with an alkyl group.

6. R １ However, phenyl, halogenophenyl, for example, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, or C １ -C ６ The pharmaceutical composition according to claim 1, wherein the alkylphenyl is selected from methylphenyls such as 2-methylphenyl, 3-methylphenyl, and 4-methylphenyl.

7. R １ However, it contains one or more halogens, cyano, hydroxy, or C １ -C ６ The pharmaceutical composition according to claim 1, wherein the heteroaryl is optionally substituted with an alkyl group.

8. R １ However, it is an optionally substituted heteroaryl selected from optionally substituted pyridinyl, optionally substituted thiophenyl, optionally substituted pyrazolyl, and optionally substituted tetrazolyl, where optionally substituted means one or more halogens, cyano, hydroxy, or C １ -C ６ The pharmaceutical composition according to claim 1, wherein substitution by alkyl is permitted.

9. R ２ The pharmaceutical composition according to claim 1, wherein is H.

10. R ３ The pharmaceutical composition according to claim 1, wherein is CN.

11. R ４ The pharmaceutical composition according to claim 1, wherein is an XR(R') group (wherein X, R, and R' are defined in any one of the preceding claims).

12. R ４ The pharmaceutical composition according to claim 1, wherein is an OR group (where R is an optionally substituted alkyl group).

13. R ４ The pharmaceutical composition according to claim 1, wherein is an SR group (where R is an optionally substituted alkyl group).

14. R ４ The pharmaceutical composition according to claim 1, wherein is selected from pyridine, pyrrolidine, and azetidine.

15. R ４ The pharmaceutical composition according to claim 1, wherein the pyridine may be substituted in some cases.

16. R ４ The pharmaceutical composition according to claim 1, wherein is an SR group (where R is an optionally substituted C1-C6 alkyl group).

17. The aforementioned compounds belong to the following group: 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(azetidine-1-yl)pyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(pyrrolidine-1-yl)pyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(2'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(2'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(3'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(4'-methyl-[1,1'-biphenyl]-3-yl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-6-(piperidine-1-yl)-4-(3-(pyridine-2-yl)phenyl)pyridine-3,5-dicarbonitric; 2-amino-6-(piperidine-1-yl)-4-(3-(pyridine-3-yl)phenyl)pyridine-3,5-dicarbonitric; 2-amino-6-(piperidine-1-yl)-4-(3-(pyridine-4-yl)phenyl)pyridine-3,5-dicarbonitric; 2-amino-6-(piperidine-1-yl)-4-(3-(thiophen-2-yl)phenyl)pyridine-3,5-dicarbonitric; 2-amino-6-(piperidine-1-yl)-4-(3-(thiophen-3-yl)phenyl)pyridine-3,5-dicarbonitric; 4-(3-(1H-pyrazole-5-yl)phenyl)-2-amino-6-(piperidine-1-yl)pyridine-3,5-dicarbonitrile; 4-(3-(1H-tetrazole-5-yl)phenyl)-2-amino-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(diethylamino)pyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-morpholinopyridine-3,5-dicarbonitride; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(bis(2-hydroxyethyl)amino)pyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(4-methylpiperazine-1-yl)pyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-((2-hydroxyethyl)amino)pyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(diisopropylamino)pyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(benzylamino)pyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(benzylthio)pyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-methoxypyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-ethoxypyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(2-hydroxyethoxy)pyridine-3,5-dicarbonitric; 4-([1,1'-biphenyl]-3-yl)-2-amino-6-(benzyloxy)pyridine-3,5-dicarbonitric; 2-amino-4-(3-(3-fluoropyridine-2-yl)phenyl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(3-(6-fluoropyridine-2-yl)phenyl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(3-(4-fluoropyridine-2-yl)phenyl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(3-(5-fluoropyridine-3-yl)phenyl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(3-(2-fluoropyridine-3-yl)phenyl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(2-fluoro-5-(pyridine-3-yl)phenyl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(2-fluoro-3-(pyridine-3-yl)phenyl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric; 2-amino-4-(3-fluoro-5-(pyridine-3-yl)phenyl)-6-(piperidine-1-yl)pyridine-3,5-dicarbonitrile, and 4-(3-(1,3,4-oxadiazole-2-yl)phenyl)-2-amino-6-(piperidine-1-yl)pyridine-3,5-dicarbonitric A pharmaceutical composition according to claim 1, selected from the following.

18. A pharmaceutical composition comprising at least one compound according to claim 4, and a pharmaceutically acceptable carrier, diluent, or excipient.

19. The pharmaceutical composition according to claim 1, to be used in combination with one or more treatments selected from radiotherapy, chemotherapy, adoptive cell therapy, anti-cancer vaccine therapy, targeted biological therapy, or immunomodulatory therapy.

20. The pharmaceutical composition according to claim 19, wherein the targeted biological therapy is based on a tumor-specific antibody, and the immunomodulatory therapy is based on an anti-cancer immunotherapy agent and / or an immune checkpoint inhibitor.

21. The pharmaceutical composition according to claim 1, wherein the cancer is selected from lung cancer, 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 preparing a compound of formula (I), the method comprising the step of reacting a cyanopyridine of formula (i) with a nucleophile compound of formula (ii) in an aprotic solvent in the presence of a base, as follows: 【Transformation 5】 A method comprising [wherein R1 to R4, X, R and R' are defined in any one of the preceding claims].

23. A method for preparing a compound of formula (I), the method comprising the step of cross-coupling intermediates of formulas (iii) and (iv) in the presence of a mineral base and a catalytic amount of palladium source, as follows: 【Transformation 6】 [In the formula, R １ , R ２ , R ３ and R ４ This is as defined in any one of the preceding claims, R ８ and R ９ A method comprising [a selected group from halogen, boronic acid, or pinacol boronic acid ester group].

24. A method for preparing a compound of formula (I) as defined in claim 1, wherein the method comprises: an aldehyde of formula (vi) in a polar protic solvent and a cyclic amine of formula (vii) (wherein R １ and R ２ The step of reacting in the presence of two equivalents of malononitrile (viiii) and an oxidizing agent (as described in any one of the preceding claims): 【Transformation 7】 [In the formula, R1 and R2 are as defined in claim 1, and R ６ and R ７ A method comprising: [together forming a heterocyclic alkyl which may be substituted in some cases].

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