Combination therapy containing A2A / A2B and PD-1 / PD-L1 inhibitors

Abstract

The present application provides a method for treating cancer using a combination of A2A and / or A2B inhibitors and PD-1 and / or PD-L1 inhibitors.

Description

Technical Field

[0001] Disclosed herein are combination therapies comprising an A2A / A2B inhibitor and a PD-1 / PD-L1 inhibitor, and methods of using the combination therapies to treat disorders (eg, cancer). Background Art

[0002] Some cancer patients have a poor long-term prognosis and / or are resistant to one or more types of treatment commonly used in the art. Therefore, there remains a need for effective cancer therapies with increased efficacy and improved safety profiles in this difficult-to-treat patient population. Summary of the invention

[0003] The present application provides, inter alia, a method of treating cancer in a subject, the method comprising administering to the subject:

[0004] (i) A2A / A2B inhibitors; and

[0005] (ii) PD-1 / PD-L1 inhibitors.

[0006] Other features, objects, and advantages of the invention will be apparent from the description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figures 1A-1C The synergistic effect of compound 9 with (1A) pembrolizumab, (1B) antibody X and (1C) compound Y in CHO-PD-L1 co-cultured with primary T cells is shown (see Example 1).

[0008] Figures 2A-2D The synergistic effect of compound 9 or compound 3A and atezolizumab was shown in PBMCs stimulated with CD3 antibody.

[0009] Figures 3A-3C Anti-tumor effects of compound 9 and anti-PD1 (clone 29F.1A12 against murine PD-1) are shown in preclinical CT26 and B16-F10 tumor models. (3A) Efficacy study of 10 mg / kg BID compound 9 as a single agent and in combination with anti-PD1 antibody in the CT26 syngeneic model. (3B) Efficacy study of 10 mg / kg BID compound 9 in the CT-26 NSG xenograft model. (3C) Efficacy study of 10 mg / kg BID compound 9 as a single agent and in combination with anti-PD-L1 antibody in the B16 syngeneic model. DETAILED DESCRIPTION

[0010] The present application provides a method for treating cancer in a subject, the method comprising administering to the subject:

[0011] (i) A2A / A2B inhibitors; and

[0012] (ii) PD-1 / PD-L1 inhibitors.

[0013] A2A / A2B inhibitors

[0014] Adenosine is an extracellular signaling molecule that can modulate immune responses in many immune cell types. Drury and Adenosine was first identified as a physiological regulator of coronary vascular tone (Sachdeva, S. and Gupta, M. Saudi Pharmaceutical Journal, 2013, 21, 245-253), however, it was not until 1970 that Sattin and Rall showed that adenosine regulates cellular function by occupying specific receptors on the cell surface (Sattin, A. and Rall, TW, 1970. Mol. Pharmacol. 6, 13-23; Hasko′, G. et al., 2007, Pharmacol. Ther. 113, 264-275).

[0015] Adenosine plays an important role in a variety of other physiological functions. It participates in the synthesis of nucleic acids when connected to three phosphate groups; it forms ATP, an integral component of the cell energy system. Adenosine can be produced by enzymatic decomposition of extracellular ATP, or it can also be released from damaged neurons and glial cells by passing through damaged plasma membranes (Tautenhahn, M. et al., Neuropharmacology, 2012, 62, 1756-1766). In the peripheral nervous system and in the central nervous system, adenosine produces a variety of pharmacological effects by acting on specific receptors located on the cell membrane (Matsumoto, T. et al., Pharmacol. Res., 2012, 65, 81-90). Alternative pathways for the production of extracellular adenosine have been described. These pathways include the production of adenosine from nicotinamide dinucleotide (NAD) rather than ATP through the synergistic action of CD38, CD203a and CD73. The CD73-independent production of adenosine can also be carried out by other phosphates (e.g., alkaline phosphatase or prostate-specific phosphatase).

[0016] There are four known subtypes of human adenosine receptors, including A1, A2A (ADORA2A), A2B (ADORA2B), and A3 receptors. A1 and A2A are high-affinity receptors, while A2B and A3 are low-affinity receptors. Adenosine and its agonists can act through one or more of these receptors and can regulate the activity of adenylate cyclase, which is responsible for increasing cyclic AMP (cAMP). Different receptors have different stimulatory and inhibitory effects on this enzyme. Increased intracellular cAMP concentrations can inhibit the activity of immune cells and inflammatory cells (Livingston, M. et al., Inflamm. Res., 2004, 53, 171-178).

[0017] A2A adenosine receptors can signal in the peripheral nervous system and CNS, with agonists used as anti-inflammatory drugs and antagonists used in neurodegenerative diseases (Carlsson, J. et al., J. Med. Chem., 2010, 53, 3748-3755). In most cell types, the A2A subtype suppresses intracellular calcium levels, while A2B enhances intracellular calcium levels. A2A receptors generally appear to suppress inflammatory responses of immune cells (Borrmann, T. et al., J. Med. Chem., 2009, 52 (13), 3994-4006).

[0018] A2B receptors are highly expressed in the gastrointestinal tract, bladder, lungs, and on mast cells (Antonioli, L. et al., Nature Reviews Cancer, 2013, 13, 842-857). Although the A2B receptor is closely related to the A2A receptor in structure and can activate adenylate cyclase, its function is different. It is assumed that this subtype can utilize signal transduction systems other than adenylate cyclase (Livingston, M. et al., Inflamm. Res., 2004, 53, 171-178). Among all adenosine receptors, the A2B adenosine receptor is considered a low-affinity receptor that is believed to remain silent under physiological conditions and to be activated by increased extracellular adenosine levels (Ryzhov, S. et al., Neoplasia, 2008, 10, 987-995). Activation of the A2B adenosine receptor can stimulate adenylate cyclase and phospholipase C by activating Gs and Gq proteins, respectively. Coupling to mitogen-activated protein kinases has also been described (Borrmann, T. et al., J. Med. Chem., 2009, 52(13), 3994-4006).

[0019] In the immune system, the involvement of adenosine signaling can be a key regulatory mechanism to protect tissues from excessive immune responses. Adenosine can negatively regulate immune responses through many immune cell types, including T cells, natural killer cells, macrophages, dendritic cells, mast cells, and myeloid-derived suppressor cells (Allard, B. et al., Current Opinion in Pharmacology, 2016, 29, 7-16).

[0020] In tumors, this pathway is hijacked by the tumor microenvironment and undermines the anti-tumor ability of the immune system, thereby promoting cancer progression. In the tumor microenvironment, adenosine is mainly produced from extracellular ATP by two ectonucleotidases, CD39 and CD73. A variety of cell types can produce adenosine by expressing CD39 and CD73. This is the case for tumor cells, T effector cells, T regulatory cells, tumor-associated macrophages, myeloid-derived suppressor cells (MDSC), endothelial cells, cancer-associated fibroblasts (CAFs), and mesenchymal stromal / stem cells (MSCs). In addition, hypoxia and inflammation, common conditions in the tumor microenvironment, induce the expression of CD39 and CD73, thereby increasing adenosine production. Therefore, adenosine levels in solid tumors are higher than normal physiological conditions.

[0021] A2A is primarily expressed on lymphoid-derived cells, including T effector cells, T regulatory cells, and natural killer (NK) cells. Blocking the A2A receptor prevents downstream immunosuppressive signals that temporarily inactivate T cells. The A2B receptor is primarily expressed on monocyte-derived cells, including dendritic cells, tumor-associated macrophages, myeloid-derived suppressor cells (MDSC), and mesenchymal stromal / stem cells (MSC). Blocking the A2B receptor in preclinical models inhibits tumor growth, blocks metastasis, and increases tumor antigen presentation.

[0022] In terms of the safety profile of ADORA2A / ADORA2B (A2A / A2B) blockade, both A2A and A2B receptor knockout (KO) mice are viable, indicating no growth abnormalities and are fertile (Allard, B. et al., Current Opinion in Pharmacology, 2016, 29, 7-16). A2A KO mice show increased levels of proinflammatory cytokines only after challenge with lipopolysaccharide (LPS) and no evidence of baseline inflammation (Antonioli, L. et al., Nature Reviews Cancer, 2013, 13, 842-857). A2B KO mice exhibit normal platelet, red blood cell and white blood cell counts, but increased baseline inflammation (e.g., TNF-α and IL-6) (Antonioli, L. et al., Nature Reviews Cancer, 2013, 13, 842-857). Further increases in the production of TNF-α and IL-6 were detected after LPS treatment. A2B KO mice also exhibit increased vascular adhesion molecules that mediate inflammation and leukocyte adhesion / rolling; enhanced mast cell activation; increased susceptibility to IgE-mediated allergic reactions, and increased vascular leakage and neutrophil influx under hypoxia (Antonioli, L. et al., Nature Reviews Cancer, 2013, 13, 842-857).

[0023] The adenosine pathway is a key immunosuppressive pathway that protects tissues from excessive immune responses (Antonioli, L. et al., Nature Review Cancer. 2013, 13, 842-857; Inflamm. Res. 2004, 53: 171-178; Allard et al., Current Opinion in Pharmacology 2016, 29: 7). The immunosuppressive activity of adenosine is mediated by two G protein-coupled receptors (GPCRs) called A2A and A2B; both receptors are found to be expressed on many immune cell types, including T cells, natural killer cells, macrophages, dendritic cells, mast cells, and myeloid-derived suppressor cells (Saudi Pharmaceutical Journal. 2013, 21: 245; Frontiers in Immunology. 2019, 10: 925; J Clin Invest. 2017, 127 (3): 929; Neoplasia. 2008, 10: 987; Neoplasia. 2013, 15: 1400). As a result of the high levels of adenosine production observed in the tumor microenvironment, it has been reported that the anti-tumor ability of the immune system is suppressed, thereby causing cancer progression.

[0024] In some embodiments, the A2A / A2B inhibitor is a compound selected from Table 1 or a pharmaceutically acceptable salt thereof.

[0025] Table 1.

[0026]

[0027]

[0028]

[0029]

[0030] In some embodiments, the A2A / A2B inhibitor is a compound of formula (I):

[0031]

[0032] or a pharmaceutically acceptable salt thereof, wherein

[0033] Cy 1 is phenyl substituted with 1 or 2 substituents independently selected from halo and CN;

[0034] Cy 2 is a 5-6 membered heteroaryl or a 4-7 membered heterocycloalkyl, wherein Cy 2 The 5-6 membered heteroaryl or 4-7 membered heterocycloalkyl of each is optionally substituted by 1, 2 or 3 groups each independently selected from the following: 1-3 Alkyl, C 1-3 Alkoxy, NH2, NH(C 1-3 Alkyl) and N(C 1-3 Alkyl)2;

[0035] R 2 Selected from phenyl-C 1-3 Alkyl-, C 3-7 Cycloalkyl-C 1-3 Alkyl-, (5-7 membered heteroaryl)-C 1-3 Alkyl-, (4-7 membered heterocycloalkyl)-C 1-3 Alkyl- and OR a2 , where R 2 Phenyl-C 1-3 Alkyl-, C 3-7 Cycloalkyl-C 1-3 Alkyl-, (5-7 membered heteroaryl)-C 1-3 Alkyl- and (4-7 membered heterocycloalkyl)-C 1-3 alkyl-each optionally substituted with 1, 2 or 3 independently selected R C Substituent substitution;

[0036] R a2 is (5-7 membered heteroaryl)-C 1-3 Alkyl-, optionally substituted with 1 or 2 independently selected R C Substituent substitution;

[0037] Each R C independently selected from halogen, C 1-6 Alkyl, C6 aryl, 5-7 membered heteroaryl, (4-7 membered heterocycloalkyl)-C 1-3 Alkyl-, OR a4 and NR c4 R d4 ;and

[0038] Each R a4 , R c4 and R d4 Independently selected from H and C 1-6 alkyl.

[0039] In some embodiments of the compound of Formula (I), Cy 2 It is a pyrimidinyl group.

[0040] In some embodiments of the compound of Formula (I), R 2 Selected from pyridin-2-ylmethyl, (2,6-difluorophenyl)(hydroxy)methyl, (5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl, (3-methylpyridin-2-yl)methoxy and (5-(1H-pyrazol-1-yl)-1H-tetrazol-1-yl)methyl.

[0041] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is 3-(5-amino-2-(pyridin-2-ylmethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 1, Table 1).

[0042] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is 3-(5-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 2, Table 1).

[0043] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is 3-(5-amino-2-((5-(pyridin-2-yl)-2H-tetrazol-2-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 3A, Table 1).

[0044] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 3B, Table 1).

[0045] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is 3-(5-amino-2-((3-methylpyridin-2-yl)methoxy)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 4, Table 1).

[0046] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is 3-(2-((5-(1H-pyrazol-1-yl)-2H-tetrazol-2-yl)methyl)-5-amino-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 21A, Table 1).

[0047] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is 3-(2-((5-(1H-pyrazol-1-yl)-1H-tetrazol-1-yl)methyl)-5-amino-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 21B, Table 1).

[0048] In some embodiments, the A2A / A2B inhibitor is selected from:

[0049] 3-(5-amino-2-(pyridin-2-ylmethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof;

[0050] 3-(5-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof;

[0051] 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof;

[0052] 3-(5-amino-2-((3-methylpyridin-2-yl)methoxy)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0053] 3-(2-((5-(1H-pyrazol-1-yl)-1H-tetrazol-1-yl)methyl)-5-amino-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof.

[0054] The synthesis and characterization of the compound of formula (I) can be found in WO2019 / 168847 and US62 / 891,685, both of which are incorporated herein by reference in their entirety.

[0055] In some embodiments, the A2A / A2B inhibitor is a compound of formula (II):

[0056]

[0057] or a pharmaceutically acceptable salt thereof, wherein

[0058] R 2 Selected from H and CN;

[0059] Cy 1 is phenyl substituted with 1 or 2 substituents independently selected from halo and CN;

[0060] L is C 1-3 wherein the alkylene group is optionally substituted with 1, 2 or 3 independently selected R 8D Substituent substitution;

[0061] Cy 4 is selected from phenyl, cyclohexyl, pyridyl, pyrrolidinone and imidazolyl, wherein phenyl, cyclohexyl, pyridyl, pyrrolidinone and imidazolyl are each optionally substituted by 1, 2 or 3 independently selected from R 8D and R 8 Substituents are substituted;

[0062] Each R 8 independently selected from halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, phenyl, C 3-7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C 1-3 Alkyl, C 3-7 Cycloalkyl-C 1-3 Alkyl, (5-6 membered heteroaryl)-C 1-3Alkyl and (4-7 membered heterocycloalkyl)-C 1-3 Alkyl, where R 8 C 1-6 Alkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, phenyl, C 3-7 Cycloalkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, phenyl-C 1-3 Alkyl, C 3-7 Cycloalkyl-C 1-3 Alkyl, (5-6 membered heteroaryl)-C 1-3 Alkyl and (4-7 membered heterocycloalkyl)-C 1-3 Each alkyl group is optionally substituted with 1, 2 or 3 independently selected R 8A Substituent substitution;

[0063] Each R 8A independently selected from halogen, C 1-6 Alkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, CN, OR a81 and NR c81 R d81 , where R 8A C 1-3 alkyl, 5-6 membered heteroaryl and 4-7 membered heterocycloalkyl are each optionally substituted with 1, 2 or 3 independently selected R 8B Substituent substitution;

[0064] Each R a81 , R c81 and R d81 Independently selected from H, C 1-6 Alkyl and 4-7 membered heterocycloalkyl, wherein R a81 , R c81 and R d81 C 1-6 The alkyl and 4-7 membered heterocycloalkyl groups are each optionally substituted with 1, 2 or 3 independently selected R 8B Substituent substitution;

[0065] Each R 8B independently selected from halo and C 1-3 Alkyl; and

[0066] Each R 8D independently selected from OH, CN, halogen, C 1-6 Alkyl and C 1-6 Halogenated alkyl.

[0067] In some embodiments, the compound of formula (II) or a pharmaceutically acceptable salt thereof is 3-(5-amino-2-(hydroxy(phenyl)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 5, Table 1).

[0068] In some embodiments, the compound of formula (II) or a pharmaceutically acceptable salt thereof is 3-(5-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile or a pharmaceutically acceptable salt thereof (see Compound 6, Table 1).

[0069] In some embodiments, the compound of formula (II) or a pharmaceutically acceptable salt thereof is 5-amino-7-(3-cyano-2-fluorophenyl)-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidine-8-carbonitrile or a pharmaceutically acceptable salt thereof (see Compound 7, Table 1).

[0070] In some embodiments, the compound of formula (II) or a pharmaceutically acceptable salt thereof is 3-(5-amino-2-((2-fluoro-6-(((1-methyl-2-oxopyrrolidin-3-yl)amino)methyl)phenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile or a pharmaceutically acceptable salt thereof (see Compound 8, Table 1).

[0071] The synthesis and characterization of the compound of formula (II) can be found in WO2019 / 222677, the entire text of which is incorporated herein by reference.

[0072] In some embodiments, the A2A / A2B inhibitor is a compound of formula (III):

[0073]

[0074] or a pharmaceutically acceptable salt thereof, wherein

[0075] Cy 1 is phenyl substituted with 1 or 2 substituents independently selected from halo and CN;

[0076] R 2 is selected from 5-6 membered heteroaryl and 4-7 membered heterocycloalkyl, wherein R 2 The 5-6 membered heteroaryl and 4-7 membered heterocycloalkyl are each optionally substituted by 1, 2 or 3 independently selected R 2A Substituent substitution;

[0077] Each R 2A are independently selected from D, halogen, C1-6 Alkyl and C 1-6 Haloalkyl;

[0078] R 4 Selected from phenyl-C 1-3 Alkyl-, C 3-7 Cycloalkyl-C 1-3 Alkyl-, (5-6 membered heteroaryl)-C 1-3 Alkyl- and (4-7 membered heterocycloalkyl)-C 1-3 Alkyl, where R 4 Phenyl-C 1-3 Alkyl-, C 3-7 Cycloalkyl-C 1-3 Alkyl-, (5-6 membered heteroaryl)-C 1-3 Alkyl- and (4-7 membered heterocycloalkyl)-C 1-3 alkyl-each optionally substituted with 1, 2 or 3 independently selected R 4A Substituent substitution;

[0079] Each R 4A independently selected from halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, CN, OR a41 and NR c41 R d41 ;and

[0080] Each R a41 , R c41 and R d41 Independently selected from H and C 1-6 alkyl.

[0081] In some embodiments, the compound of formula (III) or a pharmaceutically acceptable salt thereof is 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 9, Table 1).

[0082] In some embodiments, the compound of formula (III) or a pharmaceutically acceptable salt thereof is 3-(8-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-5-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 10, Table 1).

[0083] In some embodiments, the compound of formula (III) or a pharmaceutically acceptable salt thereof is 3-(8-amino-2-(amino(2,6-difluorophenyl)methyl)-5-(4-methyloxazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 11, Table 1).

[0084] In some embodiments, the compound of formula (III) or a pharmaceutically acceptable salt thereof is 3-(8-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-5-(2,6-dimethylpyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 12, Table 1).

[0085] The synthesis and characterization of the compound of formula (III) can be found in PCT / US2019 / 040496, the entire text of which is incorporated herein by reference.

[0086] In some embodiments, the A2A / A2B inhibitor is a compound of formula (IV):

[0087]

[0088] or a pharmaceutically acceptable salt thereof, wherein

[0089] Cy 1 is phenyl substituted with 1 or 2 substituents independently selected from halo and CN;

[0090] Cy 2 is selected from 5-6 membered heteroaryl and 4-7 membered heterocycloalkyl, wherein Cy 2 The 5-6 membered heteroaryl and 4-7 membered heterocycloalkyl are each optionally substituted by 1, 2 or 3 independently selected R 6 Substituent substitution;

[0091] Each R 6 independently selected from halogen, C 1-6 Alkyl and C 1-6 Haloalkyl;

[0092] R 2 It is phenyl-C 1-3 Alkyl- or (5-6 membered heteroaryl)-C 1-3 Alkyl-, where R 2 Phenyl-C 1-3 Alkyl- and (5-6 membered heteroaryl)-C 1-3 alkyl-each optionally substituted with 1, 2 or 3 independently selected R 2A Substituents are substituted; and

[0093] Each R2A independently selected from halogen, C 1-6 Alkyl and C 1-6 Halogenated alkyl.

[0094] In some embodiments, the compound of formula (IV) or a pharmaceutically acceptable salt thereof is 3-(4-amino-2-(pyridin-2-ylmethyl)-7-(pyrimidin-4-yl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 13, Table 1).

[0095] In some embodiments, the compound of formula (IV) or a pharmaceutically acceptable salt thereof is 3-(4-amino-2-((3-fluoropyridin-2-yl)methyl)-7-(pyrimidin-4-yl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 14, Table 1).

[0096] In some embodiments, the compound of formula (IV) or a pharmaceutically acceptable salt thereof is 3-(4-amino-2-((3-fluoropyridin-2-yl)methyl)-7-(pyridin-4-yl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof (see Compound 15, Table 1).

[0097] In some embodiments, the compound of formula (IV) or a pharmaceutically acceptable salt thereof is 3-(4-amino-7-(1-methyl-1H-pyrazol-5-yl)-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)-2-fluorobenzonitrile or a pharmaceutically acceptable salt thereof (see Compound 16, Table 1).

[0098] The synthesis and characterization of the compound of formula (IV) can be found in US 62 / 798,180, which is incorporated herein by reference in its entirety.

[0099] In some embodiments, the A2A / A2B inhibitor is a compound of formula (V):

[0100]

[0101] or a pharmaceutically acceptable salt thereof, wherein

[0102] R 2 Selected from H, D, halogen, C 1-6 Alkyl and C 1-6 Haloalkyl;

[0103] R 3 Selected from H and C 1-6 alkyl;

[0104] R 4 Selected from H and C 1-6 alkyl;

[0105] R 5 Selected from H, halogen, CN, C 1-6 alkyl;

[0106] R 6 Selected from phenyl, C 3-7 Cycloalkyl, 5-7 membered heteroaryl and 4-7 membered heterocycloalkyl, wherein R 6 The phenyl group, the C 3-7 The cycloalkyl, the 5-7 membered heteroaryl and the 4-7 membered heterocycloalkyl are optionally substituted with 1, 2 or 3 independently selected R A Substituent substitution;

[0107] Each R A are independently selected from (5-10 membered heteroaryl)-C 1-3 Alkyl- and (4-10 membered heterocycloalkyl)-C 1-3 Alkyl-, where R A (5-10 membered heteroaryl)-C 1-3 Alkyl- and (4-10 membered heterocycloalkyl)-C 1-3 Alkyl - each optionally substituted with 1 or 2 independently selected R B Substituent substitution;

[0108] Each R B independently selected from halogen, C 1-6 Alkyl and C(O)R b26 ;

[0109] R b26 Independently selected from H and C 1-3 Alkyl, where R b26 C 1-3 The alkyl group is optionally substituted with 1 or 2 independently selected R C Substituent substitution;

[0110] Each R C independently selected from halogen, C 1-6 Alkyl, CN, OR a36 and NR c36 R d36 ;and

[0111] Each R a36 , R c36 and R d36 Independently selected from H and C 1-6 alkyl.

[0112] In some embodiments, the compound of formula (V) or a pharmaceutically acceptable salt thereof is 7-(1-((5-chloropyridin-3-yl)methyl)-1H-pyrazol-4-yl)-3-methyl-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one or a pharmaceutically acceptable salt thereof (see Compound 17, Table 1).

[0113] In some embodiments, the compound of formula (V) or a pharmaceutically acceptable salt thereof is 3-methyl-7-(1-((5-methylpyridin-3-yl)methyl)-1H-pyrazol-4-yl)-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one or a pharmaceutically acceptable salt thereof (see Compound 18, Table 1).

[0114] In some embodiments, the compound of formula (V) or a pharmaceutically acceptable salt thereof is 3-methyl-9-pentyl-7-(1-(thieno[3,2-b]pyridin-6-ylmethyl)-1H-pyrazol-4-yl)-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one or a pharmaceutically acceptable salt thereof (see Compound 19, Table 1).

[0115] In some embodiments, the compound of formula (V) or a pharmaceutically acceptable salt thereof is 7-(1-((2-(2-(dimethylamino)acetyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazol-4-yl)-3-methyl-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one or a pharmaceutically acceptable salt thereof (see Compound 20, Table 1).

[0116] The synthesis and characterization of the compound of formula (V) can be found in US-2019-0337957, the entire text of which is incorporated herein by reference.

[0117] PD-1 / PD-L1 inhibitors

[0118] The immune system plays an important role in controlling and eliminating diseases (such as cancer). However, cancer cells usually produce strategies to escape or suppress the immune system to facilitate their growth. One such mechanism is to change the expression of co-stimulatory and co-inhibitory molecules expressed on immune cells (Postow et al., J.Clinical Oncology 2015, 1-9). Blocking the signaling of inhibitory immune checkpoints (such as PD-1) has been shown to be a promising and effective treatment.

[0119] Programmed death-1 ("PD-1", also known as "CD279") is an approximately 31 kD type I membrane protein member of the extended CD28 / CTLA-4 family of T cell regulators that broadly negatively regulates immune responses (Ishida, Y. et al. (1992) EMBO J. 11:3887-3895; U.S. Patent Publication Nos. 2007 / 0202100; 2008 / 0311117; and 2009 / 00110667; U.S. Patent Nos. 6,808,710; 7,101,550; 7,488,802; 7,635,757; and 7,722,868; PCT Publication No. WO 01 / 14557).

[0120] PD-1 is expressed on activated T cells, B cells and monocytes (Agata, Y. et al. (1996) Int. Immunol. 8(5):765-772; Yamazaki, T. et al. (2002) J. Immunol. 169:5538-5545), and is expressed at low levels in natural killer (NK) T cells (Nishimura, H. et al. (2000) J. Exp. Med. 191:891-898; Martin-Orozco, N. et al. (2007) Semin. Cancer Biol. 17(4):288-298).

[0121] The extracellular region of PD-1 consists of a single immunoglobulin (Ig) V domain with 23% identity to the equivalent domain in CTLA-4 (Martin-Orozco, N. et al. (2007) Semin. Cancer Biol. 17 (4): 288-298). The extracellular IgV domain is followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based conversion motif, indicating that PD-1 negatively regulates TCR signals (Ishida, Y. et al. (1992) EMBO J. 11: 3887-3895; Blank, C. et al. (2006) Immunol. Immunother. 56 (5): 739-745).

[0122] PD-1 mediates suppression of the immune system by binding to B7-H1 and B7-DC (Flies, DB et al. (2007) J. Immunother. 30(3):251-260; U.S. Pat. Nos. 6,803,192; 7,794,710; U.S. Patent Application Publication Nos. 2005 / 0059051; 2009 / 0055944; 2009 / 0274666; 2009 / 0313687; PCT Publication Nos. WO 01 / 39722; WO 02 / 086083).

[0123] The amino acid sequence of human PD-1 protein (Genbank accession number NP_005009) is: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAIS LAPKAQIKESLRAELRVTERRAEVPTAHSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ ID NO:1).

[0124] PD-1 has two ligands, PD-L1 and PD-L2 (Parry et al., Mol Cell Biol 2005, 9543-9553; Latchman et al., Nat Immunol 2001, 2, 261-268), and their expression patterns are different. PD-L1 protein is upregulated in macrophages and dendritic cells in response to lipopolysaccharide and GM-CSF treatment, and is upregulated in T cells and B cells after T cell receptor and B cell receptor signaling. PD-L1 is also highly expressed on almost all tumor cells, and expression is further increased after IFN-γ treatment (Iwai et al., PNAS 2002, 99 (19): 12293-7; Blank et al., Cancer Res 2004, 64 (3): 1140-5). Indeed, tumor PD-L1 expression status has been shown to be prognostic in multiple tumor types (Wang et al., Eur J Surg Oncol 2015; Huang et al., Oncol Rep 2015; Sabatier et al., Oncotarget 2015, 6(7):5449-5464). In contrast, PD-L2 expression is more restricted and is primarily expressed by dendritic cells (Nakae et al., J Immunol 2006, 177:566-73). PD-1 binds to its ligands PD-L1 and PD-L2 on T cells to deliver signals that inhibit IL-2 and IFN-γ production and cell proliferation induced after T cell receptor activation (Carter et al., Eur J Immunol 2002, 32(3):634-43; Freeman et al., J Exp Med 2000, 192(7):1027-34). The mechanism involves recruiting SHP-2 or SHP-1 phosphatases to inhibit T cell receptor signaling, such as Syk and Lck phosphorylation (Sharpe et al., Nat Immunol 2007, 8, 239-245). Activation of the PD-1 signaling axis also attenuates PKC-θ activation loop phosphorylation, which is required for activation of the NF-κB and AP1 pathways and cytokine production (e.g., IL-2, IFN-γ, and TNF) (Sharpe et al., Nat Immunol 2007, 8, 239-245; Carter et al., Eur J Immunol 2002, 32(3): 634-43; Freeman et al., J Exp Med 2000, 192(7): 1027-34).

[0125] Several lines of evidence from preclinical animal studies indicate that PD-1 and its ligands negatively regulate immune responses. PD-1-deficient mice have been shown to suffer from lupus-like glomerulonephritis and dilated cardiomyopathy (Nishimura et al., Immunity 1999, 11: 141-151; Nishimura et al., Science 2001, 291: 319-322). Using the LCMV model of chronic infection, it has been shown that PD-1 / PD-L1 interactions inhibit the activation, expansion and acquisition of effector functions of virus-specific CD8 T cells (Barber et al., Nature 2006, 439, 682-7). In short, these data support the development of therapeutic methods for blocking PD-1-mediated inhibitory signaling cascades to strengthen or "rescue" T cell responses. Therefore, new methods for blocking PD-1 / PD-L1 protein / protein interactions, thereby treating cancer in subjects, are needed.

[0126] In some embodiments, the PD-1 / PD-L1 inhibitor is a compound selected from the group consisting of nivolumab, BMS-936558, MDX1106 or MK-34775), pembrolizumab ( MK-3475, SCH-900475, lambrolizumab (CAS Reg. No. 1374853-91-4), atezolizumab ( CAS registration number 1380723-44-3), durvalumab, avelumab, ), cemiplimab, AMP-224, AMP-514 / MEDI-0680, atezolizumab, avelumab, BGB-A317, BMS936559, durvalumab, JTX-4014, SHR-1210, pidilizumab (CT-011), REGN2810, BGB-108, BGB-A317, SHR-1210 (HR-3012 10, SHR1210 or SHR-1210), BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and one or more PD-1 / PD-L1 blockers described in the following patents: U.S. Patent Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, or WO 8 ... No. 03042402, No. WO 2008 / 156712, No. WO 2010 / 089411, No. WO 2010 / 036959, No. WO2011 / 066342, No. WO 2011 / 159877, No. WO 2011 / 082400, No. WO No. 2011 / 161699, No. WO2017 / 070089, No. WO 2017 / 087777, No. WO 2017 / 106634, No. WO 2017 / 112730, No. WO2017 / 192961, No. WO 2017 / 205464, No. WO No. 2017 / 222976, WO 2018 / 013789, WO 2018 / 04478, WO 2018 / 119236, WO 2018 / 119266, WO 2018 / 119221, WO 2018 / 119286, WO 2018 / 119263, WO 2018 / 119224, WO 2019 / 191707 and WO 2019 / 217821, and any combination thereof. The disclosures of each of the foregoing patents, applications, and publications are incorporated herein by reference in their entirety.

[0127] In some embodiments, the PD-1 / PD-L1 inhibitor is selected from compounds disclosed in WO 2018 / 119266, for example

[0128] (S)-1-((7-chloro-2-(2'-chloro-3'-(5-(((2-hydroxyethyl)amino)methyl)picolinamido)-2-methyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl)methyl)piperidine-2-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0129] (S)-1-((7-chloro-2-(3'-(7-chloro-5-(((S)-3-hydroxypyrrolidin-1-yl)methyl)benzo[d]oxazol-2-yl)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0130] (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0131] (S)-1-((2-(2'-chloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-2-methylbiphenyl-3-yl)-7-cyanobenzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0132] (R)-1-((7-cyano-2-(2,2'-dimethyl-3'-(4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)biphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0133] (R)-1-((7-cyano-2-(3'-(5-(2-(dimethylamino)acetyl)-5,6-dihydro-4H-pyrrolo[3,4-d]thiazol-2-yl)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof; and

[0134] 1-((7-cyano-2-(3'-(5-(2-(dimethylamino)acetyl)-5,6-dihydro-4H-pyrrolo[3,4-d]thiazol-2-yl)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)piperidine-4-carboxylic acid or a pharmaceutically acceptable salt thereof.

[0135] In some embodiments, the PD-1 / PD-L1 inhibitor is (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof.

[0136] (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof is also referred to herein as Compound Y. The synthesis and characterization of Compound Y are disclosed in WO 2018 / 119266, which is incorporated herein by reference in its entirety.

[0137] In some embodiments, the PD-1 / PD-L1 inhibitor is selected from:

[0138] (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid hydrobromide;

[0139] (R)-1-((7-cyano-2-(3′-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2′-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid oxalate;

[0140] (R)-1-((7-cyano-2-(3′-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2′-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid hydrochloride;

[0141] (R)-1-((7-cyano-2-(3′-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2′-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid L-tartrate;

[0142] (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid malonate; and

[0143] (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid phosphate.

[0144] In some embodiments, the PD-1 / PD-L1 inhibitor is selected from the compounds disclosed in WO 2018 / 119224, for example

[0145] (S)-1-((2-(2'-chloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-2-methylbiphenyl-3-yl)-7-cyanobenzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0146] (R)-1-((2-(2'-chloro-3'-(6-isopropyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-2-yl)-2-methylbiphenyl-3-yl)-7-cyanobenzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0147] (S)-N-(2-chloro-3'-(5-(2-hydroxypropyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-2'-methylbiphenyl-3-yl)-5-isopropyl-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide or a pharmaceutically acceptable salt thereof;

[0148] cis-4-((2-((2,2'-dichloro-3'-(1-methyl-5-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)methyl)cyclohexane-1-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0149] trans-4-(2-(2-((2,2'-dichloro-3'-(5-(2-hydroxyethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)cyclohexane-1-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0150] trans-4-(2-(2-((2-chloro-2'-methyl-3'-(1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)cyclohexane-1-carboxylic acid or a pharmaceutically acceptable salt thereof; and

[0151] cis-4-((2-(2-chloro-3'-(5-(2-(ethyl(methyl)amino)acetyl)-5,6-dihydro-4H-pyrrolo[3,4-d]thiazol-2-yl)-2'-methylbiphenyl-3-ylcarbamoyl)-1-methyl-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methyl)cyclohexane-1-carboxylic acid or a pharmaceutically acceptable salt thereof.

[0152] In some embodiments, the PD-1 / PD-L1 inhibitor is selected from the compounds disclosed in WO 2019 / 191707, for example

[0153] (R)-1-((7-cyano-2-(3'-(7-((3-hydroxypyrrolidin-1-yl)methyl)-2-methylpyrido[3,2-d]pyrimidin-4-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)piperidine-4-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0154] (R)-1-((7-cyano-2-(3'-(7-(((S)-1-hydroxypropan-2-ylamino)methyl)-2-methylpyrido[3,2-d]pyrimidin-4-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0155] (R)-1-((7-cyano-2-(3'-(2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3,2-d]pyrimidin-4-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)piperidine-4-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0156] (R)-1-((7-cyano-2-(3'-(2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3,2-d]pyrimidin-4-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)-N,N-dimethylpiperidine-4-carboxamide or a pharmaceutically acceptable salt thereof;

[0157] (R)-1-((7-cyano-2-(3'-(2-cyclopropyl-7-(((R)-3-hydroxypyrrolidin-1-yl)methyl)pyrido[3,2-d]pyrimidin-4-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof; and

[0158] (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-6-methyl-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof.

[0159] In some embodiments, the PD-1 / PD-L1 inhibitor is selected from the compounds disclosed in WO 2019 / 217821, for example

[0160] 4-(2-(2-((2,2'-dichloro-3'-(1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0161] 4-(2-(2-((3'-(5-((1H-pyrazol-3-yl)methyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0162] (R)-4-(2-(2-((2,2'-dichloro-3'-(5-(2-hydroxypropyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0163] 4,4'-(((((2,2'-dichloro-[1,1'-biphenyl]-3,3'-diyl)bis(azanediyl))bis(carbonyl))bis(1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-2,5-diyl))bis(ethane-2,1-diyl))bis(bicyclo[2.2.1]heptane-1-carboxylic acid) or a pharmaceutically acceptable salt thereof;

[0164] 4-(2-(2-((2-chloro-2'-methyl-3'-(1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid or a pharmaceutically acceptable salt thereof;

[0165] 4-(2-(2-((2,2'-dimethyl-3'-(1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid or a pharmaceutically acceptable salt thereof; and

[0166] 4-(2-(2-((3'-(5-(trans-4-carboxy-4-methylcyclohexyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid or a pharmaceutically acceptable salt thereof.

[0167] In some embodiments, the PD-1 / PD-L1 inhibitor is pembrolizumab.

[0168] In some embodiments, the PD-1 / PD-L1 inhibitor is nivolumab.

[0169] In some embodiments, the PD-1 / PD-L1 inhibitor is atezolizumab.

[0170] In some embodiments, the PD-1 / PD-L1 inhibitor is antibody X. Antibody X as used herein is a humanized IgG4 monoclonal antibody that binds to human PD-1. See hPD-1mAb 7 (1.2) in WO2017019846, which is incorporated herein by reference in its entirety. The amino acid sequences of the mature heavy and light chains of antibody X are shown below. The complementary determining regions (CDRs) 1, 2, and 3 of the variable heavy chain (VH) domain and the variable light chain (VL) domain are shown in the order from the N-terminus to the C-terminus of the mature VL and VH sequences, underlined and bolded. The antibody consisting of the mature heavy chain (SEQ ID NO: 2) and the mature light chain (SEQ ID NO: 3) listed below is referred to as antibody X.

[0171] Mature antibody X heavy chain (HC)

[0172]

[0173]

[0174] Mature antibody X light chain (LC)

[0175]

[0176] The variable heavy chain (VH) domain of Antibody X has the following amino acid sequence:

[0177]

[0178] The variable light chain (VL) domain of Antibody X has the following amino acid sequence:

[0179]

[0180] The amino acid sequence of the VH CDR of Antibody X is listed below:

[0181] VH CDR1: SYWMN (SEQ ID NO: 6);

[0182] VH CDR2:VIHPSDSETWLDQKFKD(SEQ ID NO:7);

[0183] VH CDR3:EHYGTSPFAY(SEQ ID NO:8)

[0184] The amino acid sequence of the VL CDR of Antibody X is listed below:

[0185] VL CDR1:RASESVDNYGMSFMNW(SEQ ID NO:9);

[0186] VL CDR2: AASNQGS (SEQ ID NO: 10); and

[0187] VL CDR3: QQSKEVPYT (SEQ ID NO: 11).

[0188] As used herein, "QD" means a dose administered to a subject once a day. "QOD" means a dose administered to a subject once every other day. "QW" means a dose administered to a subject once a week. "Q2W" means a dose administered to a subject once every other week. "Q3W" means a dose administered to a subject once every three weeks. "Q4W" means a dose administered to a subject once every four weeks.

[0189] As used herein, "about" is intended to encompass variations of ±10% when referring to a measurable value (e.g., an amount, dosage, length of time, etc.). In certain embodiments, "about" may include variations of ±5%, ±1%, or ±0.1% from the specified value, and any variations therebetween, as such variations are suitable for practicing the disclosed methods.

[0190] In some embodiments, the compounds disclosed herein are the (S)-enantiomers of the compounds or pharmaceutically acceptable salts thereof. In some embodiments, the compounds are the (R)-enantiomers of the compounds or pharmaceutically acceptable salts thereof.

[0191] It should be further understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination.

[0192] The term "n-membered" (where n is an integer) generally describes the number of ring-forming atoms in a moiety where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridinyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl.

[0193] As used herein, the phrase "optionally substituted" means unsubstituted or substituted. Substituents are independently selected, and substitutions may be at any chemically accessible position. As used herein, the term "substituted" means that a hydrogen atom is removed and replaced with a substituent. A single divalent substituent (e.g., oxo) may replace two hydrogen atoms. It is understood that substitution at a given atom is limited by valence.

[0194] As used herein, the phrase "each 'variable' is independently selected from" means substantially the same as "at each occurrence the 'variable' is selected from".

[0195] Throughout this definition, the term “C n-m " indicates a range including the endpoint, where n and m are integers and indicate the number of carbons. Examples include C 1-3 , C 1-4 , C 1-6 wait.

[0196] As used herein, the term "C n-m "Alkyl" refers to a saturated hydrocarbon group having n to m carbons, which may be straight or branched. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl (Me), ethyl (Et), n-propyl (n-Pr), isopropyl (iPr), n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologues, for example, 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0197] As used herein, the term "C n-m "Alkoxy" refers to a radical of the formula -O-alkyl, wherein the alkyl radical has n to m carbons. Example alkoxy radicals include, but are not limited to, methoxy, ethoxy, propoxy (eg, n-propoxy and isopropoxy), butoxy (eg, n-butoxy and tert-butoxy), and the like.

[0198] As used herein, the term "aryl", used alone or in combination with other terms, refers to an aromatic hydrocarbon group which may be monocyclic or polycyclic (eg, having 2, 3, or 4 fused rings). n-m "Aryl" refers to an aromatic radical having n to m ring carbon atoms. Aryl includes, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, the aryl radical has 5 to 10 carbon atoms. In some embodiments, the aryl radical is phenyl or naphthyl.

[0199] In some embodiments, aryl is phenyl (ie, C6 aryl).

[0200] As used herein, "halo" or "halogen" refers to F, Cl, Br, or I. In some embodiments, halo is F, Cl, or Br. In some embodiments, halo is F or Cl.

[0201] In some embodiments, halo is F. In some embodiments, halo is Cl.

[0202] As used herein, the term "C n-m"Haloalkyl" refers to an alkyl group having from 1 to 2s+1 halogen atoms, which may be the same or different, wherein "s" is the number of carbon atoms in the alkyl group, wherein the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is only fluorinated. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Example haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCl3, CHCl2, C2Cl5, and the like.

[0203] As used herein, "cycloalkyl" refers to non-aromatic cyclic hydrocarbons, including cyclized alkyl and alkenyl. Cycloalkyl may include monocyclic or polycyclic (e.g., having 2 fused rings) groups, spirocyclic rings, and bridged rings (e.g., bridged bicycloalkyl). The ring-forming carbon atoms of the cycloalkyl may be optionally substituted with oxo or sulfide (e.g., C(O) or C(S)). The definition of cycloalkyl also includes parts having one or more aromatic rings fused to the cycloalkyl ring (i.e., having a common bond), such as benzo or thienyl derivatives of cyclopentane, cyclohexane, etc. Cycloalkyl containing fused aromatic rings may be connected by any ring-forming atom (including the ring-forming atom of the fused aromatic ring). Cycloalkyl may have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbons (i.e., C 3-10 In some embodiments, the cycloalkyl group is C 3-10 In some embodiments, the cycloalkyl group is C 3-7 In some embodiments, the cycloalkyl group is C 4-7 In some embodiments, the cycloalkyl group is C 4-10 Spirocyclic or bridged cycloalkyl (e.g., bridged bicycloalkyl). Example cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cyclohexatrienyl, norbornyl, norpinyl, norcarbyl, cubane, adamantane, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[3.1.1]heptyl, bicyclo[2.2.2]octyl, spiro[3.3]heptyl, etc. In some embodiments, the cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0204] As used herein, "heteroaryl" refers to a monocyclic or polycyclic (e.g., having 2 fused rings) aromatic heterocycle having at least one heteroatom ring member selected from N, O, S, and B. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from N, O, S, and B. In some embodiments, any ring-forming N in the heteroaryl moiety may be an N-oxide. In some embodiments, the heteroaryl is a 5-10-membered monocyclic or bicyclic heteroaryl having 1, 2, 3, or 4 heteroatom ring members independently selected from N, O, S, and B. In some embodiments, the heteroaryl is a 5-10-membered monocyclic or bicyclic heteroaryl having 1, 2, 3, or 4 heteroatom ring members independently selected from N, O, and S. In some embodiments, the heteroaryl is a 5-6 monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from N, O, S, and B. In some embodiments, heteroaryl is a 5-6 monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from N, O and S. In some embodiments, heteroaryl contains 3 to 10, 4 to 10, 5 to 10, 5 to 7, 3 to 7, or 5 to 6 ring-forming atoms. In some embodiments, heteroaryl has 1 to 4 ring-forming heteroatoms, 1 to 3 ring-forming heteroatoms, 1 to 2 ring-forming heteroatoms, or 1 ring-forming heteroatom. When heteroaryl contains more than one heteroatom ring member, the heteroatoms may be the same or different. Example heteroaryl groups include, but are not limited to, thienyl (thiophenyl), furyl (furyl), pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, 1,3,4-oxadiazolyl, and 1,2-dihydro-1,2-azaborinyl. rine), pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, oxazolyl, triazolyl, thiadiazolyl, quinolyl, isoquinolyl, indolyl, benzothiophenyl, benzofuranyl, benzisoxazolyl, imidazo[1,2-b]thiazolyl, purinyl, triazine, thieno[3,2-b]pyridinyl, imidazo[1,2-a]pyridinyl, 1,5-naphthyridinyl, 1H-pyrazolo[4,3-b]pyridinyl, triazolo[4,3-a]pyridinyl, 1H-pyrrolo[3,2-b]pyridinyl, 1H-pyrrolo[2,3-b]pyridinyl, pyrazolo[1,5-a]pyridinyl, indazolyl, and the like.

[0205] As used herein, "heterocycloalkyl" refers to a monocyclic or polycyclic heterocycle having at least one non-aromatic ring (saturated or partially unsaturated ring), wherein one or more ring-forming carbon atoms of the heterocycloalkyl are replaced by heteroatoms selected from N, O, S and B, and wherein the ring-forming carbon atoms and heteroatoms of the heterocycloalkyl may be optionally substituted by one or more oxo or thio groups (e.g., C(O), S(O), C(S) or S(O)2, etc.). When the ring-forming carbon atoms or heteroatoms of the heterocycloalkyl are optionally substituted by one or more oxo or thio groups, the O or S of the group is not included in the number of ring atoms specified herein (e.g., 1-methyl-6-oxo-1,6-dihydropyridazin-3-yl is a 6-membered heterocycloalkyl, wherein the ring-forming carbon atoms are substituted by oxo, and wherein the 6-membered heterocycloalkyl is further substituted by methyl). Heterocycloalkyl includes monocyclic and polycyclic (e.g., having 2 fused rings) systems. Monocyclic and polycyclic 3 to 10 yuan, 4 to 10 yuan, 5 to 10 yuan, 4 to 7 yuan, 5 to 7 yuan or 5 to 6 yuan heterocycloalkyl are included in heterocycloalkyl.Heterocycloalkyl can also include spiro ring and bridged ring (e.g., 5 to 10 yuan bridged diheterocycloalkyl rings replaced by heteroatoms independently selected from N, O, S and B with one or more ring carbon atoms).Heterocycloalkyl can be connected by ring carbon atoms or ring heteroatoms.In some embodiments, heterocycloalkyl contains 0 to 3 double bonds.In some embodiments, heterocycloalkyl contains 0 to 2 double bonds.

[0206] Also included in the definition of heterocycloalkyl are moieties having one or more aromatic rings fused to (i.e., having a common bond with) a non-aromatic heterocycle, such as piperidine, morpholine, aza The heterocycloalkyl group containing a fused aromatic ring may be attached via any ring-forming atom (including the ring-forming atom of the fused aromatic ring).

[0207] In some embodiments, heterocycloalkyl contains 3 to 10 ring atoms, 4 to 10 ring atoms, 3 to 7 ring atoms, or 5 to 6 ring atoms. In some embodiments, heterocycloalkyl has 1 to 4 heteroatoms, 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom. In some embodiments, heterocycloalkyl is a monocyclic 4-6 membered heterocycloalkyl having 1 or 2 heteroatoms independently selected from N, O, S, and B and having one or more oxidized ring members. In some embodiments, heterocycloalkyl is a monocyclic or bicyclic 5-10 membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from N, O, S, and B and having one or more oxidized ring members. In some embodiments, heterocycloalkyl is a monocyclic or bicyclic 5 to 10 membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S and having one or more oxidized ring members. In some embodiments, heterocycloalkyl is a monocyclic 5- to 6-membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S and having one or more oxygenated ring members.

[0208] Example heterocycloalkyl groups include pyrrolidin-2-one (or 2-oxopyrrolidinyl), 1,3-isoxazolidin-2-one, pyranyl, tetrahydropyran, oxetanyl, azetidinyl, morpholinyl, thiomorpholinyl, piperidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, 1,2,3,4-tetrahydroisoquinoline, Benzazacycloheptenyl, azabicyclo[3.1.0]hexyl, diazabicyclo[3.1.0]hexyl, oxobicyclo[2.1.1]hexyl, azabicyclo[2.2.1]heptyl, diazabicyclo[2.2.1]heptyl, azabicyclo[3.1.1]heptyl, diazabicyclo[3.1.1]heptyl, azabicyclo[3.2.1]octyl, diazabicyclo[3.2.1]octyl, oxobicyclo[2.2. 2]octyl, azabicyclo[2.2.2]octyl, azaadamantyl, diazaadamantyl, oxo-adamantyl, azaspiro[3.3]heptyl, diazaspiro[3.3]heptyl, oxo-azaspiro[3.3]heptyl, azaspiro[3.4]octyl, diazaspiro[3.4]octyl, oxo-azaspiro[3.4]octyl, azaspiro[2.5]octyl, diazaspiro[2.5]octyl, azaspiro[4.4]nonyl 4- cyclohexyl, ...

[0209]

[0210] As used herein, “C o-p Cycloalkyl-C n-m "Alkyl-" refers to a radical of the formula cycloalkyl-alkylene- wherein the cycloalkyl has o to p carbon atoms and the alkylene linking group has n to m carbon atoms.

[0211] As used herein, “C o-p Aryl-C n-m "Alkyl-" refers to a radical of the formula aryl-alkylene- wherein the aryl has o to p carbon atoms and the alkylene linking group has n to m carbon atoms.

[0212] As used herein, "heteroaryl-C n-m "Alkyl-" refers to a radical of the formula heteroaryl-alkylene- wherein the alkylene linking group has n to m carbon atoms.

[0213] As used herein, "heterocycloalkyl-C n-m "Alkyl-" refers to a radical of the formula heterocycloalkyl-alkylene- wherein the alkylene linking group has n to m carbon atoms.

[0214] In some places, definitions or embodiments refer to specific rings (e.g., azetidine rings, pyridine rings, etc.). Unless otherwise indicated, these rings may be attached to any ring member, provided that the valence of the atom is not exceeded. For example, the azetidine ring may be attached at any position of the ring, while the pyridin-3-yl ring is attached at the 3-position.

[0215] As used herein, the term "oxo" refers to an oxygen atom (ie, =0) as a divalent substituent, forming a carbonyl group (e.g., C=O or C(O)) when attached to carbon or a nitrogen or sulfur heteroatom to form a nitroso, sulfinyl, or sulfonyl group.

[0216] As used herein, the term "independently selected" means that each occurrence of a variable or substituent is independently selected from the applicable list at each occurrence.

[0217] The compounds described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise indicated, all stereoisomers (such as enantiomers and diastereomers) are intended to be referred to. Compounds containing asymmetrically substituted carbon atoms disclosed herein can be separated in optically active or racemic forms. Methods for preparing optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, etc. may also be present in the compounds described herein, and all such stable isomers are encompassed in the present invention. Cis and trans geometric isomers of the compounds disclosed herein are described, and they can be separated into mixtures of isomers or into separate isomeric forms. In some embodiments, the compound has an (R)-configuration. In some embodiments, the compound has an (S)-configuration. The formulas provided herein (e.g., formula (I), formula (II), etc.) include stereoisomers of the compound.

[0218] The resolution of the racemic mixture of a compound can be carried out by any of a variety of methods known in the art. Example methods include fractional recrystallization using chiral resolution acid, which is an optically active salified organic acid. The resolving agent suitable for fractional recrystallization method is, for example, an optically active acid, such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or various optically active camphorsulfonic acids (such as β-camphorsulfonic acid) in the form of D and L. Other resolving agents suitable for fractional crystallization method include α-methylbenzylamine (for example, S and R forms, or diastereoisomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, etc.

[0219] The resolution of the racemic mixture can also be carried out by eluting on a column packed with an optically active resolving agent (eg, dinitrobenzoylphenylglycine). The composition of the suitable elution solvent can be determined by those skilled in the art.

[0220] Compounds provided herein also include tautomeric forms. Tautomeric forms are produced by the exchange of a single bond with an adjacent double bond and the accompanying proton migration. Tautomeric forms include proton transfer tautomers, which are isomeric protonation states with the same empirical formula and total charge. Examples of proton transfer tautomers include keto-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and annular forms in which protons can occupy two or more positions of a heterocyclic system, such as 1H- and 3H-imidazoles, 1H-, 2H- and 4H-1,2,4-triazoles, 1H- and 2H-isoindoles, 2-hydroxypyridines and 2-pyridones and 1H- and 2H-pyrazoles. Tautomeric forms can be in equilibrium or spatially locked into a form by appropriate substitution.

[0221] All compounds and pharmaceutically acceptable salts thereof may be found together with other substances (eg, water and solvents) (eg, hydrates and solvates) or may be isolated.

[0222] In some embodiments, preparation of the compounds may involve the addition of an acid or base to effect, for example, catalysis of a desired reaction or the formation of a salt form (eg, an acid addition salt).

[0223] In some embodiments, the compounds provided herein or their salts are substantially separated. "Substantially separated" means that the compound is at least partially or substantially separated from the environment in which it is formed or detected. Partial separation can include, for example, a composition rich in the compound provided herein. Substantially separating can include a composition containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of the compound provided herein or its salt by weight. Methods for separating compounds and their salts are conventional in the industry.

[0224] The term "compound" as used herein is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structure. Unless otherwise indicated, a compound identified herein by name or structure as one particular tautomeric form is intended to include the other tautomeric forms.

[0225] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0226] The present application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds, wherein the parent compound is modified by converting the acid or base moiety present into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues (such as amines); alkaline or organic salts of acidic residues (such as carboxylic acids); and the like. Pharmaceutically acceptable salts disclosed herein include conventional non-toxic salts of the parent compound, for example, formed by non-toxic inorganic or organic acids. Pharmaceutically acceptable salts disclosed herein can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of a suitable base or acid in water or in an organic solvent or in a mixture of the two; typically, non-aqueous media such as ether, ethyl acetate, alcohols (e.g., methanol, ethanol, isopropanol or butanol) or acetonitrile (ACN) are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

[0227] The compounds described herein, including salts thereof, can be prepared using known organic synthesis techniques, and can be synthesized according to any of a variety of possible synthetic routes.

[0228] The reactions for preparing the compounds described herein can be carried out in suitable solvents that can be readily selected by those skilled in the art of organic synthesis. Suitable solvents may be substantially non-reactive with starting materials (reactants), intermediates or products at the temperature at which the reaction is carried out (e.g., a temperature that may range from the freezing point of the solvent to the boiling point of the solvent). A given reaction may be carried out in one solvent or a mixture of more than one solvent. Depending on the specific reaction step, a solvent suitable for a specific reaction step may be selected by one skilled in the art.

[0229] The preparation of compounds described herein may involve the protection and deprotection of multiple chemical groups. One skilled in the art can easily determine the need for protection and deprotection and the selection of suitable protecting groups. The chemistry of protecting groups can be found, for example, in TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd Edition, Wiley & Sons, Inc., New York (1999), which is incorporated herein by reference in its entirety.

[0230] The reaction can be monitored by any suitable method known in the art. For example, the reaction can be monitored by spectroscopic methods (e.g., nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV visible light), mass spectrometry) or by chromatographic methods (e.g., high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS) or thin layer chromatography (TLC)) to monitor product formation. Compounds can be purified by a variety of methods by those skilled in the art, including high performance liquid chromatography (HPLC) ("Preparative LC-MS Purification: Improved Compound Specific Method Optimization" Karl F. Blom et al., J. Combi. Chem. 2004, 6 (6), 874-883, all of which are incorporated herein by reference) and normal phase silica chromatography.

[0231] The compounds described herein can modulate the activity of one or more of a variety of GPCRs (including, for example, A2A / A2B). The term "modulation" means the ability to increase or decrease the activity of one or more members of the A2A / A2B family. Therefore, the compounds described herein can be used in methods for modulating A2A / A2B by contacting A2A / A2B with any one or more of the compounds or compositions described herein. In some embodiments, the compounds of the present invention can be used as inhibitors of one or both of A2A and A2B. In other embodiments, the compounds described herein can be used to modulate the activity of A2A / A2B in individuals who need to modulate receptors by administering a modulating amount of a compound described herein or a pharmaceutically acceptable salt thereof. In some embodiments, modulation is inhibition.

[0232] Given that cancer cell growth and survival are affected by multiple signaling pathways, the present invention can be used to treat disease states characterized by drug resistance mutants. In addition, different GPCR inhibitors that exhibit different preferences in GPCRs can be used in combination, which modulate the activity of GPCRs. This approach may prove to be highly effective in treating disease states by targeting multiple signaling pathways, reducing the likelihood of drug resistance in cells, and reducing the toxicity of disease treatments.

[0233] GPCRs that are bound to and / or modulated (eg, inhibited) by the compounds of the present invention include any member of the A2A / A2B family.

[0234] In some embodiments, more than one compound described herein is used to inhibit the activity of one GPCR (eg, A2A).

[0235] In some embodiments, more than one compound described herein is used to inhibit more than one GPCR, such as at least two GPCRs (eg, A2A and A2B).

[0236] In some embodiments, one or more compounds are used in combination with another GPCR antagonist to inhibit the activity of one GPCR (eg, A2A or A2B).

[0237] The A2A / A2B inhibitors described herein may be selective. "Selectivity" means that the compound binds or inhibits a GPCR with greater affinity or potency, respectively, compared to at least one other GPCR. In some embodiments, the compounds described herein are selective inhibitors of A2A or A2B. In some embodiments, the compounds described herein are selective inhibitors of A2A (e.g., relative to A2B). In some embodiments, the compounds described herein are selective inhibitors of A2B (e.g., relative to A2A). In some embodiments, the selectivity may be at least about 2 times, 5 times, 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 500 times, or at least about 1000 times. Selectivity can be measured by conventional methods in the art. In some embodiments, selectivity can be tested for biochemical affinity for each GPCR. In some embodiments, the selectivity of the compounds described herein can be determined by cell analysis associated with specific A2A / A2B GPCR activity.

[0238] As used herein, the term "contacting" refers to bringing the indicated moieties together into an in vitro system or an in vivo system. For example, "contacting" A2A / A2B with a compound described herein includes administering a compound of the invention to an individual or patient (e.g., a human) having A2A / A2B, as well as, for example, introducing a compound described herein into a sample containing cells or purified preparations containing A2A / A2B.

[0239] As used herein, the terms "subject" or "patient" are used interchangeably and refer to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, most preferably humans.

[0240] As used herein, the phrase "therapeutically effective amount" refers to that amount of an active compound or pharmaceutical agent that elicits the biological or medical response of a tissue, system, animal, subject or human that is being sought by a researcher, veterinarian, physician or other clinician.

[0241] As used herein, the terms "treating" or "treatment" refer to one or more of the following: (1) preventing a disease; e.g., preventing a disease, illness, or condition in an individual who may be susceptible to the disease, disorder, or condition but does not yet experience or display symptoms or symptoms of the disease; (2) inhibiting a disease; e.g., inhibiting a disease, disorder, or condition in an individual who is experiencing or displaying symptoms or symptoms of the disease, disorder, or condition (i.e., preventing further development of the symptoms and / or symptoms); and (3) ameliorating a disease; e.g., ameliorating a disease, disorder, or condition in an individual who is experiencing or displaying symptoms or symptoms of the disease, disorder, or condition (i.e., reversing the symptoms and / or symptoms), such as reducing the severity of the disease. In some embodiments, the terms "treating" or "treatment" refer to inhibiting or ameliorating a disease.

[0242] Dosing and administration

[0243] In some embodiments, the A2A / A2B inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose of about 0.1 mg to about 1000 mg based on the free base. In some embodiments, the A2A / A2B inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose of about 1 mg to about 500 mg based on the free base. In some embodiments, the A2A / A2B inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose of about 5 mg to about 250 mg based on the free base. In some embodiments, the A2A / A2B inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose of about 10 mg to about 100 mg based on the free base.

[0244] In some embodiments, the A2A / A2B inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose selected from about 0.5 mg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg based on the free base. , about 155mg, about 160mg, about 165mg, about 170mg, about 175mg, about 180mg, about 185mg, about 190mg, about 195mg, about 200mg, about 205mg, about 210mg, about 215mg, about 220mg, about 225mg, about 230mg, about 235mg, about 240mg, about 245mg, about 250mg, about 255mg, about 260mg, about 265mg, about 270mg, about 275mg, about 280mg, about 285mg, about 290mg, about 295mg, about 300mg, about 305mg, about 310mg, about 315mg, about 320mg, about 325mg, about 330 mg, about 335mg, about 340mg, about 345mg, about 350mg, about 355mg, about 360mg, about 365mg, about 370mg, about 375mg, about 380mg, about 385mg, about 390mg, about 395mg, about 400mg, about 405mg, about 410mg, about 415mg, about 420mg, about 425mg, about 430mg, about 435mg, about 440mg, about 445mg, about 450mg, about 455mg, about 460mg, about 465mg, about 470mg, about 475mg, about 480mg, about 485mg, about 490mg, about 495mg, about 500mg, about 505mg, about 5 10mg, about 515mg, about 520mg, about 525mg, about 530mg, about 535mg, about 540mg, about 545mg, about 550mg, about 555mg, about 560mg, about 565mg, about 570mg, about 575mg, about 580mg, about 585mg, about 590mg, about 595mg, about 600mg, about 605mg, about 610mg, about 615mg, about 620mg, about 625mg, about 630mg, about 635mg, about 640mg, about 645mg, about 650mg, about 655mg, about 660mg, about 665mg, about 670mg, about 675mg, about 680mg, about 685mg,about 690mg, about 695mg, about 700mg, about 705mg, about 710mg, about 715mg, about 720mg, about 725mg, about 730mg, about 735mg, about 740mg, about 745mg, about 750mg, about 755mg, about 760mg, about 765mg, about 770mg, about 775mg, about 780mg, about 785mg, about 790mg, about 795mg, about 800mg, about 805mg, about 810mg, about 815mg, about 820mg, about 825mg, about 830mg, about 835mg, about 840mg, about 845 In some embodiments, the A2A / A2B inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose ranging from about 0.1 mg to about 500 mg based on the free base, or any dose value therebetween. In some embodiments, the A2A / A2B inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose ranging from about 1 mg to about 100 mg based on the free base, or any dose value therebetween.

[0245] In some embodiments, the A2A / A2B inhibitor or a pharmaceutically acceptable salt thereof is administered to the subject once a day, every other day, once a week, or any time interval therebetween. In some embodiments, the A2A / A2B inhibitor or a pharmaceutically acceptable salt thereof is administered to the subject once a day. In some embodiments, the A2A / A2B inhibitor or a pharmaceutically acceptable salt thereof is administered to the subject every other day. In some embodiments, the A2A / A2B inhibitor or a pharmaceutically acceptable salt thereof is administered to the subject once a week.

[0246] In some embodiments, each dose is administered as a single once-daily dose. In some embodiments, each dose is administered as a single once-daily oral dose.

[0247] In some embodiments, the PD-1 / PD-L1 inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose of about 0.1 mg to about 1000 mg based on the free base. In some embodiments, the PD-1 / PD-L1 inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose of about 1 mg to about 500 mg based on the free base. In some embodiments, the PD-1 / PD-L1 inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose of about 5 mg to about 250 mg based on the free base. In some embodiments, the PD-1 / PD-L1 inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose of about 10 mg to about 100 mg based on the free base.

[0248] In some embodiments, the PD-1 / PD-L1 inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose selected from about 0.5 mg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 156 mg, about 157 mg, about 158 ​​mg, about 161 mg, about 162 mg, about 164 mg, about 165 mg, about 166 mg, about 167 mg, about 168 mg, about 169 mg, about 170 mg, about 171 mg, about 172 mg, about 173 mg, about 174 mg, about 175 mg 50mg, about 155mg, about 160mg, about 165mg, about 170mg, about 175mg, about 180mg, about 185mg, about 190mg, about 195mg, about 200mg, about 205mg, about 210mg, about 215mg, about 220mg, about 225mg, about 230mg, about 235mg, about 240mg, about 245mg, about 250mg, about 255mg, about 260mg, about 265mg, about 270mg, about 275mg, about 280mg, about 285mg, about 290mg, about 295mg, about 300mg, about 305mg, about 310mg, about 315mg, about 320mg, about 325mg , about 330mg, about 335mg, about 340mg, about 345mg, about 350mg, about 355mg, about 360mg, about 365mg, about 370mg, about 375mg, about 380mg, about 385mg, about 390mg, about 395mg, about 400mg, about 405mg, about 410mg, about 415mg, about 420mg, about 425mg, about 430mg, about 435mg, about 440mg, about 445mg, about 450mg, about 455mg, about 460mg, about 465mg, about 470mg, about 475mg, about 480mg, about 485mg, about 490mg, about 495mg, about 500mg, about 50 5mg, about 510mg, about 515mg, about 520mg, about 525mg, about 530mg, about 535mg, about 540mg, about 545mg, about 550mg, about 555mg, about 560mg, about 565mg, about 570mg, about 575mg, about 580mg, about 585mg, about 590mg, about 595mg, about 600mg, about 605mg, about 610mg, about 615mg, about 620mg, about 625mg, about 630mg, about 635mg, about 640mg, about 645mg, about 650mg, about 655mg, about 660mg, about 665mg, about 670mg, about 675mg, about 680mg,about 685mg, about 690mg, about 695mg, about 700mg, about 705mg, about 710mg, about 715mg, about 720mg, about 725mg, about 730mg, about 735mg, about 740mg, about 745mg, about 750mg, about 755mg, about 760mg, about 765mg, about 770mg, about 775mg, about 780mg, about 785mg, about 790mg, about 795mg, about 800mg, about 805mg, about 810mg, about 815mg, about 820mg, about 825mg, about 830mg, about 835mg, about 840mg, In some embodiments, the PD-1 / PD-L1 inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose ranging from about 0.1 mg to about 500 mg based on the free base, or any dose value therebetween. In some embodiments, the PD-1 / PD-L1 inhibitor or a pharmaceutically acceptable salt thereof is administered to a subject at a dose ranging from about 1 mg to about 100 mg based on the free base or any dose value therebetween.

[0249] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to a subject at a dose of about 1 mg / kg to about 10 mg / kg. In some embodiments, the PD-1 / PD-L1 inhibitor is administered to a subject at a dose of about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg. In some embodiments, the PD-1 / PD-L1 inhibitor is administered to a subject at a dose of about 200 mg to about 1000 mg. In some embodiments, the PD-1 / PD-L1 inhibitor is administered to a subject at a dose of about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, or about 1000 mg.

[0250] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject once a day, every other day, once a week, or any time interval therebetween. In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject once a day. In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject every other day. In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject once a week.

[0251] In some embodiments, each dose is administered as a single once-daily dose. In some embodiments, each dose is administered as a single once-daily oral dose.

[0252] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject every two weeks, every three weeks, or every four weeks. In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject monthly or quarterly. In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject by intravenous administration.

[0253] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dose of 1 mg / kg Q2W.

[0254] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dose of 3 mg / kg Q2W.

[0255] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dose of 3 mg / kg Q4W.

[0256] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dose of 10 mg / kg Q2W.

[0257] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dose of 10 mg / kg Q4W.

[0258] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dose of 200 mg Q3W.

[0259] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dose of 250 mg Q3W.

[0260] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dose of 375 mg Q3W.

[0261] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dose of 500 mg Q4W.

[0262] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dose of 750 mg Q4W.

[0263] In some embodiments, the PD-1 / PD-L1 inhibitor is antibody X. In some embodiments, antibody X is administered to a subject at a dose of about 250 mg to about 850 mg. In some embodiments, antibody X is administered to a subject at a dose of about 375 mg to about 850 mg. In some embodiments, antibody X is administered to a subject at a dose of about 450 mg to about 850 mg. In some embodiments, antibody X is administered to a subject at a dose of about 500 mg to about 750 mg. In some embodiments, antibody X is administered to a subject at a dose of about 500 mg. In some embodiments, antibody X is administered to a subject at a dose of about 750 mg. In some embodiments, antibody X is administered to a subject every four weeks. In some embodiments, antibody X is administered to a subject by intravenous administration.

[0264] In some embodiments, Antibody X is administered to a subject at a dose of 1 mg / kg Q2W.

[0265] In some embodiments, Antibody X is administered to a subject at a dose of 3 mg / kg Q2W.

[0266] In some embodiments, Antibody X is administered to a subject at a dose of 3 mg / kg Q4W.

[0267] In some embodiments, Antibody X is administered to a subject at a dose of 10 mg / kg Q2W.

[0268] In some embodiments, Antibody X is administered to a subject at a dose of 10 mg / kg Q4W.

[0269] In some embodiments, Antibody X is administered to a subject at a dose of 200 mg Q3W.

[0270] In some embodiments, Antibody X is administered to a subject at a dose of 250 mg Q3W.

[0271] In some embodiments, Antibody X is administered to a subject at a dose of 375 mg Q3W.

[0272] In some embodiments, Antibody X is administered to a subject at a dose of 500 mg Q4W.

[0273] In some embodiments, Antibody X is administered to a subject at a dose of 750 mg Q4W.

[0274] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0275] (i) an A2A / A2B inhibitor which is 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0276] (ii) a PD-1 / PD-L1 inhibitor, which is antibody X.

[0277] In some embodiments, the A2A / A2B inhibitor is administered to a subject at a dose of about 0.1 mg to about 500 mg based on the free base, wherein the A2A / A2B inhibitor is administered once a day or every other day.

[0278] In some embodiments, Antibody X is administered to a subject at a dose of about 100 mg to about 1000 mg Q4W.

[0279] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0280] (i) an A2A / A2B inhibitor which is 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0281] (ii) a PD-1 / PD-L1 inhibitor, which is antibody X;

[0282] wherein the A2A / A2B inhibitor is administered to the subject at a dose of about 0.1 mg to about 500 mg based on the free base, wherein the A2A / A2B inhibitor is administered once a day or every other day; and

[0283] Antibody X is administered to the subject at a dose of about 100 mg to about 1000 mg Q4W.

[0284] In some embodiments, antibody X is administered to a subject at a dose of about 375 mg Q4 W. In some embodiments, antibody X is administered to a subject at a dose of about 500 mg Q4 W. In some embodiments, antibody X is administered to a subject at a dose of about 750 mg Q4 W.

[0285] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0286] (i) an A2A / A2B inhibitor which is 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0287] (ii) PD-1 / PD-L1 inhibitor, which is pembrolizumab.

[0288] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0289] (i) an A2A / A2B inhibitor which is 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0290] (ii) PD-1 / PD-L1 inhibitor, which is pembrolizumab.

[0291] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0292] (i) an A2A / A2B inhibitor which is 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0293] (ii) PD-1 / PD-L1 inhibitor, which is atezolizumab.

[0294] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0295] (i) an A2A / A2B inhibitor which is 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0296] (ii) PD-1 / PD-L1 inhibitor, which is atezolizumab.

[0297] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0298] (i) an A2A / A2B inhibitor which is 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0299] (ii) a PD-1 / PD-L1 inhibitor, which is (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof (Compound Y).

[0300] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0301] (i) an A2A / A2B inhibitor which is 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0302] (ii) a PD-1 / PD-L1 inhibitor, which is (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof (Compound Y).

[0303] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0304] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-2H-tetrazol-2-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0305] (ii) a PD-1 / PD-L1 inhibitor, which is antibody X.

[0306] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0307] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0308] (ii) a PD-1 / PD-L1 inhibitor, which is antibody X.

[0309] In some embodiments, the A2A / A2B inhibitor is administered to a subject at a dose of about 0.1 mg to about 500 mg based on the free base, wherein the A2A / A2B inhibitor is administered once a day or every other day.

[0310] In some embodiments, Antibody X is administered to a subject at a dose of about 100 mg to about 1000 mg Q4W.

[0311] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0312] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-2H-tetrazol-2-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0313] (ii) a PD-1 / PD-L1 inhibitor, which is antibody X;

[0314] wherein the A2A / A2B inhibitor is administered to the subject at a dose of about 0.1 mg to about 500 mg based on the free base, wherein the A2A / A2B inhibitor is administered once a day or every other day; and

[0315] Antibody X is administered to the subject at a dose of about 100 mg to about 1000 mg Q4W.

[0316] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0317] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0318] (ii) a PD-1 / PD-L1 inhibitor, which is antibody X;

[0319] wherein the A2A / A2B inhibitor is administered to the subject at a dose of about 0.1 mg to about 500 mg based on the free base, wherein the A2A / A2B inhibitor is administered once a day or every other day; and

[0320] Antibody X is administered to the subject at a dose of about 100 mg to about 1000 mg Q4W.

[0321] In some embodiments, antibody X is administered to a subject at a dose of about 375 mg Q4 W. In some embodiments, antibody X is administered to a subject at a dose of about 500 mg Q4 W. In some embodiments, antibody X is administered to a subject at a dose of about 750 mg Q4 W.

[0322] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0323] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-2H-tetrazol-2-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0324] (ii) PD-1 / PD-L1 inhibitor, which is pembrolizumab.

[0325] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0326] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0327] (ii) PD-1 / PD-L1 inhibitor, which is pembrolizumab.

[0328] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0329] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-2H-tetrazol-2-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0330] (ii) PD-1 / PD-L1 inhibitor, which is pembrolizumab.

[0331] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0332] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0333] (ii) PD-1 / PD-L1 inhibitor, which is pembrolizumab.

[0334] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0335] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-2H-tetrazol-2-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0336] (ii) PD-1 / PD-L1 inhibitor, which is atezolizumab.

[0337] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0338] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0339] (ii) PD-1 / PD-L1 inhibitor, which is atezolizumab.

[0340] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0341] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-2H-tetrazol-2-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0342] (ii) PD-1 / PD-L1 inhibitor, which is atezolizumab.

[0343] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0344] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0345] (ii) PD-1 / PD-L1 inhibitor, which is atezolizumab.

[0346] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0347] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-2H-tetrazol-2-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0348] (ii) a PD-1 / PD-L1 inhibitor, which is (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof (Compound Y).

[0349] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject:

[0350] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0351] (ii) a PD-1 / PD-L1 inhibitor, which is (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof (Compound Y).

[0352] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0353] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-2H-tetrazol-2-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0354] (ii) a PD-1 / PD-L1 inhibitor, which is (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof (Compound Y).

[0355] In some embodiments, provided herein is a method of treating a cancer in a subject selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merkel cell carcinoma, the method comprising administering to the subject:

[0356] (i) an A2A / A2B inhibitor which is 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile or a pharmaceutically acceptable salt thereof; and

[0357] (ii) a PD-1 / PD-L1 inhibitor, which is (R)-1-((7-cyano-2-(3'-(3-(((R)-3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-ylamino)-2,2'-dimethylbiphenyl-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof (Compound Y).

[0358] In some embodiments, the A2A / A2B inhibitor and the PD-1 / PD-L1 inhibitor are administered simultaneously.

[0359] In some embodiments, the A2A / A2B inhibitor and the PD-1 / PD-L1 inhibitor are administered sequentially.

[0360] When the PD-1 / PD-L1 inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof, it can be administered to a subject, such as a subject in need, such as a human subject, by a variety of methods. The methods and dosages discussed herein are applicable to all anti-PD-1 antibodies or antigen-binding fragments thereof, including antibody X. For many applications, the route of administration is one of the following: intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP), or intramuscular injection. Intraarticular delivery can also be used. Other parenteral modes of administration can also be used. Examples of such modes include: intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and epidural and intrasternal injections. In some cases, administration may be oral.

[0361] The route and / or mode of administration of the antibody or antigen-binding fragment thereof may also be tailored to the individual case, such as by monitoring the subject, for example, using tomographic imaging, e.g., to visualize tumors.

[0362] The antibody or its antigen-binding fragment can be administered at a fixed dose or mg / kg dose. The dose can also be selected to reduce or avoid antibody production against the anti-PD-1 antibody. The dosage regimen is adjusted to provide a desired response, such as a therapeutic response or a combined therapeutic effect. Generally, a dose of an anti-PD-1 antibody (and an optional second dose) can be used to provide a bioavailable amount of the agent to the subject. For example, a dose in the range of 0.1-100 mg / kg, 0.5-100 mg / kg, 1 mg / kg-100 mg / kg, 0.5-20 mg / kg, 0.1-10 mg / kg or 1-10 mg / kg can be administered. Other doses can also be used. In a specific embodiment, a subject who needs to be treated with an anti-PD-1 antibody is administered a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 35 mg / kg or 40 mg / kg of the antibody.

[0363] The composition may comprise about 1 mg / mL to 100 mg / ml, or about 10 mg / mL to 100 mg / ml, or about 50 to 250 mg / mL, or about 100 to 150 mg / ml, or about 100 to 250 mg / ml of an anti-PD-1 antibody or antigen-binding fragment thereof.

[0364] Dosage unit form or "fixed dose" as used herein refers to physically discrete units suitable as unit doses for subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the required pharmaceutical carrier and, optionally, another agent. Single or multiple doses may be administered. Alternatively or additionally, the antibody may be administered via continuous infusion. Exemplary fixed doses include 375 mg, 500 mg, and 750 mg.

[0365] The dose of the anti-PD-1 antibody or antigen-binding fragment thereof may be administered, for example, at periodic intervals, for example, once or twice a day, or about 1 to 4 times a week, or preferably weekly, biweekly, every two weeks, every three weeks, monthly, for example, for about 1 to 12 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, even more preferably for about 4 weeks, 5 weeks or 6 weeks. Factors that may affect the dose and time required to effectively treat a subject include, for example, the severity of the disease or disorder, formulation, route of delivery, previous treatment, the general health and / or age of the subject, and other diseases present. In addition, treating a subject with a therapeutically effective amount of a compound may include a single treatment, or preferably may include a series of treatments.

[0366] A pharmaceutical composition may contain a "therapeutically effective amount" of an agent described herein. Such an effective amount may be determined based on the effect of the agent administered or the combined effect of the agents when more than one agent is used. The therapeutically effective amount of an agent may also vary according to factors such as the disease state, age, sex, and weight of the subject and the ability of the compound to induce a desired response in the individual (e.g., improving at least one parameter of the condition or improving at least one symptom of the condition). A therapeutically effective amount is also an amount in which the therapeutically beneficial effects of the composition outweigh any toxic or deleterious effects.

[0367] Pharmaceutical preparations

[0368] When used as a drug, the compounds of the present disclosure can be administered in the form of a pharmaceutical composition. These compositions can be prepared in a manner well known in the pharmaceutical field, and can be administered by a variety of routes, depending on whether local treatment or systemic treatment is desired and on the area to be treated. Administration can be topical (including transdermal administration, epidermal administration, ocular administration, and mucosal administration, including intranasal delivery, vaginal delivery, and rectal delivery), pulmonary administration (e.g., by inhalation or insufflation of powder or aerosol, including by a nebulizer; intratracheal or intranasal), oral or parenteral administration. Parenteral administration includes intravenous administration, intraarterial administration, subcutaneous administration, intraperitoneal administration, intramuscular administration, or injection or infusion; or intracranial administration, such as intrathecal administration or intraventricular administration. Parenteral administration can be in the form of a single bolus dose, or can be, for example, by a continuous infusion pump. Pharmaceutical compositions and preparations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0369] The present disclosure also includes pharmaceutical compositions, which contain a combination of a compound of the present disclosure or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers (excipients) as an active ingredient. In some embodiments, the composition is suitable for topical application. In preparing the composition of the present disclosure, the active ingredient is usually mixed with an excipient, diluted with an excipient or encapsulated in such a carrier in the form of, for example, a capsule, a sachet, paper or other container. When an excipient is used as a diluent, it can be a solid, semisolid or liquid material, which is used as a vehicle, carrier or medium for the active ingredient. Therefore, the composition can be in the form of tablets, pills, powders, diamond-shaped tablets, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), ointments containing, for example, up to 10% by weight of active compounds, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

[0370] In preparing the formulation, the active compound may be ground to provide a suitable particle size before mixing with the other ingredients. If the active compound is substantially insoluble, it may be ground to a particle size of less than 200 mesh. If the active compound is substantially water-soluble, the particle size may be adjusted by grinding to provide a substantially uniform distribution in the formulation, for example, about 40 mesh.

[0371] The compounds of the present disclosure may be milled using known milling procedures (eg, wet milling) to obtain particle sizes suitable for tablet formation and other formulation types. Finely divided (nanoparticulate) formulations of the compounds of the present disclosure may be prepared by methods known in the art, for example, see International Application No. WO2002 / 000196.

[0372] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose. The formulation may additionally include: lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl hydroxybenzoate and propyl hydroxybenzoate; sweeteners; and flavoring agents. The compositions of the present disclosure can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to the patient by using procedures known in the art.

[0373] The compositions may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unit dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[0374] To prepare solid compositions (e.g., tablets), the main active ingredient is mixed with a pharmaceutical excipient to form a solid preformulated composition containing a homogeneous mixture of the disclosed compounds. When these preformulated compositions are referred to as homogeneous, the active ingredient is generally evenly dispersed throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation is then subdivided into the above-mentioned types of unit dosage forms.

[0375] Tablets or pills of the present disclosure may be coated or otherwise compounded to provide a dosage form with the advantage of prolonged action. For example, tablets or pills may include an inner dosage component and an outer dosage component, the latter being in the form of a film coating on the former. The two components may be separated by an enteric layer, which is used to resist disintegration in the stomach and allows the inner component to enter the duodenum intact or to delay release. A variety of materials may be used for such enteric layers or coatings, including a mixture of a variety of polymeric acids and polymeric acids with materials such as shellac, hexadecanol, and cellulose acetate.

[0376] The compounds and compositions of the present disclosure may be incorporated into liquid forms for oral administration or administration by injection, including aqueous solutions, appropriately flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0377] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the composition is administered by oral or nasal respiratory route to obtain local or systemic effects. The composition may be atomized by the use of an inert gas. The atomized solution may be breathed directly from the atomizing device, or the atomizing device may be connected to a mask, tent, or intermittent positive pressure breathing machine. Solutions, suspensions, or powder compositions may be administered orally or nasally from a device that delivers the formulation in an appropriate manner.

[0378] The topical preparation may contain one or more conventional carriers. In some embodiments, the ointment may contain water and one or more hydrophobic carriers, and the one or more hydrophobic carriers are selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white vaseline, etc. The carrier composition of the cream may be based on a combination of water with glycerol and one or more other components (such as glyceryl monostearate, PEG-glyceryl monostearate and cetearyl alcohol). Gels may be prepared using isopropyl alcohol and water in combination with other components (such as glycerol, hydroxyethyl cellulose, etc.). In some embodiments, the topical preparation contains at least about 0.1% by weight, at least about 0.25% by weight, at least about 0.5% by weight, at least about 1% by weight, at least about 2% by weight or at least about 5% by weight of the compound of the present disclosure. The topical preparation may be suitably packaged in, for example, a 100g tube, which is optionally associated with the instructions for treating selected indications (such as psoriasis or other skin disorders).

[0379] The amount of the compound or composition administered to a patient will vary depending on the substance being administered, the purpose of the administration (such as prevention or treatment), the state of the patient, the mode of administration, etc. In therapeutic applications, the composition can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. The effective dose will depend on the disease condition being treated and the judgment of the attending clinician, which depends on factors such as the severity of the disease, the age, weight and general condition of the patient.

[0380] The composition administered to the patient may be in the form of the above-mentioned pharmaceutical composition. These compositions can be sterilized by conventional sterilization techniques, or can be aseptically filtered. The aqueous solution can be packaged for use as is, or lyophilized, and the lyophilized preparation is combined with a sterile aqueous carrier before administration. The pH of the compound preparation will generally be between 3 and 11, more preferably 5 to 9, and most preferably 7 to 8. It should be understood that the use of some of the above-mentioned excipients, carriers or stabilizers will result in the formation of pharmaceutical salts.

[0381] The therapeutic dose of the compounds of the present disclosure may vary according to, for example, the specific use for which the treatment is to be performed, the mode of administration of the compounds, the health and condition of the patient, and the judgment of the prescribing physician. The ratio or concentration of the compounds of the present disclosure in the pharmaceutical composition may vary according to many factors, including dosage, chemical characteristics (e.g., hydrophobicity), and route of administration. For example, the compounds of the present disclosure may be provided for parenteral administration in an aqueous physiological buffer solution containing about 0.1% to about 10% w / v of the compound.

[0382] The compositions of the present disclosure may further comprise one or more other pharmaceutical agents, such as chemotherapeutic agents, steroids, anti-inflammatory compounds, or immunosuppressants, examples of which are listed herein.

[0383] In certain embodiments, the anti-PD-1 antibody can be prepared with a carrier that will protect the compound from rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations have been patented or are generally known. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York (1978).

[0384] Solid Tumors and Cancer

[0385] Examples of cancers that may be treated using the methods and regimens of the present disclosure include, but are not limited to, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, carcinoma of the endometrium, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, Disease), non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or urethral cancer, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer (including asbestos-induced cancer) and combinations of the cancers. The methods disclosed herein can also be used to treat metastatic cancers, especially metastatic cancers expressing PD-L1.

[0386] In some embodiments, cancers treatable with the methods of the present disclosure include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), squamous cell head and neck cancer, urothelial carcinoma (e.g., bladder), and cancers with high microsatellite instability (MSI high). Additionally, the present disclosure includes refractory or recurrent malignancies whose growth can be inhibited using the methods of the present disclosure.

[0387] In some embodiments, cancers that can be treated using the methods of the present disclosure include, but are not limited to, solid tumors (e.g., prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.), blood cancers (e.g., lymphoma, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, non-Hodgkin's lymphoma (including relapsed or refractory NHL and relapsed follicular NHL), Hodgkin's lymphoma, or multiple myeloma), and combinations of such cancers.

[0388] In some embodiments, cancers that may be treated using the methods of the present disclosure include, but are not limited to, biliary tract cancer, bile duct cancer, triple negative breast cancer, rhabdomyosarcoma, small cell lung cancer, leiomyosarcoma, hepatocellular carcinoma, Ewing's sarcoma, brain cancer, brain tumor, astrocytoma, neuroblastoma, neurofibroma, basal cell carcinoma, chondrosarcoma, epithelioid sarcoma, eye cancer, fallopian tube cancer, gastrointestinal cancer, gastrointestinal stromal tumor, hairy cell leukemia, intestinal cancer, islet cell carcinoma, oral cancer, mouth cancer, laryngeal cancer, laryngeal cancer, lip cancer, mesothelioma, neck cancer, nasal cancer, eye cancer, eye melanoma, pelvic cancer, rectal cancer, renal cell carcinoma, salivary gland cancer, sinus cancer, spinal cancer, tongue cancer, tubular cancer, urethral cancer, and ureteral cancer.

[0389] In some embodiments, the cancer is selected from lung cancer (e.g., non-small cell lung cancer), melanoma, pancreatic cancer, breast cancer, prostate cancer, liver cancer, colon cancer, endometrial cancer, bladder cancer, skin cancer, uterine cancer, ovarian cancer, head and neck cancer, thyroid cancer, kidney cancer, gastric cancer, and sarcoma. In some embodiments, the cancer is selected from acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, polycythemia vera, spontaneous thrombocythemia, chronic myeloid leukemia, myelofibrosis, primary myelofibrosis, polycythemia vera / myelofibrosis after spontaneous thrombocythemia, myelofibrosis after spontaneous thrombocythemia, and myelofibrosis after polycythemia vera. In some embodiments, the cancer is selected from melanoma, endometrial cancer, lung cancer, renal cell carcinoma, urothelial carcinoma, bladder cancer, breast cancer, and pancreatic cancer.

[0390] In some embodiments, the cancer is selected from bladder cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer, or lung metastasis), melanoma (e.g., metastatic melanoma), breast cancer, cervical cancer, ovarian cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, esophageal cancer, prostate cancer, kidney cancer, skin cancer, thyroid cancer, liver cancer, uterine cancer, head and neck cancer, renal cell carcinoma, endometrial cancer, anal cancer, biliary tract cancer, oral cancer, non-melanoma skin cancer, and Merkel cell carcinoma.

[0391] In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).

[0392] In some embodiments, the colorectal cancer is colorectal carcinoma (CRC).

[0393] In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer), melanoma, pancreatic cancer, breast cancer, head and neck squamous cell carcinoma, prostate cancer, liver cancer, colon cancer, endometrial cancer, bladder cancer, skin cancer, uterine cancer, kidney cancer, stomach cancer, or sarcoma. In some embodiments, the sarcoma is Askin's tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystic sarcoma phyllodes, dermatofibrosarcoma protuberans, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, or undifferentiated pleomorphic sarcoma.

[0394] In some embodiments, the cancer is mesothelioma or adrenal cancer. In some embodiments, the disease or condition is mesothelioma. In some embodiments, the cancer is adrenal cancer.

[0395] MDSC (myeloid-derived suppressor cells) are heterogeneous immune cell populations (cell families derived from bone marrow stem cells) from the bone marrow lineage. MDSC can be strongly amplified due to changes in hematopoiesis in pathological conditions such as chronic infection and cancer. MDSC is different from other bone marrow cell types in that they have strong immunosuppressive activity rather than immunostimulatory properties. Similar to other bone marrow cells, MDSC interacts with other immune cell types (including T cells, dendritic cells, macrophages, and natural killer cells) to regulate their functions. In some embodiments, the compounds described herein, etc. can be used in methods associated with cancer tissues (e.g., tumors) with high MDSC infiltration, including solid tumors with high basal levels of macrophages and / or MDSC infiltration. In some embodiments, the combination therapy described herein can be used in methods associated with cancer tissues (e.g., tumors) with tumors or tumor-infiltrating lymphocytes (TILs) expressing PD-1 or PD-L1.

[0396] In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (eg, colon cancer), melanoma, ovarian cancer, bladder cancer, renal cell carcinoma, liver cancer, or hepatocellular carcinoma.

[0397] In some embodiments, the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, anal cancer, endometrial cancer, kidney cancer, oral cancer, head and neck cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, and Merk cell carcinoma.

[0398] In some embodiments, the cancer is selected from melanoma, endometrial cancer, lung cancer, kidney cancer, bladder cancer, breast cancer, pancreatic cancer, and colon cancer.

[0399] In some embodiments, the cancer is selected from endometrial cancer, anal cancer, and biliary tract cancer.

[0400] In some embodiments, the cancer is a tumor that exhibits high adenosine levels in the tumor microenvironment. These tumors can be enriched by gene expression signatures, or by high expression levels of CD73 and / or other alkaline phosphatases, including tissue nonspecific alkaline phosphatases (i.e., TNAP and PAP).

[0401] In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is endometrial cancer. In some embodiments, the endometrial cancer is endometrioid adenocarcinoma. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is selected from non-small cell lung cancer and small cell lung cancer. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is urothelial carcinoma. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the breast cancer is triple negative breast cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma. In some embodiments, the cancer is sarcoma. In some embodiments, the sarcoma is selected from the group consisting of Atkin's tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystic sarcoma phyllodes, dermatofibrosarcoma protuberans, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and undifferentiated pleomorphic sarcoma.

[0402] Labeled compounds and analytical methods

[0403] The present disclosure further includes isotopically labeled compounds of the present disclosure. An "isotopically labeled" or "radiolabeled" compound is a compound of the present disclosure in which one or more atoms are replaced or substituted with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature (i.e., native). Suitable radionuclides that may be incorporated into the compounds of the present disclosure include, but are not limited to: 2 H (also written as deuterium, D), 3 H (also written as tritium, T), 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 18 F. 35 S. 36 Cl, 82 Br, 75 Br, 76 Br, 77 Br, 123 I. 124 I. 125 I and 131 I. For example, one or more hydrogen atoms in the disclosed compounds may be replaced with a deuterium atom (eg, one or more hydrogen atoms of an alkyl group of a compound described herein may be optionally replaced with a deuterium atom, such as replacing -CH3 with -CD3).

[0404] One or more constituent atoms of the compounds presented herein may be substituted or replaced by isotopes of atoms of natural or non-natural abundance. In some embodiments, the compound comprises at least one deuterium atom. In some embodiments, the compound comprises two or more deuterium atoms. In some embodiments, the compound comprises 1-2, 1-3, 1-4, 1-5 or 1-6 deuterium atoms. In some embodiments, all hydrogen atoms in the compound may be substituted or replaced by deuterium atoms.

[0405] In some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms attached to carbon atoms of the compounds described herein are optionally replaced with deuterium atoms.

[0406] Synthetic methods for incorporating isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry, Alan F. Thomas (New York, NY, Appleton-Century-Crofts, 1971; The Renaissance of H / D Exchange, Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew Chem. Int. 2007 edition, 7744-7765; The Organic Chemistry of Isotopic Labelling, James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds are useful in a variety of studies, such as NMR spectroscopy, metabolic experiments and / or analysis.

[0407] Substitution with heavier isotopes (e.g., deuterium) may provide certain therapeutic advantages derived from greater metabolic stability, such as extended in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances (see, e.g., A. Kerekes et al., J. Med. Chem. 2011, 54, 201-210; R. Xu et al., J. Label Compd. Radiopharm. 2015, 58, 308-312). In particular, substitution at one or more metabolic sites may provide one or more therapeutic advantages.

[0408] The radionuclide incorporated into the radiolabeled compounds of the invention will depend on the specific application of the radiolabeled compound. For example, for in vitro A2A / A2B labeling and competition assays, incorporation of 3 H. 14 C. 82 Br, 125 I. 131 I or 35 For radioactive imaging applications, compounds of S can be used 11 C. 18 F. 125 I. 123 I. 124 I. 131 I. 75 Br, 76 Br or 77 Br.

[0409] It is understood that a "radiolabeled" or "labeled compound" is a compound that has incorporated at least one radionuclide. In some embodiments, the radionuclide is selected from 3 H. 14 C. 125 I. 35 S and 82 The group composed of Br.

[0410] The present disclosure may further include synthetic methods for incorporating radioisotopes into compounds of the present disclosure.Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and those skilled in the art will readily recognize methods applicable to the compounds of the present disclosure.

[0411] Methods of producing antibodies

[0412] Antibodies can be produced in bacteria or eukaryotic cells. Some antibodies (e.g., Fab) can be produced in bacterial cells (e.g., E. coli cells). Antibodies can also be produced in eukaryotic cells (e.g., transformed cell lines (e.g., CHO, 293E, COS)). In addition, antibodies (e.g., scFv) can be expressed in yeast cells (e.g., Pichia (Pichia) (see, e.g., Powers et al., J Immunol Methods. 251: 123-35 (2001)), Hanseula or Saccharomyces). To produce an antibody of interest, a polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells and recover antibodies.

[0413] If the antibody is to be expressed in bacterial cells (e.g., Escherichia coli), the expression vector should have the feature of allowing the vector to be amplified in bacterial cells. In addition, when using Escherichia coli (e.g., JM109, DH5α, HB101, or XL1-Blue) as a host, the vector must have a promoter that can be allowed to be effectively expressed in Escherichia coli, such as lacZ promoter (Ward et al., 341:544-546 (1989)), araB promoter (Better et al., Science, 240:1041-1043 (1988)) or T7 promoter. Examples of such vectors include, for example, M13 series vectors, pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIA expression system" (QIAGEN), pEGFP, and pET (when using such expression vectors, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. For production in the periplasm of E. coli, the pelB signal sequence (Lei et al., J. Bacteriol., 169:4379 (1987)) can be used as a signal sequence for antibody secretion. For bacterial expression, the expression vector can be introduced into bacterial cells using the calcium chloride method or electroporation method.

[0414] If the antibody is to be expressed in animal cells (e.g., CHO, COS, and NIH3T3 cells), the expression vector includes a promoter required for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277: 108 (1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res., 18: 5322 (1990)), or CMV promoter. In addition to the nucleic acid sequence encoding the immunoglobulin or its domain, the recombinant expression vector may carry other sequences, such as sequences that regulate the replication of the vector in the host cell (e.g., an origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, the selectable marker gene typically confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Examples of vectors containing selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0415] In one embodiment, the antibody is produced in a mammalian cell. Exemplary mammalian host cells for expressing the antibody include Chinese hamster ovary cells (CHO cells) (including the dhfr described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220). - CHO cells, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocytic cell lines (e.g., NS0 myeloma cells and SP2 cells), and cells from transgenic animals (e.g., transgenic mammals). For example, the cells are mammary epithelial cells.

[0416] In an exemplary system for antibody expression, a recombinant expression vector encoding the antibody heavy chain and antibody light chain of an anti-PD-1 antibody (eg, Antibody X) is introduced into the dhfr - In Chinese hamster ovary celI cells.In the recombinant expression vector, antibody heavy chain and light chain genes are each operably connected to enhancer / promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus, etc., such as CMV enhancer / AdMLP promoter regulatory elements or SV40 enhancer / AdMLP promoter regulatory elements) to drive high-level gene transcription.The recombinant expression vector also carries the DHFR gene, which allows the use of methotrexate selection / amplification to select the CHO cells transfected with the vector.Cultivate the selected transformed host cells to allow expression of antibody heavy and light chains and recover the antibody from the culture medium.

[0417] Antibodies can also be produced by transgenic animals. For example, U.S. Pat. No. 5,849,992 describes a method for expressing antibodies in the mammary gland of a transgenic mammal. A transgene is constructed comprising a milk-specific promoter and a nucleic acid encoding an antibody of interest and a secretory signal sequence. The milk produced by females of such transgenic mammals includes the antibody of interest secreted therein. The antibodies can be purified from the milk, or used directly for some applications. Animals comprising one or more nucleic acids described herein are also provided.

[0418] The antibodies disclosed herein can be separated and purified as substantially pure and homogeneous antibodies from the inside or outside (e.g., culture medium) of the host cell. The separation and purification methods commonly used in antibody purification can be used for separation and purification of antibodies, and are not limited to any particular method. Antibodies can be separated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Daniel R. Marshak et al., ed., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be implemented using liquid chromatography (e.g., HPLC and FPLC). Columns for affinity chromatography include protein A columns and protein G columns. Examples of columns using protein A columns include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The disclosure also includes antibodies highly purified using these purification methods.

[0419] Antibodies (e.g., Antibody X) can be made, for example, by preparing and expressing a synthetic gene encoding the listed amino acid sequence, or by mutating a human germline gene to provide a gene encoding the listed amino acid sequence. In addition, such antibodies and other anti-PD-1 antibodies can be obtained, for example, using one or more of the following methods.

[0420] Humanized antibodies can be produced by replacing Fv variable region sequences that are not directly involved in antigen binding with equivalent sequences of human Fv variable regions. General methods for producing humanized antibodies are provided in Morrison, SL, Science, 229: 1202-1207 (1985); Oi et al., BioTechniques, 4: 214 (1986); and US 5,585,089; US 5,693,761; US ​​5,693,762; US 5,859,205; and US 6,407,213. Those methods include isolating, manipulating and expressing nucleic acid sequences encoding all or part of the immunoglobulin Fv variable region of at least one of the heavy chain or light chain. The source of such nucleic acids is well known to those skilled in the art, and can be obtained, for example, from hybridomas producing antibodies to a predetermined target as described above, from germline immunoglobulin genes, or from synthetic constructs. The recombinant DNA encoding the humanized antibody can then be cloned into an appropriate expression vector.

[0421] For example, human germline sequences are disclosed in Tomlinson, IA et al., J. Mol. Biol., 227: 776-798 (1992); Cook, GP et al., Immunol. Today, 16: 237-242 (1995); Chothia, D. et al., J. Mol. Biol. 227: 799-817 (1992); and Tomlinson et al., EMBO J., 14: 4628-4638 (1995). The VBASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, IA et al., MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, such as framework regions and CDRs. Consensus human framework regions can also be used, for example, as described in U.S. Patent No. 6,300,064.

[0422] Other methods for humanizing antibodies can also be used. For example, other methods can explain the three-dimensional structure of the antibody, the three-dimensional framework position near the binding determinant, and the immunogenic peptide sequence. See, for example, WO 90 / 07861; U.S. Patent Nos. 5,693,762; 5,693,761; 5,585,089; 5,530,101; and 6,407,213; Tempest et al. (1991) Biotechnology 9: 266-271. Another method is called "humaneering" and is described, for example, in US2005-008625.

[0423] The antibody may include a human Fc region, such as a wild-type Fc region or an Fc region including one or more changes. In one embodiment, the constant region is changed (e.g., mutated) to improve the properties of the antibody (e.g., increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function). For example, the human IgG1 constant region may be mutated at one or more residues (e.g., one or more of residues 234 and 237 (based on Kabat numbering)). The antibody may have a mutation in the CH2 region of the heavy chain that reduces or changes effector function (e.g., Fc receptor binding and complement activation). For example, the antibody may have mutations, such as those described in U.S. Pat. Nos. 5,624,821 and 5,648,260. The antibody may also have a mutation that stabilizes the disulfide bond between the two heavy chains of the immunoglobulin, such as a mutation in the hinge region of IgG4, as disclosed in the art (e.g., Angal et al. (1993) Mol. Immunol. 30: 105-08). See also, e.g., US2005-0037000.

[0424] The anti-PD-1 antibody may be in the form of a full-length antibody, or in the form of a low molecular weight form of an anti-PD-1 antibody (e.g., a biologically active antibody fragment or mini-antibody), such as Fab, Fab', F(ab')2, Fv, Fd, dAb, scFv, and sc(Fv)2. Other anti-PD-1 antibodies encompassed by the present disclosure include single domain antibodies (sdAbs) containing a single variable chain (e.g., VH or VL) or biologically active fragments thereof. See, e.g., Moller et al., J. Biol. Chem., 285(49):38348-38361 (2010); Harmsen et al., Appl. Microbiol. Biotechnol., 77(1):13-22 (2007); US2005 / 0079574 and Davies et al. (1996) Protein Eng., 9(6):531-7. Like intact antibodies, sdAbs are capable of selectively binding to specific antigens. The molecular weight of sdAb is only 12-15 kDa, which is much smaller than ordinary antibodies, and even smaller than Fab fragments and single-chain variable fragments.

[0425] Provided herein are compositions comprising a mixture of an anti-PD-1 antibody or an antigen-binding fragment thereof and one or more acidic variants thereof, for example, wherein the amount of the acidic variant is less than about 80%, 70%, 60%, 60%, 50%, 40%, 30%, 30%, 20%, 10%, 5% or 1%. Also provided are compositions comprising an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the anti-PD-1 antibody or an antigen-binding fragment thereof comprises at least one deamidation site, wherein the pH of the composition is about 5.0 to about 6.5, such that, for example, at least about 90% of the anti-PD-1 antibody is not deamidated (i.e., less than about 10% of the antibody is deamidated). In certain embodiments, less than about 5%, 3%, 2% or 1% of the antibody is deamidated. The pH may be 5.0 to 6.0, for example 5.5 or 6.0. In certain embodiments, the pH of the composition is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

[0426] An "acidic variant" is a variant of a polypeptide of interest that is more acidic (eg, as determined by cation exchange chromatography) than the polypeptide of interest. An example of an acidic variant is a deamidated variant.

[0427] A "deamidated" variant of a polypeptide molecule is one in which one or more asparagine residues of the original polypeptide have been converted to aspartic acid, ie, the neutral amide side chain has been converted to a residue having an overall acidic character.

[0428] The term "mixture" as used herein to refer to a composition comprising an anti-PD-1 antibody or an antigen-binding fragment thereof means that both the desired anti-PD-1 antibody or an antigen-binding fragment thereof and one or more acidic variants thereof are present. The acidic variants may comprise primarily deamidated anti-PD-1 antibodies and minor amounts of other acidic variants.

[0429] In certain embodiments, the binding affinity (K) of an antibody mutated to eliminate deamidation is D ), association rate (K D Association) and / or dissociation rate (K D The binding affinity, on-rate and / or off-rate of the antibody may be similar to that of the wild-type antibody, e.g., less than about 5-fold, 2-fold, 1-fold (100%), 50%, 30%, 20%, 10%, 5%, 3%, 2% or 1%.

[0430] Antibody fragments

[0431] Antibody fragments (e.g., Fab, Fab', F(ab')2, Facb, and Fv) can be prepared by proteolytic digestion of intact antibodies. For example, antibody fragments can be obtained by treating intact antibodies with enzymes (e.g., papain, pepsin, or plasmin). Papain digestion of intact antibodies produces F(ab)2 or Fab fragments; pepsin digestion of intact antibodies produces F(ab')2 or Fab'; and plasmin digestion of intact antibodies produces Facb fragments.

[0432] Alternatively, antibody fragments can be produced recombinantly. For example, a nucleic acid encoding an antibody fragment of interest can be constructed, introduced into an expression vector, and expressed in a suitable host cell. See, e.g., Co, MS et al., J. Immunol., 152:2968-2976 (1994); Better, M. and Horwitz, AH, Methods in Enzymology, 178:476-496 (1989); Plueckthun, A. and Skerra, A., Methods in Enzymology, 178:476-496 (1989); Lamoyi, E., Methods in Enzymology, 121:652-663 (1989); Rousseaux, J. et al., Methods in Enzymology, (1989) 121:663-669 (1989); and Bird, RE et al., TIBTECH, 9:132-137 (1991). Antibody fragments can be expressed in and secreted from E. coli, so large quantities of these fragments can be easily produced. Antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab)2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). According to another method, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Fab and F(ab')2 fragments with extended in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046.

[0433] Microantibodies

[0434] Minibodies of anti-PD-1 antibodies include bivalent antibodies, single-chain (scFv), and single-chain (Fv)2 (sc(Fv)2).

[0435] "Diabodies" are bivalent miniantibodies constructed by gene fusion (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); EP 404,097; WO 93 / 11161). Diabodies are dimers consisting of two polypeptide chains. The VL and VH domains of each polypeptide chain of the bivalent antibody are bound by a connector. The number of amino acid residues constituting the connector may be between 2 and 12 residues (e.g., 3-10 residues or 5 or about 5 residues). The connectors of the polypeptides in the bivalent antibody are usually too short for VL and VH to bind to each other. Therefore, the VL and VH encoded in the same polypeptide chain cannot form single-chain variable region fragments, but form dimers with different single-chain variable region fragments. Therefore, the bivalent antibody has two antigen binding sites.

[0436] scFv is a single-chain polypeptide antibody obtained by connecting VH and VL with a linker (see, for example, Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Plickthun, "The Pharmacology of onoclonal Antibodies", Vol. 113, Resenburg and Moore, eds., Springer Verlag, New York, pp. 269-315, (1994)). The order in which VH and VL are connected is not particularly limited, and they can be arranged in any order. Examples of the arrangement include: [VH] linker [VL]; or [VL] linker [VH]. The H chain V region and the L chain V region in the scFv can be derived from any anti-PD-1 antibody or antigen-binding fragment thereof described herein.

[0437] sc(Fv)2 is a mini antibody in which two VH and two VL are connected by a linker to form a single chain (Hudson et al., J. Immunol. Methods, (1999) 231: 177-189 (1999)). sc(Fv)2 can be prepared, for example, by linking scFv with a linker. The sc(Fv)2 of the present invention includes antibodies in which two VH and two VL are preferably arranged in the following order: starting from the N-terminus of the single-chain polypeptide, VH, VL, VH and VL ([VH] linker [VL] linker [VH] linker [VL]); however, the order of two VH and two VL is not limited to the above arrangement, and they can be arranged in any order.

[0438] Bispecific Antibodies

[0439] Bispecific antibodies are antibodies that have binding specificity for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the PD-1 protein. Other such antibodies can combine the PD-1 binding site with the binding site of another protein. Bispecific antibodies can be prepared as full-length antibodies or low-molecular-weight forms thereof (e.g., F(ab')2 bispecific antibodies, sc(Fv)2 bispecific antibodies, bivalent antibody bispecific antibodies).

[0440] The production of traditional full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). In different methods, antibody variable domains with desired binding specificities are fused to immunoglobulin constant domain sequences. DNA encoding immunoglobulin heavy chain fusions and (if necessary) immunoglobulin light chains are inserted into separate expression vectors and co-transfected into suitable host cells. This provides greater flexibility in adjusting the ratio of the three polypeptide fragments. However, when at least two polypeptide chains are expressed in equal ratios to produce high yields, the coding sequences of two or all three polypeptide chains can be inserted into a single expression vector.

[0441] According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferably, the interface comprises C H3 At least a portion of a domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of size identical or similar to the larger side chains are produced on the interface of the second antibody molecule by replacing the larger amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). This provides a mechanism for increasing the heterodimer yield relative to other undesirable final products (e.g., homodimers).

[0442] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate may be coupled to avidin and the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking method.

[0443] "Diabody" technology provides an alternative mechanism for making bispecific antibody fragments. The fragments contain VH linked to VL by a linker that is too short to allow pairing between the two domains on the same chain. Therefore, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.

[0444] Multivalent Antibodies

[0445] Cells expressing antigens to which the antibodies bind may internalize (and / or degrade) multivalent antibodies faster than bivalent antibodies. The antibodies described herein may be multivalent antibodies (e.g., tetravalent antibodies) having three or more antigen binding sites, which can be easily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. Multivalent antibodies may comprise a dimerization domain and three or more antigen binding sites. Exemplary dimerization domains comprise (or consist of) an Fc region or a hinge region. Multivalent antibodies may comprise (or consist of) three to about eight (e.g., four) antigen binding sites. Multivalent antibodies optionally comprise at least one polypeptide chain (e.g., at least two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, a polypeptide chain may comprise VD1-(X1) n -VD2-(X2) n -Fc, wherein VD1 is the first variable domain, VD2 is the second variable domain, Fc is the polypeptide chain of the Fc region, X1 and X2 represent amino acids or peptide spacers, and n is 0 or 1.

[0446] Binding Antibody

[0447] The antibodies disclosed herein may be conjugated antibodies that bind to various molecules, including macromolecular substances, such as polymers (e.g., polyethylene glycol (PEG), polyethyleneimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer), hyaluronic acid, radioactive materials (e.g., 90 Y. 131 I), fluorescent substances, luminescent substances, haptens, enzymes, metal chelates, drugs and toxins (e.g. calcheamicin, Pseudomonas exotoxin A, ricin (e.g. deglycosylated ricin A chain)).

[0448] In one embodiment, to improve the cytotoxic effects of anti-PD-1 antibodies and thus improve their therapeutic effectiveness, antibodies are combined with highly toxic substances (including radioisotopes and cytotoxic agents). These conjugates can selectively deliver toxic loads to target sites (i.e., cells expressing antigens recognized by the antibody) while retaining cells that are not recognized by the antibody. To minimize toxicity, conjugates are usually engineered based on molecules with short serum half-lives (thus, using murine sequences and IgG3 or IgG4 isotypes).

[0449] In certain embodiments, the anti-PD-1 antibody or its antigen-binding fragment is modified with a portion that improves its stability and / or retention in the circulation (e.g., in the blood, serum, or other tissues) by, for example, at least 1.5 times, 2 times, 5 times, 10 times, or 50 times. For example, the anti-PD-1 antibody or its antigen-binding fragment can be associated (e.g., bound) with a polymer (e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or polyethylene oxide). Suitable polymers vary widely by weight. Polymers with a number average molecular weight ranging from about 200 to about 35,000 Daltons (or about 1,000 to about 15,000 and 2,000 to about 12,500) can be used. For example, the anti-PD-1 antibody or its antigen-binding fragment can be bound to a water-soluble polymer, such as a hydrophilic polyvinyl polymer, such as polyvinyl alcohol or polyvinyl pyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers, such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylated polyols, copolymers thereof, and block copolymers thereof, provided that the water solubility of the block copolymer is maintained. Other useful polymers include polyoxyalkylenes, such as polyethylene oxide, polypropylene oxide, and block copolymers of polyethylene oxide and polypropylene oxide; polymethacrylates; carbomers; and branched or unbranched polysaccharides.

[0450] The above-mentioned binding antibodies can be prepared by chemically modifying the antibodies described herein or their lower molecular weight forms. Methods for modifying antibodies are well known in the art (eg, US 5057313 and US 5156840).

[0451] Pill Box

[0452] The present disclosure also includes pharmaceutical kits useful, for example, for treating or preventing the A2A / A2B-related diseases or conditions described herein, which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure. Such kits may further include, if desired, one or more of a variety of conventional pharmaceutical kit components, such as a container containing one or more pharmaceutically acceptable carriers, other containers, etc., as will be readily apparent to those skilled in the art. Instructions in the form of inserts or labels indicating the amounts of the components to be administered, administration guidelines, and / or guidelines for mixing the components may also be included in the kit.

[0453] The present invention will be described in more detail by specific examples. The following examples are provided for illustrative purposes and are not intended to limit the present invention in any way. Those skilled in the art will readily appreciate that a variety of non-critical parameters can be altered or modified to produce substantially the same results. It should be appreciated that certain features of the present invention described in the context of a separate embodiment for clarity may also be provided in combination in a single embodiment. On the contrary, a plurality of features of the present invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination.

[0454] In addition to those modifications described herein, those skilled in the art will appreciate various modifications of the present invention based on the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The full text of each reference cited in this disclosure (including all patents, patent applications and publications) is incorporated herein by reference.

[0455] Example

[0456] Example 1: In vitro CHO-PD-L1 co-culture analysis

[0457] In an in vitro CHO-PD-L1 co-culture assay, T cells were treated with PD-1 antibodies in the presence of CHO-PD-L1 cells, and adenosine signaling was activated using the adenosine mimetic agent 5′-N-ethylcarboxamide adenosine (NECA). Under these conditions, compound 9 could restore T cell function using anti-PD1 agents.

[0458] Under 2 μM NECA treatment, anti-PD1 agents tested in this system included: (A) pembrolizumab, (B) antibody X, and (C) compound Y, as shown in FIG1 .

[0459] plan :

[0460] On day 0, 10,000 CHO PDL1+ cells were plated in 100 ul of CHO medium without antibiotics in a 96-well tissue culture flat bottom plate. On day 1, T cells were thawed and cultured at 1×10 6100ul T cell culture medium.Individual cells / ml are resuspended in T cell culture medium.Remove culture medium from CHO PDL1+ cell plate and add 130ul T cell culture medium.198ul T cell culture medium is added to compound plate with 2ul or 1:100 dilution, then resuspended.20ul compound from compound plate is added to CHO cell plate with 1:1000 final compound dilution.50ul T cells (50,000 cells) are added to the plate containing CHO cells to make a total volume of 200ul, and cultivate 72hr at 37 ℃.After 3 days of cultivation, supernatant is collected, and hIFNg and hIL2 cytokine analysis operation (manufacturer's scheme) is performed using ProCartaplex 2plex kit (Life Technologies catalog number PPX-02) for hIFNg and hIL2.Cytokine analysis is run using ProCartaplex kit on Flex Map 3D Luminex multiplexing platform.

[0461] Example 2: In vitro mixed lineage response analysis

[0462] In another in vitro assay, the mixed lineage response assay (MLR), PBMCs from healthy donors were stimulated with CD3 antibodies and treated with atezolizumab, compound 9, or compound 3A in the presence of 10 μM adenosine mimetic agent NECA ( Figures 2A-2D ).

[0463] plan:

[0464] At day 0, 10,000 PBMCs from a healthy donor were co-cultured with 10,000 gamma-irradiated PBMCs from another healthy donor. The cells were plated in 200ul RPMI-1640 medium supplemented with 10% FBS in a 96-well tissue culture round bottom plate, and treated with or without 10μM NECA, 5ng / ml CD3 antibody (clone OKT3) and indicated concentrations of compounds / antibodies. The cells were incubated at 37°C for 4 days. IFN-γ in the supernatant of each well was measured by HTRF kit (Cisbio, 62HIFNGPEH), and the fluorescence signal was detected on a Pherastar FS plate reader (BMG Labtech) on day 4.

[0465] Compound 9 was able to significantly increase IFNγ production when combined with an anti-PD-L1 antibody (i.e., atezolizumab) Figure 2A-2B Compound 3A was also able to significantly increase IFNγ production when combined with an anti-PD-L1 antibody (i.e., atezolizumab) ( Figure 2C-2D ).

[0466] Example 3: In vivo efficacy study in a mouse synergy model

[0467] Compound 9 was evaluated for inhibition of tumor growth in two different models. CT-26 murine colon carcinomas have been shown to have high adenosine levels in the tumor microenvironment and reflect high adenosine tumors selected for clinical studies (Mosely et al., Cancer Immunol Res; 5(1) January 2017, pp. 29-41). As a single agent, at 10 mg / kg BID, compound 9 significantly slowed tumor growth with a tumor growth inhibition (TGI) of 52% relative to vehicle control, and additionally showed an additive effect in combination with anti-PD-1 antibody (77% TGI relative to vehicle) ( Figure 3A ). In contrast, no single-dose efficacy was observed when the same regimen was applied to a model hosted by NSG mice, which lack T and NK cells through which compound 9 is thought to exert most of its therapeutic effects ( Figure 3B ).

[0468] The ability of compound 9 to disrupt immune checkpoint resistance was further evaluated in the immunologically cold B16 melanoma model. Both compound 9 and anti-PD-L1 had modest but nonsignificant single-agent activity, but synergized in combination to produce 54% tumor growth inhibition ( Figure 3C These data suggest that compound 9 can alter the microenvironment in high-adenosine tumors and cooperate with other tumor immunity agents to drive effective anti-tumor responses.

[0469] Example A: Activity of A2A / A2B Inhibitors

[0470] I.A2A HTRF analysis

[0471] The assay was performed in a black small volume 384-well polystyrene plate (Greiner 784076-25) in a final volume of 10 μL. The test compound was first serially diluted in DMSO and 100 nl was added to the wells before the other reaction components were added. The final concentration of DMSO was 1%. Adenosine A2A labeled cells (CisBio C1TT1A2A) were diluted 1:5 into Tag-lite buffer (CisBio LABMED) and spun at 1200 g for 5 min. The pellet was resuspended in Tag-lite buffer at a volume of 10.4X the initial cell suspension volume, and the adenosine A2A receptor red antagonist fluorescent ligand (CisBio L0058RED) was added to a final concentration of 12.5 nM. 10 ul of the cell and ligand mixture was added to the assay wells and incubated at room temperature for 45 minutes before reading on a PHERAstar FS plate reader (BMGLabtech) equipped with a HTRF 337 / 620 / 665 optical module. The percentage of binding of the fluorescent ligand was calculated; where 100 nM of the A2A antagonist control ZM 241385 (Tocris 1036) displaced 100% of the ligand, and 1% DMSO had 0% displacement. The data of % binding versus log inhibitor concentration were fit to a one-site competitive binding model (GraphPad Prism version 7.02) with ligand constant = 12.5 nM and ligand Kd = 1.85 nM. The K obtained via this method was i The data are shown in Table 2.

[0472] II. Adenosine A2B receptor cyclic AMP GS analysis

[0473] HEK-293 cells (Perkin Elmer) stably transfected expressing human adenosine A2B receptors were maintained in MEM medium (Life Technologies) containing 10% FBS and 100 μg / ml Geneticin. Geneticin was removed from the culture 18 to 24 hours before analysis. The cisbiocAMP-GS dynamic kit using FRET (fluorescence resonance energy transfer) technology was used to measure cAMP accumulation in cells. The appropriate concentration of the compounds of the present disclosure was mixed with 10,000 cells / well in a white 96-well half-area plate (Perkin Elmer) and gently shaken for 30 minutes at RT. 12nM of the agonist NECA (R&D Technologies) was added to each well and gently shaken for 60 minutes at RT. The detection reagent d2-labeled cAMP (receptor) and anti-cAMP cryptate (donor) were added to each well and gently shaken for 60 minutes at RT. Plates were read on a Pherastar (BMG Labtech), the fluorescence ratio 665 / 620 was calculated, and EC values ​​were performed by fitting a curve of % control versus log compound concentration using GraphPad Prism. 50 The EC obtained by this method 50 The data are shown in Table 2.

[0474] Table 2. A 2A _Ki data (Example A(I)) and A 2B _cAMP_EC 50 Data (Example A(II)).

[0475]

[0476]

[0477] Instructions A 2A _K i or A 2B _cAMP_EC 50 ≤10nM,

[0478] Instructions A 2A _K i or A 2B _cAMP_EC 50 >10nM but ≤100nM,

[0479] Instructions A 2A _K i or A 2B _cAMP_EC 50 >100nM but ≤1μM,

[0480] Instructions A 2A _K i or A 2B _cAMP_EC 50 Greater than 1μM.

[0481] Example A1: Synthesis of 3-(5-amino-2-(pyridin-2-ylmethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (Compound 1)

[0482]

[0483] Step 1: 3-(2-amino-6-chloropyrimidin-4-yl)benzonitrile

[0484]

[0485] A mixture of 4,6-dichloropyrimidine-2-amine (2.5 g, 15.2 mmol), (3-cyanophenyl)boronic acid (2.02 g, 13.7 mmol), tetrakis(triphenylphosphine)palladium(0) (1.06 g, 0.92 mmol) and sodium carbonate (3.23 g, 30.5 mmol) in 1,4-dioxane (60 mL) and water (5 mL) was degassed with nitrogen, and the resulting mixture was heated and stirred at 60 ° C for two days. After cooling to room temperature (rt), the mixture was concentrated, diluted with water, and extracted with DCM (30 mL×3). The combined organic layers were dried over MgSO4, filtered, and concentrated. The resulting residue was purified by flash chromatography on a silica gel column eluting with 8% EtOAc in dichloromethane to provide the desired product. For C 11 H8ClN4(M+H) + LCMS calculated value: 231.0. Found value: 231.0.

[0486] Step 2: 2-(Pyridin-2-yl)acetohydrazide

[0487]

[0488] At rt, hydrazine (4.15 mL, 132 mmol) was added to a solution of methyl 2-(pyridin-2-yl)acetate (10 g, 66.2 mmol) in ethanol (66 mL). The mixture was heated and stirred at 85 °C for 4 h and then cooled to rt. A white solid was formed upon standing, which was collected by filtration and used in the next step without further purification. For C7H 10 N3O(M+H) + LCMS calculated value: 152.1. Found value: 152.0.

[0489] Step 3: 3-(5-amino-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0490]

[0491] At rt, 2-(pyridin-2-yl)acetohydrazide (2.62g, 17.34mmol) is added to the ethanol (35mL) solution of 3-(2-amino-6-chloropyrimidin-4-yl)benzonitrile (4.00g, 17.34mmol). After heating and stirring for 2h under reflux, the reaction mixture is cooled to rt and concentrated. The obtained residue is dissolved in N, O-bis(trimethylsilyl)acetamide (20mL) and stirred for 7h at 120°C. Then the mixture is cooled to rt, poured onto ice, and stirred for 1h at rt. The obtained solid is collected by filtration, and dissolved in 20mL 1N HCl solution. The obtained mixture is stirred for 1h at rt, filtered, and the water layer is neutralized by adding saturated NaHCO3 solution. The obtained precipitation is collected by filtration, and dried to obtain the desired product as a brown solid. For C 18 H 14 N7(M+H) + LCMS calculated value: 328.1; found value: 328.1.

[0492] Step 4: 3-(5-amino-8-bromo-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0493]

[0494] To a mixture of 3-(5-amino-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (2 g, 6.11 mmol) in DMF (12 mL) was added NBS (1.09 g, 6.11 mmol) portionwise at -30 °C. The reaction mixture was slowly warmed to 0 °C to produce a homogeneous solution. After stirring at 0 °C for 1 h, the reaction mixture was diluted with saturated NaHCO3 solution and the resulting solid was collected by filtration. The solid was then purified by flash chromatography on a silica gel column eluting with 0 to 10% MeOH in DCM to provide the desired product. For C 18 H 13 BrN7(M+H) + LCMS calcd: 406.0; found: 406.0.

[0495] Step 5: 3-(5-amino-2-(pyridin-2-ylmethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0496] Pd(Ph3P)4(284mg, 0.246mmol) was added to a mixture of 4-(tributyltinyl)pyrimidine (1090mg, 2.95mmol), 3-(5-amino-8-bromo-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (1000mg, 2.46mmol) and copper(I) chloride (244mg, 2.46mmol) in 1,4-dioxane (12mL). The reaction mixture was purged with N2 and stirred at 80°C for 7h. The resulting mixture was cooled to rt, concentrated, diluted with DCM (50mL) and washed with saturated NH4OH solution. The organic layer was dried over Na2SO4, concentrated, and purified by preparative LC-MS (pH 2, acetonitrile / water containing TFA) to provide the product in the form of a TFA salt. For C 22 H 16 N9(M+H) + LCMS calcd: 406.2; found: 406.2. 1 H NMR (500MHz, DMSO) δ8.95 (s, 1H), 8.83 (d, J = 5.3Hz, 1H), 8.59 (d, J = 5.1Hz, 1H), 7.96 (m, 1H), 7.88 (d, J = 5.1Hz, 1H),7.82(d,J=7.6Hz,1H),7.76(s,1H),7.60-7.53(m,2H),7.53-7.48(m,1H),7.48-7.42(m,1H),4.49(s,2H).

[0497] Example A2: Synthesis of 3-(5-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (Compound 2)

[0498]

[0499] Step 1: Methyl 2-(2,6-difluorophenyl)-2-hydroxyacetate

[0500]

[0501] At 0 ° C, concentrated sulfuric acid (1.42 mL, 27 mmol) was added to a solution of 2,6-difluoromandelic acid (5 g, 27 mmol) in methanol (45 mL). The mixture was stirred at rt for 4 h and then concentrated. Saturated NaHCO3 solution (30 mL) was added to the resulting slurry. The resulting mixture was extracted with DCM (3×20 mL). The combined organic layers were washed with water, dried over Mg2SO4, filtered, and concentrated to provide a crude product, which was used in the next step without further purification. For C 11 H 12 F2NO3(M+H+MeCN) + LC-MS calculated value: m / z = 244.1; found value: 244.2.

[0502] Step 2: 3-(5-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0503] This compound was prepared using a similar procedure as described for Example A1, substituting methyl 2-(2,6-difluorophenyl)-2-hydroxyacetate for methyl 2-(pyridin-2-yl)acetate in step 2. The two enantiomers were separated by chiral SFC using a Phenomenex Lux Cellulose-1 column (21.2×250 mm, 5 μm particle size) eluting with an isocratic mobile phase of 25% MeOH in CO2 at a flow rate of 80 mL / min. Peak 1 was isolated and further purified by preparative LCMS (pH=2, MeCN / water containing TFA) to obtain the desired product as a TFA salt. For C 23 H 15 F2N8O(M+H) + LC-MS calculated value: m / z = 457.1; found value: 457.1. 1 H NMR (500MHz, DMSO) δ8.94(d,J=1.3Hz,1H),8.81(d,J=5.2Hz,1H),7.85(dd,J=5.3,1.4Hz,1H),7.81(dt,J=7.4,1.5Hz,1H),7.76(t ,J=1.7Hz,1H),7.55(dt,J=7.8,1.5Hz,1H),7.49(t,J=7.8Hz,1H),7.44(tt,J=8.4,6.4Hz,1H),7.09(t,J=8.3Hz,2H),6.27(s,1H).

[0504] Example A3: Synthesis of 3-(5-amino-2-((5-(pyridin-2-yl)-2H-tetrazol-2-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (Compound 3A) and 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (Compound 3B)

[0505]

[0506] Step 1: 3-(5-amino-2-(hydroxymethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0507]

[0508] At rt, 2-hydroxyacetic acid hydrazide (2.34g, 26.01mmol) is added to an ethanol (35mL) solution of 3-(2-amino-6-chloropyrimidin-4-yl) benzonitrile (4.00g, 17.34mmol) (Example A1, step 1). After heating and stirring for 2h under reflux, the reaction mixture is cooled to rt and concentrated. The resulting residue is dissolved in N, O-bis (trimethylsilyl) acetamide (20mL) and stirred at 120 ° C for 7h. The mixture is then cooled to rt, poured onto ice, and stirred at rt for 1h. The resulting solid is collected by filtration and dissolved in 20mL 1N HCl solution. The resulting mixture is stirred at rt for 1h, filtered, and the water layer is neutralized by adding a saturated NaHCO3 solution. The resulting precipitate is collected by filtration and dried to obtain the desired product as a brown solid. For C 13 H 11 N6O(M+H) + LCMS calculated value: 267.1; found value: 267.1.

[0509] Step 2: 3-(5-amino-8-bromo-2-(hydroxymethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0510]

[0511] To a mixture of 3-(5-amino-2-(hydroxymethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (1.0 g, 3.76 mmol) in DMF (12 mL) was added NBS (0.67 g, 3.76 mmol) portionwise at -30 °C. The reaction mixture was slowly warmed to 0 °C to produce a homogeneous solution. After stirring at 0 °C for 1 h, the reaction mixture was diluted with saturated NaHCO3 solution, the desired product was collected by filtration and dried. For C 13 H 10 BrN6O(M+H) + LCMS calcd: 345.0; found: 345.0.

[0512] Step 3: 3-(5-amino-2-(hydroxymethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0513]

[0514] Tetrakis(triphenylphosphine)palladium(0) (0.067 g, 0.058 mmol) was added to a mixture of 4-(tributylstannyl)pyrimidine (0.321 g, 0.869 mmol), 3-(5-amino-8-bromo-2-(hydroxymethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (0.20 g, 0.579 mmol), CsF (0.176 g, 1.159 mmol) and copper(I) iodide (0.022 g, 0.116 mmol) in 1,4-dioxane (5.0 mL). The reaction mixture was purged with N2 and stirred at 80 °C for 7 h. The resulting mixture was cooled to rt, concentrated and purified by flash column chromatography eluting with 0% to 10% methanol in DCM to provide the product. For C 17 H 13 N8O(M+H) + LC-MS calculated value: 345.1; found value: 345.1.

[0515] Step 4: 3-(5-amino-2-(chloromethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0516]

[0517] To a mixture of 3-(5-amino-2-(hydroxymethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (0.1 g, 0.290 mmol) in acetonitrile (10 ml) was added thionyl chloride (0.212 ml, 2.90 mmol) at rt. The reaction mixture was stirred at rt for 5 h, concentrated, and purified by flash chromatography eluting with 0% to 5% methanol in DCM to provide the product. For C 17 H 12 ClN8(M+H) + LC-MS calculated value: 363.1; found value: 363.1.

[0518] Step 5: A mixture of 3-(5-amino-2-((5-(pyridin-2-yl)-2H-tetrazol-2-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (Compound 3A) and 3-(5-amino-2-((5-(pyridin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (Compound 3B)

[0519] A mixture of 3-(5-amino-2-(chloromethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (10 mg, 0.028 mmol), 2-(1H-tetrazol-5-yl)pyridine (8.1 mg, 0.055 mmol) and Cs2CO3 (20.7 mg, 0.064 mmol) in DMF (1 mL) was stirred at 100 °C for 10 min. The reaction mixture was then cooled to rt, diluted with methanol (4 mL), and purified by preparative LC-MS (pH 2, acetonitrile / water containing TFA) to provide the product as a TFA salt. For C 23 H 16 N 13 (M+H) + LCMS calcd: 474.2; found: 474.2.

[0520] Compound 3A: 1H NMR (500MHz, DMSO) δ8.99(d,J=1.4Hz,1H),8.85(d,J=5.3Hz,1H),8.80-8.71(m,1H),8.71-8.39(b,2H),8.18(d,J=7.7,1.1 Hz,1H),8.04(t,J=7.8,1.8Hz,1H),7.85(m,2H),7.80-7.76(m,1H),7.62-7.55(m,2H),7.53(t,J=7.8Hz,1H),6.39(s,2H).

[0521] Example A4: Synthesis of 3-(5-amino-2-((3-methylpyridin-2-yl)methoxy)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (Compound 4)

[0522]

[0523] Step 1: 6-Chloro-N 2 ,N 2 -Bis(4-methoxybenzyl)pyrimidine-2,4-diamine

[0524]

[0525] To a solution of 2,6-dichloropyrimidine-4-amine (5.0 g, 31 mmol) in 2-propanol (31 mL) was added N,N-diisopropylethylamine (6.4 ml, 37 mmol) and bis(4-methoxybenzyl)amine (7.9 g, 31 mmol). The resulting solution was stirred at 100 °C for 16 h, cooled to rt, diluted with water (100 mL), and extracted with EtOAc (100 mL). The organic layer was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was used in the next step without further purification. For C 20 H 22 ClN4O2(M+H) + LC-MS calculated value: 385.1; found value: 385.1.

[0526] Step 2: 7-Chloro-N 5 ,N 5 -Bis(4-methoxybenzyl)-[1,2,4]triazolo[1,5-c]pyrimidine-2,5-diamine

[0527]

[0528] At rt, ethoxycarbonyl isothiocyanate (3.1 mL, 26 mmol) was added to 6-chloro-N 2 ,N2 -bis(4-methoxybenzyl)pyrimidine-2,4-diamine (1.0 g, 2.6 mmol) in 1,4-dioxane (5.0 mL). The reaction mixture was then stirred at 90 ° C overnight, cooled to rt, and concentrated. The resulting material was dissolved in methanol (12 mL) and ethanol (12 mL), and N,N-diisopropylethylamine (0.91 mL, 5.2 mmol) was added, followed by hydroxylamine hydrochloride (0.54 g, 7.8 mmol). The reaction mixture was stirred at 45 ° C for 2 h, cooled to rt, and concentrated. The resulting material was dissolved in EtOAc, washed with water, dried over anhydrous sodium sulfate, and concentrated. The crude material was then purified by silica gel chromatography, eluting with 0% to 50% EtOAc in hexane to provide the product. For C 21 H 22 ClN6O2(M+H) + LC-MS calculated value: 425.1; found value: 425.2.

[0529] Step 3: 3-(2-amino-5-(bis(4-methoxybenzyl)amino)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0530]

[0531] Chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (330 mg, 0.42 mmol) was added to (3-cyanophenyl)boronic acid (460 mg, 3.2 mmol), 7-chloro-N 5 ,N 5 -bis(4-methoxybenzyl)-[1,2,4]triazolo[1,5-c]pyrimidine-2,5-diamine (890 mg, 2.1 mmol) and sodium carbonate (890 mg, 8.4 mmol) in a mixture of 1,4-dioxane (8.8 mL) and water (1.8 mL). The mixture was purged with N2 and stirred at 95 °C overnight. The reaction mixture was then cooled to rt, concentrated, and purified by silica gel chromatography, eluting with 0% to 50% EtOAc in DCM to provide the desired product. For C 28 H 26 N7O2(M+H) + LC-MS calculated value: 492.2; found value: 492.2.

[0532] Step 4: 3-(2-amino-5-(bis(4-methoxybenzyl)amino)-8-bromo-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0533]

[0534] To a solution of 3-(2-amino-5-(bis(4-methoxybenzyl)amino)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (330 mg, 0.66 mmol) in DMF (1.4 mL) was slowly added NBS (120 mg, 0.66 mmol) at 0°C. The reaction mixture was then stirred at rt for 30 min before water (10 mL) was added. The resulting solid was collected by filtration and dried to obtain the desired product. For C 28 H 25 BrN7O2(M+H) + LC-MS calculated value: m / z = 570.1; found value: 570.2.

[0535] Step 5: 3-(2-amino-5-(bis(4-methoxybenzyl)amino)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0536]

[0537] A mixture of 3-(2-amino-5-(bis(4-methoxybenzyl)amino)-8-bromo-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (350 mg, 0.61 mmol), 4-(tributylstannyl)pyrimidine (210 μL, 0.67 mmol), tetrakis(triphenylphosphine)palladium(0) (70 mg, 0.060 mmol), copper(I) iodide (23 mg, 0.12 mmol) and cesium fluoride (180 mg, 1.2 mmol) in dioxane (4.7 mL) was heated and stirred at 140 ° C in a microwave reactor for 30 min. The reaction mixture was then cooled to rt, filtered through a celite plug (washed with DCM), and concentrated. The resulting material was purified by silica gel column chromatography eluting with 0-20% MeOH / DCM to obtain the desired product. For C 32 H 28 N9O2(M+H) + LC-MS calculated value: m / z = 570.2; found value: 570.3.

[0538] Step 6: 3-(5-(bis(4-methoxybenzyl)amino)-2-bromo-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0539]

[0540] To a mixture of copper (II) bromide (91 mg, 0.407 mmol) and tert-butyl nitrite (0.054 ml, 0.407 mmol) in acetonitrile (3 mL) was added dropwise 3-(2-amino-5-(bis(4-methoxybenzyl)amino)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (100 mg, 0.203 mmol) in acetonitrile (3 mL) under nitrogen at 50 ° C. The mixture was stirred at 50 ° C for 2 hours. After cooling to room temperature, 1N NH4OH aqueous solution (20 mL) was added and the mixture was extracted three times with CH2Cl2 (20 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated. The crude material was purified by silica gel column chromatography eluting with 50%-100% ethyl acetate / hexane to obtain the desired product. For C 32 H 26 BrN8O2(M+H) + LC-MS calculated value: m / z = 633.1; found value: 633.2.

[0541] Step 7: 3-(5-amino-2-((3-methylpyridin-2-yl)methoxy)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0542] A suspension of sodium hydride (60% in mineral oil, 3.8 mg, 0.095 mmol), 3-(5-(bis(4-methoxybenzyl)amino)-2-bromo-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (20 mg, 0.032 mmol) and (3-methylpyridin-2-yl)methanol (9.1 μL, 0.095 mmol) in 1,4-dioxane (1 mL) was heated at 110 °C under nitrogen and stirred overnight. The reaction mixture was then cooled to rt, concentrated, and TFA (1.0 mL) was added. The resulting mixture was then stirred at 110 °C for 30 min, cooled to rt, diluted with acetonitrile, filtered and purified by preparative LC-MS (pH 2, acetonitrile / water with TFA) to obtain the desired product as a TFA salt. For C 23 H 18 N9O(M+H) + LC-MS calculated value: m / z = 436.2; found value: 436.2. 1H NMR (600MHz, DMSO) δ8.97(d,J=1.4Hz,1H),8.88(d,J=5.2Hz,1H),8.58-8.52(m,1H),7.97(d,J=7.8Hz,1H),7.88(dd,J=5.4,1.4 Hz,1H),7.85(dt,J=7.5,1.5Hz,1H),7.78(t,J=1.8Hz,1H),7.60-7.54(m,2H),7.53(t,J=7.8Hz,1H),5.69(s,2H),2.48(s,3H).

[0543] Example A5: Synthesis of 3-(5-amino-2-(hydroxy(phenyl)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (Compound 5)

[0544]

[0545] Step 1: 3-(2-amino-6-chloropyrimidin-4-yl)benzonitrile

[0546]

[0547] A mixture of 4,6-dichloropyrimidine-2-amine (2.5 g, 15.24 mmol), (3-cyanophenyl)boronic acid (2.016 g, 13.72 mmol), tetrakis(triphenylphosphine)palladium(0) (1.057 g, 0.915 mmol) and sodium carbonate (3.23 g, 30.5 mmol) in 1,4-dioxane (60 mL) and water (5 mL) was degassed with nitrogen, and the resulting mixture was heated at 60 ° C for two days. After cooling to room temperature (RT), the mixture was concentrated, then diluted with water and extracted with dichloromethane (DCM, 3×30 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated. The residue was purified by flash chromatography on a silica gel column containing 8% ethyl acetate (EtOAc) in dichloromethane to provide the desired product. For C 11 H8ClN4(M+H) + LCMS calculated value: 231.0. Found value: 231.0.

[0548] Step 2: 3-(5-amino-2-(hydroxy(phenyl)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile

[0549] A solution of 3-(2-amino-6-chloropyrimidin-4-yl)benzonitrile (100mg, 0.434mmol) and 2-hydroxy-2-phenylacetohydrazide (108mg, 0.650mmol) in ethanol (2ml) was heated at 95°C and stirred for 3h. After cooling to RT, the reaction mixture was concentrated to dryness, dissolved in N, O-bis(trimethylsilyl)acetamide (1mL) and stirred at 120°C for 7h. The resulting mixture was cooled to RT, poured onto ice, and stirred for 1h. The resulting suspension was extracted three times with DCM. The combined organic layers were dried over MgSO4, filtered, and concentrated. The residue was dissolved in methanol (MeOH) and purified by preparative LC-MS (pH 2, acetonitrile / water containing TFA) to provide a product in the form of a TFA salt. For C 19 H 15 N6O(M+H) + LCMS calculated value: 343.1; found value: 343.1.

[0550] Example A6: Synthesis of 3-(5-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile (Compound 6)

[0551]

[0552] Step 1: 3-(2-amino-6-chloropyrimidin-4-yl)-2-fluorobenzonitrile

[0553]

[0554] i-PrMgCl LiCl complex (70.4 mL, 91 mmol) in THF (1.3 M) was added to a solution of 3-bromo-2-fluorobenzonitrile (18.3 g, 91 mmol) in THF (60 mL) cooled to 0 ° C in 20 min. The mixture was stirred at 0 ° C for 50 min, and then zinc chloride (48.1 mL, 91 mmol) in 2-MeTHF (1.9 M) was added at 0 ° C. The reaction was stirred at rt for 25 min, and 4,6-dichloropyrimidine-2-amine (10 g, 61.0 mmol) was added at once. The solution was stirred for 10 min. Tetrakis (triphenylphosphine) palladium (1.41 g, 1.22 mmol) was added to the mixture, and the reactant was stirred at rt for 16 h. After completion, 2,4,6-trimercaptotriazine silica gel (2 g) was added to the reaction solution. The mixture was stirred for 1 h and filtered. The solid was washed with ethyl acetate until the desired product was completely eluted (as detected by LCMS). The filtrate was washed with saturated ammonium chloride solution and water. The organics were concentrated to provide the crude product. Water was added to the crude material and the resulting precipitate was collected by filtration and dried under a stream of nitrogen. The crude material was subsequently used without additional purification. 11 H7ClFN4(M+H) + LC-MS calculated value: m / z = 249.0; found value: 249.0.

[0555] Step 2: Methyl 2-(2,6-difluorophenyl)-2-hydroxyacetate

[0556]

[0557] At 0 ° C, concentrated sulfuric acid (1.4 mL, 27 mmol) was added to a solution of 2,6-difluoromandelic acid (5.0 g, 27 mmol) in methanol (45 mL). The mixture was stirred at rt for 4 h and then concentrated. Saturated NaHCO3 solution was added to the resulting slurry. The resulting mixture was extracted with DCM. The combined organic layers were washed with water, dried over MgSO4, filtered, and concentrated to provide a crude product, which was used in the next step without further purification. For C 11 H 12 F2NO3(M+H+MeCN) + LC-MS calculated value: m / z = 244.1; found value: 244.2.

[0558] Step 3: 2-(2,6-difluorophenyl)-2-hydroxyacetohydrazide

[0559]

[0560] Hydrazine (3.0 mL, 96 mmol) was added to a solution of methyl 2-(2,6-difluorophenyl)-2-hydroxyacetate (10.8 g, 53 mmol) in ethanol (90 mL) at RT. The reaction mixture was stirred at 100 °C for 2 h, cooled to RT, concentrated, and used in the next step without further purification. For C8H9F2N2O2 (M+H) + LC-MS calculated value: 203.1; found value: 203.2.

[0561] Step 4: 3-(5-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile

[0562] The title compound was prepared using a procedure similar to that described for Example A5, Step 2, replacing 3-(2-amino-6-chloropyrimidin-4-yl)-2-fluorobenzonitrile with 3-(2-amino-6-chloropyrimidin-4-yl)benzonitrile and replacing 2-hydroxy-2-phenylacetohydrazide with 2-(2,6-difluorophenyl)-2-hydroxyacetohydrazide. The two enantiomers were separated by chiral SFC using a Phenomenex (R,R)-Whelk-O1 column (21.2×250 mm, 5 μm particle size) eluted with an isocratic mobile phase of 15% MeOH in CO2 at a flow rate of 85 mL / min. The retention times of peaks 1 and 2 were 3.8 min and 5.3 min, respectively. After concentration, peak 2 was purified by preparative LCMS (pH=2, MeCN / water containing TFA) to obtain the desired product in the form of a TFA salt. For C 19 H 12 F3N6O(M+H) + LC-MS calculated value: 397.1; found value: 397.1.

[0563] Example A7: Synthesis of 5-amino-7-(3-cyano-2-fluorophenyl)-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidine-8-carbonitrile (Compound 7)

[0564]

[0565] Step 1: 3-(5-amino-8-bromo-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile

[0566]

[0567] This compound was prepared using a procedure similar to that described for Example A1, Step 4, substituting 3-(5-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile (from Example A6) for 3-(5-amino-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile. 19 H 11 BrF3N6O(M+H) + LCMS calcd: 475.0; found: 475.0.

[0568] Step 2: 5-amino-7-(3-cyano-2-fluorophenyl)-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidine-8-carbonitrile

[0569] A mixture of 3-(5-amino-8-bromo-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile (0.12 g, 0.25 mmol), ZnCN2 (0.060 g, 0.51 mmol) and tBuXPhos Pd G3 (0.020 g, 0.025 mmol) in 1,4-dioxane (0.63 mL) and water (0.63 mL) was purged with N2 and stirred at 100°C for 1 h. After cooling to rt, the reaction was diluted with saturated NaHCO3 and the organics were extracted with EtOAc (3×). The combined organics were dried over MgSO4 and concentrated. The two enantiomers were separated by chiral HPLC using a Phenomenex Lux Celluose-4 column (21.2×250 mm, 5 μm particle size) with an isocratic mobile phase of 60% EtOH in hexane at a flow rate of 20 mL / min. The retention times of peak 1 and peak 2 were 4.9 min and 7.2 min, respectively. After concentration, peak 1 was purified by preparative LC-MS (pH 2, acetonitrile / water containing TFA) to obtain the desired product in the form of a TFA salt. For C 20 H 11 F3N7O(M+H) + LC-MS calculated value: 422.1; found value: 422.1.

[0570] Example A8: Synthesis of 3-(5-amino-2-((2-fluoro-6-(((1-methyl-2-oxopyrrolidin-3-yl)amino)methyl)phenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile (Compound 8)

[0571]

[0572] Step 1: Methyl 2-(2-fluoro-6-vinylphenyl)acetate

[0573]

[0574] A mixture of methyl 2-(2-bromo-6-fluorophenyl)acetate (6.0 g, 24 mmol), tripotassium phosphate (15.5 g, 73 mmol), palladium(II) acetate (0.55 g, 2.4 mmol) and SPhos (1.0 g, 2.4 mmol) was added to a 500 mL pressure vessel. Then, 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (6.4 ml, 36 mmol) in dioxane (150 mL) and water (15 mL) was added, the reaction mixture was purged with N2, and stirred at 80 °C for 16 h. The reaction mixture was then cooled to RT, concentrated, and extracted with EtOAc (×3). The combined organic layers were dried over MgSO4, concentrated, and purified by column chromatography (0 to 50% EtOAc in DCM). For C 11 H 12 FO2(M+H) + LC-MS calculated value: 195.1; found value: 195.1.

[0575] Step 2: Methyl 2-(2-fluoro-6-vinylphenyl)-2-hydroxyacetate

[0576]

[0577] Methyl 2-(2-fluoro-6-vinylphenyl)acetate (2.5 g, 12.9 mmol) was dissolved in THF (130 mL) and cooled to -78 °C. LDA (16.7 mL, 16.7 mmol) in THF (1.0 M) was added dropwise, and the resulting solution was stirred at -78 °C for 30 min. Then, 9,9-dimethyltetrahydro-4H-4a,7-methanobenzo[c][1,2]oxazireno[2,3-b]isothiazole 3,3-dioxide (4.7 g, 20.6 mmol) was added dropwise in THF (0.5 M). After 30 min at -78 °C, the reaction mixture was warmed to 0 °C and stirred for 1 h. The reaction was quenched with saturated NH4Cl. The aqueous layer was extracted with DCM (3×). The combined organics were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography eluting with 0 to 50% ethyl acetate in hexanes to afford the desired product. 11 H 11 FO3Na(M+Na) +LCMS calculated value: 233.1; found value: 233.1.

[0578] Step 3: 2-(2-Fluoro-6-vinylphenyl)-2-hydroxyacetohydrazide

[0579]

[0580] This compound was prepared using a procedure similar to that described for Example A6, Step 3, substituting methyl 2-(2-fluoro-6-vinylphenyl)-2-hydroxyacetate for methyl 2-(2,6-difluorophenyl)-2-hydroxyacetate. 10 H 12 FN2O2(M+H) + LCMS calculated value: 211.1; found value: 211.1.

[0581] Step 4: 3-(5-amino-2-((2-fluoro-6-vinylphenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile

[0582]

[0583] This compound was prepared using a procedure similar to that described for Example A6, Step 4, substituting 2-(2-fluoro-6-vinylphenyl)-2-hydroxyacetohydrazide for 2-(2,6-difluorophenyl)-2-hydroxyacetohydrazide. 21 H 15 F2N6O(M+H) + LCMS calculated value: 405.1; found value: 405.1.

[0584] Step 5: 3-(5-amino-2-((2-fluoro-6-formylphenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile

[0585]

[0586] Osmium tetroxide (4% w / w, 0.36 mL, 0.12 mmol) in water was added to a solution of 3-(5-amino-2-((2-fluoro-6-vinylphenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile (930 mg, 2.30 mmol) in THF (18 mL) and water (4.6 mL). The reaction mixture was stirred at RT for 5 min, then sodium periodate (2.5 g, 11.5 mmol) was added. After stirring for 1 h, the mixture was diluted with sodium metabisulfite in saturated aqueous NaHCO3 (5% w / w, 20 mL) and extracted with EtOAc (×3). The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. The crude material was purified by column chromatography eluting with 0 to 100% ethyl acetate in hexanes to provide the desired product. For C 20 H 13 F2N6O2(M+H) + LCMS calculated value: 407.1; found value: 407.1.

[0587] Step 6: 3-(5-amino-2-((2-fluoro-6-(((1-methyl-2-oxopyrrolidin-3-yl)amino)methyl)phenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile

[0588] A solution of 3-amino-1-methylpyrrolidin-2-one (63 mg, 0.55 mmol) and 3-(5-amino-2-((2-fluoro-6-formylphenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile (150 mg, 0.37 mmol) in 1,2-dichloroethane (1.9 mL) was stirred at 40 °C for 2 h. Sodium triacetoxyborohydride (160 mg, 0.74 mmol) was then added and the reaction mixture was stirred at room temperature for 16 h. The reaction was diluted with saturated NaHCO3 and the organics were extracted with EtOAc (3×). The combined organics were dried over MgSO4 and concentrated. The diastereomers were separated by chiral HPLC using a Phenomenex LuxCelluose-4 column (21.2×250mm, 5μm particle size) with an isocratic mobile phase of 45% EtOH in hexane at a flow rate of 20mL / min. The retention times of peak 1 and peak 2 were 14.9min and 17.5min, respectively. After concentration, peak 2 was further separated by chiral HPLC using a Phenomenex Lux Celluose-1 column (21.2×250mm, 5μm particle size) with an isocratic mobile phase of 30% EtOH in hexane at a flow rate of 20mL / min. The retention times of peak 1 and peak 2 were 11.0min and 15.5min, respectively. After concentration, peak 1 was purified by preparative LC-MS (pH=2, MeCN / water containing TFA) to obtain the desired product in the form of a TFA salt. For C 25 H 23 F2N8O2(M+H) + LC-MS calculated value: 505.2; found value: 505.2.

[0589] Example A9: Synthesis of 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (Compound 9)

[0590]

[0591] Step 1: 3-Bromo-1-(2-(3-cyanophenyl)-2-oxoethyl)-1H-1,2,4-triazole-5-carboxylic acid methyl ester

[0592]

[0593] To a solution of 3-bromo-1H-1,2,4-triazole-5-carboxylic acid methyl ester (5.0 g, 24.3 mmol) and 3-(2-bromoacetyl)benzonitrile (5.44 g, 24.3 mmol) in DMF (100 mL) was added potassium carbonate (3.35 g, 24.3 mmol). The reaction mixture was stirred at ambient temperature for 2 h. The reaction mixture was then diluted with water and DCM. The organic layer was separated, washed with brine, dried over Na2SO4, filtered and concentrated. The resulting residue was purified by flash chromatography to obtain the desired product as a white solid (5.2 g, 61%). For C 13 H 10 BrN4O3(M+H) + LC-MS calculated value: m / z = 349.0; found value: 349.0.

[0594] Step 2: 3-(2-Bromo-8-oxo-7,8-dihydro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0595]

[0596] Methyl 3-bromo-1-(2-(3-cyanophenyl)-2-oxoethyl)-1H-1,2,4-triazole-5-carboxylate (10.5 g, 30.1 mmol) was dissolved in acetic acid (100 mL), and ammonium acetate (23.18 g, 301 mmol) was added. The mixture was stirred at 110 °C for 12 h. After cooling to room temperature, the reaction mixture was diluted with water. The resulting precipitate was collected by filtration, washed with water, and dried under vacuum to provide the product (8.4 g, 88%). For C 12 H7BrN5O(M+H) + LC-MS calculated value: m / z = 316.0; found value: 316.0.

[0597] Step 3: 3-(2-Bromo-8-chloro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0598]

[0599] A mixture of 3-(2-bromo-8-oxo-7,8-dihydro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (8.4 g, 26.6 mmol) and POCl3 (49.5 mL, 531 mmol) was stirred at 110°C overnight. After cooling to room temperature, the reaction mixture was slowly added to a flask containing ice and sodium bicarbonate. The resulting precipitate was collected, washed with water, and dried to provide the product (8.8 g, 99%). For C 12 H6BrClN5(M+H)+ LC-MS calculated value: m / z = 333.9; found value: 334.0.

[0600] Step 4. 3-(8-(Bis(4-methoxybenzyl)amino)-2-bromo-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0601]

[0602] A mixture of 3-(2-bromo-8-chloro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (8.99 g, 26.9 mmol), bis(4-methoxybenzyl)amine (10.37 g, 40.3 mmol) and DIPEA (9.4 mL, 53.7 mmol) in DMF (134 mL) was stirred at 85 °C overnight. The reaction mixture was cooled to room temperature and diluted with water. The resulting precipitate was collected by filtration and dried to provide the product (14.1 g, 94%). For C 28 H 24 BrN6O2(M+H) + LC-MS calculated value: m / z = 555.1; found value: 555.1.

[0603] Step 5: 3-(8-(bis(4-methoxybenzyl)amino)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0604]

[0605] To a solution of 2-methylpyridine (0.050 g, 0.540 mmol) in THF (0.5 mL) was added 2.5 M n-butyllithium (0.216 mL, 0.540 mmol) at -78° C. The resulting solution was stirred at the same temperature for 1 h, then 1.9 M zinc chloride in 2-methyltetrahydrofuran (0.284 mL, 0.540 mmol) was added, and the resulting mixture was stirred at room temperature for 10 min.

[0606] A microwave vial charged with 3-(8-(bis(4-methoxybenzyl)amino)-2-bromo-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (0.15 g, 0.270 mmol), palladium acetate (1.1 mg, 4.7 μmol) and 2'-(dicyclohexylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6-diamine (4.1 mg, 9.5 μmol) was evacuated under high vacuum and backfilled with nitrogen. THF (2.0 mL) and toluene (0.5 mL) were then added to the reaction vial. The mixture was cooled to 0°C and the zinc reagent prepared from the previous step was slowly added via a syringe. The reaction mixture was then stirred at 60°C overnight, cooled to room temperature, and partitioned between ethyl acetate and saturated NH4Cl solution. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over MgSO4, and concentrated. The resulting residue was purified via flash chromatography to provide the product (0.11 g, 71%). 34 H 30 N7O2(M+H) + LC-MS calculated value: m / z = 568.2; found value: 568.3.

[0607] Step 6. 3-(8-amino-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0608]

[0609] A mixture of 3-(8-(bis(4-methoxybenzyl)amino)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (110 mg, 0.194 mmol) and TFA (746 μL, 9.69 mmol) was stirred at 80° C. for 30 min, cooled to room temperature, and concentrated. The resulting residue was purified by preparative LCMS (pH 2) to give the product (TFA salt) as a white solid (57 mg, 90%). 18 H 14 N7(M+H) + LC-MS calculated value: m / z = 328.1; found value: 328.1.

[0610] Step 7. 3-(8-amino-5-bromo-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0611]

[0612] To a solution of 3-(8-amino-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (TFA salt) (35 mg, 0.079 mmol) in DMF (0.5 mL) / DCM (0.5 mL) was added NBS (14.1 mg, 0.079 mmol). The reaction mixture was then stirred at room temperature for 1 h and concentrated to afford the crude product, which was used in the next step without further purification. For C 18 H 13 BrN7(M+H) + LC-MS calculated value: m / z = 406.0; found value: 406.0.

[0613] Step 8. 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0614] A mixture of 6-chloro-2-methylpyridazin-3(2H)-one (30 mg, 0.21 mmol), bis(pinacol)diboron (53 mg, 0.21 mmol), chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (15.7 mg, 0.02 mmol) (XPhos Pd G2) and potassium acetate (61.7 mg, 0.63 mmol) in 1,4-dioxane (1 mL) was stirred at 100° C. for 1 h. Then 3-(8-amino-5-bromo-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (10 mg, 0.025 mmol), cesium carbonate (37.6 mg, 0.116 mmol) and water (0.2 mL) were added to the reaction mixture. The resulting mixture was heated at 90 ° C for 1 h. The mixture was concentrated and purified by preparative LCMS (pH 2, acetonitrile / water containing TFA) to provide the desired product in the form of a TFA salt. For C 23 H 18 N9O(M+H) + LCMS calculated value: 436.2; found value: 436.2.

[0615] 1H NMR (500MHz, DMSO) δ8.66-8.62(d,J=5.1Hz,1H),8.09-8.02(d,J=1.8Hz,1H),7.88-7.85(t,J=1.8Hz,1H),7.85-7.81(m ,3H),7.78-7.72(d,J=9.6Hz,1H),7.66-7.51(m,4H),7.10-7.06(d,J=9.6Hz,1H),4.59-4.48(s,2H),3.53-3.43(s,3H).

[0616] Example A10: Synthesis of 3-(8-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-5-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (Compound 10)

[0617]

[0618] Step 1: 3-Bromo-1-(2-(3-cyanophenyl)-2-oxoethyl)-1H-1,2,4-triazole-5-carboxylic acid methyl ester

[0619]

[0620] To a solution of 3-bromo-1H-1,2,4-triazole-5-carboxylic acid methyl ester (5.0 g, 24.3 mmol) and 3-(2-bromoacetyl)benzonitrile (5.44 g, 24.3 mmol) in DMF (100 mL) was added potassium carbonate (3.35 g, 24.3 mmol). The reaction mixture was stirred at ambient temperature for 2 h. The reaction mixture was then diluted with water and DCM. The organic layer was separated, washed with brine, dried over Na2SO4, filtered and concentrated. The resulting residue was purified by flash chromatography to obtain the desired product as a white solid (5.2 g, 61%). For C 13 H 10 BrN4O3(M+H) + LC-MS calculated value: m / z = 349.0; found value: 349.0.

[0621] Step 2: 3-(2-Bromo-8-oxo-7,8-dihydro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0622]

[0623] 3-Bromo-1-(2-(3-cyanophenyl)-2-oxoethyl)-1H-1,2,4-triazole-5-carboxylic acid methyl ester (10.5 g, 30.1 mmol) was dissolved in acetic acid (100 mL), and ammonium acetate (23.18 g, 301 mmol) was added. The mixture was stirred at 110 °C for 12 h. After cooling to room temperature, the reaction mixture was diluted with water. The resulting precipitate was collected by filtration, washed with water, and dried under vacuum to provide the product (8.4 g, 88%). For C 12 H7BrN5O(M+H) + LC-MS calculated value: m / z = 316.0; found value: 316.0.

[0624] Step 3: 3-(2-Bromo-8-chloro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0625]

[0626] A mixture of 3-(2-bromo-8-oxo-7,8-dihydro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (8.4 g, 26.6 mmol) and POCl3 (49.5 mL, 531 mmol) was stirred at 110°C overnight. After cooling to room temperature, the reaction mixture was slowly added to a flask containing ice and sodium bicarbonate. The resulting precipitate was collected by filtration, washed with water, and dried to provide the product (8.8 g, 99%). For C 12 H6BrClN5(M+H) + LC-MS calculated value: m / z = 336.0; found value: 336.0.

[0627] Step 4: 3-(8-(bis(4-methoxybenzyl)amino)-2-bromo-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0628]

[0629] A mixture of 3-(2-bromo-8-chloro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (8.99 g, 26.9 mmol), bis(4-methoxybenzyl)amine (10.37 g, 40.3 mmol) and DIPEA (9.4 mL, 53.7 mmol) in DMF (134 mL) was stirred at 65 °C overnight. The reaction mixture was cooled to room temperature and diluted with water. The resulting precipitate was collected by filtration and dried to provide the product (14.1 g, 94%). For C 28 H 24 BrN6O2(M+H) +LC-MS calculated value: m / z = 555.1; found value: 555.1.

[0630] Step 5: 3-(8-(bis(4-methoxybenzyl)amino)-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0631]

[0632] A mixture of 3-(8-(bis(4-methoxybenzyl)amino)-2-bromo-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (10.0 g, 18.0 mmol), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (3.88 g, 25.2 mmol), tripotassium phosphate (9.55 g, 45.0 mmol) and chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (567 mg, 0.72 mmol) in 1,4-dioxane (200 mL) and water (50 mL) was stirred at 85° C. for 2 hr. The reaction mixture was cooled to room temperature and most of the 1,4-dioxane was removed. The resulting precipitate was collected by filtration, washed with water and dried to afford the crude product (9.1 g), which was used directly in the next step. 30 H 27 N6O2(M+H) + LC-MS calculated value: m / z = 503.2; found value: 503.1.

[0633] Step 6. 3-(8-(Bis(4-methoxybenzyl)amino)-5-bromo-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0634]

[0635] To a solution of 3-(8-(bis(4-methoxybenzyl)amino)-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (717 mg, 1.43 mmol) in 10 mL of dichloromethane at 0°C was added 1-bromopyrrolidine-2,5-dione (254 mg, 1.43 mmol). The resulting mixture was stirred for 4 hrs and directly purified by silica gel column to afford the desired product (780 mg, 94%). For C 30 H 26 BrN6O2(M+H) + LC-MS calculated value: m / z = 581.1; found value: 581.2.

[0636] Step 7: 3-(8-(bis(4-methoxybenzyl)amino)-5-(pyrimidin-4-yl)-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0637]

[0638] A mixture of 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (260 mg, 0.45 mmol), 4-(tributylstannyl)pyrimidine (215 mg, 0.58 mmol), lithium chloride (28.4 mg, 0.67 mmol), copper(I) chloride (67 mg, 0.67 mmol) and tetrakis(triphenylphosphine)palladium(0) (52 mg, 0.045 mmol) in THF (5 mL) was stirred at 90 °C for 45 min. The reaction mixture was quenched with water and extracted with dichloromethane. The combined organic layers were concentrated and purified by silica gel column to provide the desired product (176 mg, 67%). For C 34 H 29 N8O2(M+H) + LC-MS calculated value: m / z = 581.2; found value: 581.1.

[0639] Step 8: 3-(8-(bis(4-methoxybenzyl)amino)-2-formyl-5-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0640]

[0641] A mixture of 3-(8-(bis(4-methoxybenzyl)amino)-5-(pyrimidin-4-yl)-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (176 mg, 0.3 mmol), osmium(VIII) oxide (3 mg in 0.3 mL water, 0.015 mmol) and sodium periodate (292 mg, 1.36 mmol) in THF / water (1:1, 6 mL) was stirred at 65 °C for 1 h. The reaction mixture was cooled to room temperature and extracted with dichloromethane. The combined organic layers were concentrated and purified by silica gel column to provide the desired product (130 mg, 74%). For C 33 H 27 N8O3(M+H) + LC-MS calculated value: m / z = 583.2; found value: 583.2.

[0642] Step 9: 3-(8-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-5-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0643] Preparation of Grignard reagent: To a solution of 1,3-difluoro-2-iodobenzene (142 mg, 0.6 mmol) in tetrahydrofuran (1 mL) was added isopropylmagnesium chloride solution (296 μl, 2 M) at -10° C. The resulting mixture was stirred for 1 h and used directly in the next step.

[0644] To a solution of 3-(8-(bis(4-methoxybenzyl)amino)-2-formyl-5-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (120 mg, 0.2 mmol) in THF (2 mL) at -10 °C was added the Grignard reagent freshly prepared from the previous step. The reaction mixture was stirred for 30 min, quenched with ammonium chloride solution (4 mL), and extracted with dichloromethane. The combined organic layers were concentrated under vacuum. The resulting material was dissolved in TFA (5 mL) and stirred at 80 °C for 20 min. The reaction mixture was then cooled to room temperature, concentrated, and basified by the addition of aqueous NaHCO3.

[0645] The crude material was directly purified by silica gel column to afford the desired product (60 mg, 64%) as a racemic mixture. The product was then isolated by chiral HPLC using a chiral column (Phenomenex Lux 5um Cellulose-4, 21.2x250 mm) and a 75% EtOH (20 mL / min) solvent system in hexanes.

[0646] Peak 2 was isolated and further purified via preparative LC / MS (pH=2, acetonitrile / water containing TFA) to afford the desired product as a TFA salt. 23 H 15 F2N8O(M+H) + LC-MS calculated value: m / z = 457.1; found value: 457.0.

[0647] 1H NMR (600MHz, DMSO-d6) δ9.14(d,J=1.3Hz,1H),8.95(d,J=5.2Hz,1H),7.90(dd,J=5.2,1.4Hz,1H),7.88(s,1H),7.78(dt,J= 7.6,1.4Hz,1H),7.74(t,J=1.4Hz,1H),7.54(dt,J=7.9,1.3Hz,1H),7.51-7.40(m,2H),7.09(t,J=8.4Hz,2H),6.27(s,1H).

[0648] Example A11: Synthesis of 3-(8-amino-2-(amino(2,6-difluorophenyl)methyl)-5-(4-methyloxazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (Compound 11)

[0649]

[0650] Step 1: 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0651]

[0652] To a solution of 3-(8-(bis(4-methoxybenzyl)amino)-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (Example A10 Step 5; 241 mg, 0.48 mmol) in DCM (5 mL) was added NBS (84.6 mg, 0.48 mmol). The reaction mixture was then stirred at room temperature for 1 h and concentrated to provide the crude product, which was used in the next step without further purification. For C 30 H 26 BrN6O2(M+H) + LC-MS calculated value: m / z = 581.1; found value: 581.1.

[0653] Step 2: 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-formyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0654]

[0655] A mixture of 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (174 mg, 0.3 mmol), osmium(VIII) oxide (3 mg in 0.3 mL water, 0.015 mmol) and sodium periodate (292 mg, 1.36 mmol) in THF / water (1:1, 6 mL) was stirred at 65 °C for 1 h. The reaction mixture was cooled to room temperature and extracted with dichloromethane. The combined organic layers were concentrated and purified by silica gel column to provide the desired product. For C 29 H 24 N6O3Br(M+H) + LC-MS calculated value: m / z = 583.1; found value: 583.1.

[0656] Step 3: 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0657]

[0658] Preparation of Grignard reagent: To a solution of 1,3-difluoro-2-iodobenzene (142 mg, 0.6 mmol) in tetrahydrofuran (1 mL) was added isopropylmagnesium chloride solution (296 μl, 2 M) at -10° C. The resulting mixture was stirred for 1 h and used directly in the next step.

[0659] To a solution of 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-formyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (120 mg, 0.2 mmol) in THF (2 mL) at -10 °C was added the Grignard reagent freshly prepared from the previous step. The reaction mixture was stirred for 30 min, quenched with ammonium chloride solution (4 mL), and extracted with dichloromethane. The combined organic layers were concentrated under vacuum and purified by silica gel column to afford the desired product as a racemic mixture. For C 35 H 28 N6O3BrF2(M+H) + LC-MS calculated value: m / z = 697.1; found value: 697.1.

[0660] Step 4: 3-(8-(bis(4-methoxybenzyl)amino)-2-((2,6-difluorophenyl)(hydroxy)methyl)-5-(4-methyloxazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0661]

[0662] A mixture of 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (382 mg, 0.55 mmol), 4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)oxazole (137 mg, 0.65 mmol), dicyclohexyl(2',4',6'-triisopropylbiphenyl-2-yl)phosphine-(2'-aminobiphenyl-2-yl)(chloro)palladium (1:1) (17 mg, 21.6 μmol) and Cs2CO3 (356 mg, 1.09 mmol) in 1,4-dioxane (2 mL) and water (200 μl) was purged with N2 and heated at 95°C for 7 h. The mixture was concentrated and purified via flash chromatography to afford the desired product as a colorless oil. 39 H 32 N7O4F2(M+H) + LCMS calculated value: 700.2; found value: 700.2.

[0663] Step 5: 3-(8-(bis(4-methoxybenzyl)amino)-2-(chloro(2,6-difluorophenyl)methyl)-5-(4-methyloxazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0664]

[0665] To a solution of 3-(8-(bis(4-methoxybenzyl)amino)-2-((2,6-difluorophenyl)(hydroxy)methyl)-5-(4-methyloxazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (201 mg, 0.29 mmol) in 2 mL of dichloromethane was added thionyl chloride (105 μl, 1.435 mmol) at rt. The resulting mixture was stirred for 4 h, concentrated, and used in the next step without any further purification. For C 39 H 31 N7O3ClF2(M+H) + LC-MS calculated value: m / z = 718.2; found value: 718.2.

[0666] Step 6: 3-(8-amino-2-(amino(2,6-difluorophenyl)methyl)-5-(4-methyloxazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile

[0667] Ammonia solution (1 mL) was added to a solution of 3-(8-(bis(4-methoxybenzyl)amino)-2-(chloro(2,6-difluorophenyl)methyl)-5-(4-methyloxazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrazine-6-yl)benzonitrile (40 mg, 0.084 mmol) in 1 mL DMSO. The mixture was heated at 100 ° C for 10 h using microwave conditions, then diluted with water and extracted with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO4, and concentrated. The resulting residue was dissolved in TFA (1 mL) and stirred at 80 ° C for 20 min. The reaction mixture was then cooled to room temperature, concentrated, and alkalized by adding NaHCO3 aqueous solution. The crude material was directly purified by silica gel column to provide the desired product in the form of a racemic mixture. The product was then separated using a chiral column (AM-1) and a 45% EtOH (20 mL / min) solvent system in hexane using chiral HPLC. Peak 1 was isolated and further purified via preparative LC / MS (pH=2, acetonitrile / water containing TFA) to afford the desired product as a TFA salt. 23 H 17 F2N8O(M+H) + LC-MS calculated value: m / z = 459.1; found value: 459.0.

[0668] Example A12: Synthesis of 3-(8-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-5-(2,6-dimethylpyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (Compound 12)

[0669]

[0670] To 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile (Example A11, Step 3; 0.518 g, 0.638 mmol), 2,6-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane- To a solution of 2',4',6'-triisopropylbiphenyl-2-yl)pyridine (0.346 g, 1.48 mmol) and dicyclohexyl (2',4',6'-triisopropylbiphenyl-2-yl)phosphine-(2'-aminobiphenyl-2-yl)(chloro)palladium (1:1) (0.058 g, 0.074 mmol) in dioxane (3.0 mL) and water (0.60 mL) was added potassium phosphate (0.472 g, 2.23 mmol). The reaction mixture was stirred at 90 ° C for 1 h. The reaction mixture was then diluted with water and DCM. The layers were separated, the aqueous layer was extracted with DCM, and the combined organic fractions were dried over MgSO4, filtered and concentrated. The crude material was dissolved in TFA (5 mL), heated to 80 ° C and kept for 20 minutes. The reaction mixture was then cooled to room temperature, concentrated, and alkalized by adding NaHCO3 aqueous solution. The crude material was directly purified by silica gel column to provide the desired product (257 mg, 72%) in the form of a racemic mixture.

[0671] The product was then isolated using chiral HPLC using a chiral column (Phenomenex Lux 5um Cellulose-2, 21.1×250 mm) and a 35% EtOH (20 mL / min) solvent system in hexane. Peak 2 was isolated and further purified using preparative LC / MS (pH=2, acetonitrile / water containing TFA) to obtain the desired product as a TFA salt. For C 26 H 20 F2N7O(M+H) + LC-MS calculated value: m / z = 484.2; found value: 484.2. 1 H NMR(500MHz,DMSO-d6)δ7.92(s,2H),7.85(s,1H),7.83(d,J=7.6Hz,1H),7.56(d, J=8.0Hz,1H),7.53-7.40(m,4H),7.10(t,J=8.4Hz,2H),6.27(s,1H),2.51(s,6H).

[0672] Example A13: Synthesis of 3-(4-amino-2-(pyridin-2-ylmethyl)-7-(pyrimidin-4-yl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile (Compound 13)

[0673]

[0674] Step 1. 4,6-Dichloro-3H-[1,2,3]triazolo[4,5-c]pyridine

[0675]

[0676] At 0 ° C, a solution of NaNO2 (3.88 g, 56.2 mmol) in water (3 mL) was added to a solution of 2,6-dichloropyridine-3,4-diamine (10 g, 56 mmol) in 37% hydrochloric acid (5 mL). The solution was stirred for 30 min. Water (20 mL) was added and the white precipitate was filtered, washed with water, and dried to obtain the desired product. LC-MS calculated for C5H3Cl2N4: 189.0 (M+H) + ; Exp. value: 189.0 (M+H) + .

[0677] Step 2. 6-Chloro-N-(2,4-dimethoxybenzyl)-3H-[1,2,3]triazolo[4,5-c]pyridin-4-amine

[0678]

[0679] A mixture of 4,6-dichloro-3H-[1,2,3]triazolo[4,5-c]pyridine (600 mg, 3.17 mmol), (2,4-dimethoxyphenyl)methanamine (0.53 mL, 3.49 mmol) and triethylamine (0.53 mL, 3.81 mmol) in 1,4-dioxane (10 mL) was stirred at 110 °C for 3 days. Direct purification on a silica gel column afforded the desired product (875 mg, 86%). 14 H 15 LC-MS calculated value of ClN5O2: 320.1 (M+H) + ; Experimental value: 320.3 (M+H) + .

[0680] Step 3. 6-Chloro-N-(2,4-dimethoxybenzyl)-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-4-amine

[0681]

[0682] To a mixture of 6-chloro-N-(2,4-dimethoxybenzyl)-3H-[1,2,3]triazolo[4,5-c]pyridin-4-amine (875 mg, 2.74 mmol), pyridin-2-ylmethanol (0.317 mL, 3.28 mmol) and triphenylphosphine (1436 mg, 5.47 mmol) in DCM (20 mL) at 0°C was added diisopropyl azodicarboxylate (0.647 mL, 3.28 mmol). The resulting mixture was stirred at 0°C for 1 h. Direct purification on a silica gel column provided the desired product (375 mg, 33.4% yield). For C 20 H 20 LC-MS calculated value of ClN6O2: 411.1 (M+H) + ; Exp. value: 411.2 (M+H) + .

[0683] Step 4. 3-(4-((2,4-dimethoxybenzyl)amino)-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile

[0684]

[0685] To a mixture of 6-chloro-N-(2,4-dimethoxybenzyl)-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-4-amine (375 mg, 0.913 mmol) and (3-cyanophenyl)boronic acid (268 mg, 1.825 mmol) in 1,4-dioxane (10 mL) and water (1.00 mL) was added cesium carbonate (595 mg, 1.825 mmol). The resulting mixture was purged with N2, and then chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (71.8 mg, 0.091 mmol) was added. The reaction mixture was stirred at 120°C for 90 min under microwave irradiation. The reaction was quenched with 20 mL of ethyl acetate and 20 mL of water. The organic phase was separated and the aqueous solution was extracted twice with ethyl acetate. The combined extracts were dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified on a silica gel column to afford the desired product (300 mg, 68.9%). 27 H 24 LC-MS calculated value of N7O2: 478.2 (M+H) + ; Exp. value: 478.3 (M+H) + .

[0686] Step 5. 3-(4-amino-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile

[0687]

[0688] A solution of 3-(4-((2,4-dimethoxybenzyl)amino)-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile (300.3 mg, 0.629 mmol) in TFA (5 mL) was stirred at 100 ° C for 30 min. TFA was evaporated under reduced pressure, and then 20 mL of saturated NaHCO3 aqueous solution and 20 mL of ethyl acetate were added. The organic phase was separated and the aqueous solution was extracted twice with ethyl acetate. The combined extracts were dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified on a silica gel column to provide the desired product (175 mg, 85%). For C 18 H 14 LC-MS calculated value of N7: 328.1 (M+H) + ; Exp. value: 328.2 (M+H) + .

[0689] Step 6. 3-(4-amino-7-bromo-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile

[0690]

[0691] A mixture of 3-(4-amino-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile (175 mg, 0.535 mmol) and 1-bromopyrrolidine-2,5-dione (100 mg, 0.561 mmol) in THF (10 mL) was stirred at 0 ° C for 30 min and then quenched with saturated aqueous NaHCO3. The organic phase was separated, dried over Na2SO4, filtered and evaporated under reduced pressure. The resulting residue was purified on a silica gel column to provide the desired product (135 mg, 62.2%). For C 18 H 13 LC-MS calculated value of BrN7: 406.0 (M+H) + and 408.0(M+H) + ; Experimental value: 406.1 (M+H) + and 408.2(M+H) + .

[0692] Step 7. 3-(4-amino-2-(pyridin-2-ylmethyl)-7-(pyrimidin-4-yl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile

[0693]

[0694] A mixture of 3-(4-amino-7-bromo-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile (182 mg, 0.448 mmol), 4-(tributylstannyl)pyrimidine (496 mg, 1.344 mmol) and copper(I) chloride (53.2 mg, 0.538 mmol), lithium chloride (22.79 mg, 0.538 mmol) and tetrakis(triphenylphosphine)palladium(0) (51.8 mg, 0.045 mmol) in THF (1 ml) was first purged with N2 and then heated and stirred at 90°C for 2 h. The reaction was diluted with methanol and purified by preparative LCMS (pH=2) to obtain the desired product. For C 22 H 16 LC-MS calculated value of N9: 406.2 (M+H) + ; Experimental value: 406.2 (M+H) + .

[0695] Example A14: Synthesis of 3-(4-amino-2-((3-fluoropyridin-2-yl)methyl)-7-(pyrimidin-4-yl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile (Compound 14)

[0696]

[0697] Step 1. 6-Chloro-N-(2,4-dimethoxybenzyl)-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-4-amine

[0698]

[0699] To a mixture of 6-chloro-N-(2,4-dimethoxybenzyl)-3H-[1,2,3]triazolo[4,5-c]pyridin-4-amine (Example A13 Step 2; 1000 mg, 3.13 mmol), (3-fluoropyridin-2-yl)methanol (477 mg, 3.75 mmol) and triphenylphosphine (1641 mg, 6.25 mmol) in DCM (1.7 mL) at 0°C was added diisopropyl azodicarboxylate (739 μl, 3.75 mmol). The reaction mixture was stirred at 0°C for 1 h. Direct purification on a silica gel column afforded the desired product (433 mg, 32%). For C 20 H 19 LC-MS calculated value of ClFN6O2: 429.1 (M+H) + ; Exp. value: 429.3 (M+H) + .

[0700] Step 2. 3-(4-((2,4-dimethoxybenzyl)amino)-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile

[0701]

[0702] Cesium carbonate (658 mg, 2.019 mmol) was added to a mixture of 6-chloro-N-(2,4-dimethoxybenzyl)-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-4-amine (433 mg, 1.010 mmol) and (3-cyanophenyl)boronic acid (297 mg, 2.019 mmol) in 1,4-dioxane (10.0 mL) and water (1.0 mL). The resulting mixture was filled with N2 for 2 min and (SP-4-4)-[2'-amino[1,1'-biphenyl]-2-yl]chloro[dicyclohexyl[2',4',6'-tris(1-methylethyl)[1,1'-biphenyl]-2-yl]phosphine]palladium (79 mg, 0.101 mmol) was added. The reaction mixture was stirred at 120 ° C for 1.5 h under microwave irradiation. The reaction was quenched with 20 mL of ethyl acetate and 20 mL of water. The organic phase was separated and the aqueous solution was extracted twice with ethyl acetate. The combined extracts were dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified on a silica gel column to provide the desired product (357 mg, 71%). For C 27 H 23 LC-MS calculated value of FN7O2: 496.2 (M+H) + ; Exp. value: 496.3 (M+H) + .

[0703] Step 3. 3-(4-amino-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile

[0704]

[0705] A solution of 3-(4-((2,4-dimethoxybenzyl)amino)-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile (357.3 mg, 0.721 mmol) in TFA (5 mL) was stirred at 100 °C for 1 h. TFA was evaporated under reduced pressure, and then 20 mL of saturated aqueous NaHCO3 and 20 mL of ethyl acetate were added. The organic phase was separated and the aqueous solution was extracted twice with ethyl acetate. The combined extracts were dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified on a silica gel column to provide the desired product (213 mg, 61%). For C 18 H 13 LC-MS m / z calculated for FN7: 346.1 (M+H) + ; Exp. value: 346.3 (M+H) + .

[0706] Step 4. 3-(4-amino-7-bromo-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile

[0707]

[0708] A mixture of 3-(4-amino-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile (213 mg, 0.617 mmol) and 1-bromopyrrolidine-2,5-dione (220 mg, 1.234 mmol) in THF (5 mL) was stirred at 0 °C for 1 h. Direct purification on silica gel afforded the desired product (175 mg, 67%). 18 H 12 LC-MS calculated value of BrFN7: 424.0 (M+H) + and 426.0(M+H) + ; Exp. value: 424.3 (M+H) + and 426.3 (M+H) + .

[0709] Step 5. 3-(4-amino-2-((3-fluoropyridin-2-yl)methyl)-7-(pyrimidin-4-yl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile

[0710]

[0711] A mixture of 3-(4-amino-7-bromo-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile (220 mg, 0.519 mmol), 4-(tributylstannyl)pyrimidine (383 mg, 1.037 mmol) and copper(I) chloride (61.6 mg, 0.622 mmol), lithium chloride (26.4 mg, 0.622 mmol) and tetrakis(triphenylphosphine)palladium(0) (59.9 mg, 0.052 mmol) in THF (1 ml) was first purged with N2 and then heated and stirred at 90°C for 2 h. The reaction was diluted with methanol and purified by preparative LCMS (pH = 2) to obtain the desired product. For C 22 H 15 LC-MS calculated value of FN9: 424.1 (M+H) + ; Exp. value: 424.3 (M+H) + . 1 H NMR (500 MHz, DMSO- )ppm 8.98(s,1H),8.77(d,J=5.02Hz,1H),8.38(dd,J1=4.60Hz,J2=1.32Hz,1H),7.90-8.30(bs,2 H),7.76-7.89(m,3H),7.66(dd,J1=5.25Hz,J2=1.25Hz,1H),7.45-7.58(m,3H),6.25(s,2H).

[0712] Example A15: Synthesis of 3-(4-amino-2-((3-fluoropyridin-2-yl)methyl)-7-(pyridin-4-yl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile (Compound 15)

[0713]

[0714] Cesium carbonate (46.1 mg, 0.141 mmol) was added to a mixture of 3-(4-amino-7-bromo-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile (30 mg, 0.071 mmol) and pyridin-4-ylboronic acid (17.38 mg, 0.141 mmol) in 1,4-dioxane (2 mL) and water (0.2 mL). The resulting mixture was filled with N22 min and chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (5.56 mg, 7.07 μmol) was added. The reaction mixture was stirred at 120° C. for 1.5 h under microwave irradiation. The reaction mixture was diluted with methanol. Direct purification on preparative HPLC afforded the desired product. 23 H 16 LC-MS calculated value of FN8: 423.1 (M+H) + ; Exp. value: 423.3 (M+H) + .

[0715] Example A16: Synthesis of 3-(4-amino-7-(1-methyl-1H-pyrazol-5-yl)-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)-2-fluorobenzonitrile (Compound 16)

[0716]

[0717] Step 1. 3-(4-amino-7-bromo-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)-2-fluorobenzonitrile

[0718]

[0719] This compound was prepared by following a procedure similar to Example A13 Step 1 to Step 6, substituting (3-cyano-2-fluorophenyl)boronic acid for (3-cyanophenyl)boronic acid in Step 4. 18 H 12 LC-MS calculated value of BrFN7: 424.0 (M+H) + and 426.0(M+H) + ; Exp. value: 424.3 (M+H) + and 426.3 (M+H) + .

[0720] Step 2. 3-(4-amino-7-(1-methyl-1H-pyrazol-5-yl)-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)-2-fluorobenzonitrile

[0721]

[0722] This compound was prepared by following a procedure similar to that in Example A15, substituting (1-methyl-1H-pyrazol-5-yl)boronic acid for pyridin-4-ylboronic acid and substituting 3-(4-amino-7-bromo-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)-2-fluorobenzonitrile for 3-(4-amino-7-bromo-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile. 22 H 17 LC-MS calculated value of FN9: 426.2 (M+H) + ; Exp. value: 426.3 (M+H) + .

[0723] Example A17: Synthesis of 7-(1-((5-chloropyridin-3-yl)methyl)-1H-pyrazol-4-yl)-3-methyl-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (Compound 17)

[0724]

[0725] Step 1: Ethyl 3-(pentylamino)-1H-pyrrole-2-carboxylate

[0726]

[0727] At room temperature, ethyl 3-amino-1H-pyrrole-2-carboxylate (5 g, 32.4 mmol), valeraldehyde (3.79 ml, 35.7 mmol) and sodium cyanoborohydride (2.038 g, 32.4 mmol) were mixed in methanol (64.9 ml) overnight. The reaction mixture was concentrated under reduced pressure. The crude residue was purified by flash chromatography (0 to 100% EtOAc in hexane) to obtain the desired product (4.4 g, 61%). For C 12 H 21 LCMS calculated for N2O2 (M+H): 225.2. Found: 225.1.

[0728] Step 2: Ethyl 3-(3-(ethoxycarbonyl)-1-pentylthioureido)-1H-pyrrole-2-carboxylate

[0729]

[0730] The bottle was charged with ethyl 3-(pentylamino)-1H-pyrrole-2-carboxylate (4.4 g, 19.62 mmol), dichloromethane (39.2 ml) and ethoxycarbonyl isothiocyanate (2.78 ml, 23.54 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was quenched with water (40 ml) and the layers were separated. The aqueous layer was extracted with dichloromethane (3×40 mL) and the combined organic fractions were dried over MgSO4, filtered, and concentrated. The crude material was used in the next step without further purification (7.3 g, quantitative). For C 16 H 26 LCMS calculated for N3O4S (M+H): 356.2. Found: 356.1.

[0731] Step 3: 1-Pentyl-2-thione-2,3-dihydro-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one

[0732]

[0733] A microwave vial was charged with ethyl 3-(3-(ethoxycarbonyl)-1-pentylthioureido)-1H-pyrrole-2-carboxylate (7.31 g, 20.57 mmol) and sodium ethoxide (21% w / w, 8.45 ml, 22.62 mmol) solution. The vial was capped and heated in a microwave reactor at 120 °C for 10 minutes. The reaction mixture was brought to neutral pH by adding 1 M HCl solution, and the solid product (3.1 g, 64%) was filtered and dried. 11 H 16 LCMS Calcd. for N3OS (M+H): 238.1. Found: 238.1.

[0734] Step 4: 2-Hydrazono-1-pentyl-2,3-dihydro-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one

[0735]

[0736] A bottle was charged with 1-pentyl-2-thione-2,3-dihydro-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one (3.13 g, 13.19 mmol) and hydrazine hydrate (20 mL). The reaction mixture was stirred at 100 °C overnight. The solid formed was filtered and washed with water to obtain the desired product (2.2 g, 70%). 11 H 18 LCMS calcd for N5O (M+H): 236.1. Found: 236.1.

[0737] Step 5: 3-Methyl-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one

[0738]

[0739] The bottle was charged with (E)-2-hydrazono-1-pentyl-2,3-dihydro-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one (4.8 g, 20.40 mmol), a drop of trifluoroacetic acid and triethyl orthoacetate (20 mL). The reaction mixture was heated to 110°C for 3 hours. The suspension was filtered, washed with hexanes and dried (4.0 g, 76%). For C 13 H 18 LCMS calculated for N5O (M+H): 260.1. Found: 260.2.

[0740] Step 6: 3-methyl-9-pentyl-6-(phenylsulfonyl)-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one

[0741]

[0742] A bottle was charged with 3-methyl-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (from step 1) (4 g, 15.43 mmol), dichloromethane (40 mL), dimethylaminopyridine (0.188 g, 1.543 mmol), triethylamine (3.23 ml, 23.14 mmol), and benzenesulfonyl chloride (2.187 ml, 16.97 mmol). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with water and the layers were separated. The aqueous layer was extracted with dichloromethane (3×40 mL) and the combined organic fractions were dried over MgSO4, filtered, and concentrated. The crude material was used in the next step without further purification (6.1 g, quantitative). For C 19 H 22 LCMS Calcd. for N5O3S (M+H): 400.1. Found: 400.1.

[0743] Step 7: 7-Bromo-3-methyl-9-pentyl-6-(phenylsulfonyl)-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one

[0744]

[0745] The bottle was charged with 3-methyl-9-pentyl-6-(phenylsulfonyl)-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (1 g, 2.503 mmol), anhydrous THF (30 mL), and the mixture was cooled to -78 °C. A solution of lithium diisopropylamide (1 M in hexane / THF, 3.13 ml, 3.13 mmol) was added dropwise. The reaction mixture was maintained at -78 °C for 1.5 hours. A solution of 1,2-dibromo-1,1,2,2-tetrachloroethane (1.223 g, 3.75 mmol) in anhydrous THF (3 ml) was added dropwise to the reaction mixture, and the reaction mixture was maintained at -78 °C for another 1.5 hours. The reaction mixture was quenched with saturated NH4Cl aqueous solution (30 mL) and diluted with dichloromethane (30 mL). The layers were separated and the aqueous layer was extracted with DCM (3×30 mL). The combined organic fractions were dried over MgSO 4 , filtered, and concentrated. The crude residue was purified by automated flash chromatography (0 to 100% EtOAc in DCM) to obtain the desired product (0.84 g, 70%). 19 H 21 LCMS calcd for BrN5O3S (M+H): 478.1. Found: 478.1.

[0746] Step 8: 3-Chloro-5-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)pyridine

[0747]

[0748] The bottle was charged with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.5 g, 2.58 mmol), 3-(bromomethyl)-5-chloropyridine hydrobromide (0.741 g, 2.58 mmol), cesium carbonate (2.52 g, 7.73 mmol) and DMF (6.44 ml). The reaction mixture was stirred at 60 ° C for 1 hour. The reaction mixture was quenched with water (10 ml) and diluted with dichloromethane (10 ml). The layers were separated and the aqueous layer was extracted with dichloromethane (3×10 mL). The combined dichloromethane extracts were dried over MgSO4, filtered, and concentrated. Purification by automated flash chromatography (0 to 100% EtOAc in DCM) provided the product (0.548 g, 67%). For C 15 H 20 LCMS calculated for BClN3O2 (M+H): 320.1, 322.1. Found: 320.1, 322.1.

[0749] Step 9: 7-(1-((5-chloropyridin-3-yl)methyl)-1H-pyrazol-4-yl)-3-methyl-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one

[0750] A bottle was charged with 7-bromo-3-methyl-9-pentyl-6-(phenylsulfonyl)-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (0.01 g, 0.021 mmol), 3-chloro-5-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)pyridine (0.013 g, 0.042 mmol), chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (5.00 mg, 0.006 mmol), and tripotassium phosphate (0.016 g, 0.074 mmol). 1,4-dioxane (0.35ml) and water (0.07ml) were added and nitrogen was charged to the reaction mixture for 5 minutes, then stirred at 90°C for 2 hours. The reaction mixture was cooled to room temperature and sodium hydroxide (10mg) was added. The reaction mixture was stirred at 40°C for 60 minutes. The reaction mixture was cooled to room temperature and diluted with DMF (5ml). Purification by preparative HPLC (pH 2, acetonitrile / water containing TFA) provided the product in the form of a TFA salt (2mg, 21%). For C 22 H 24 LCMS calculated for ClN8O (M+H): 451.2, 453.2. Found: 451.2, 453.2.

[0751] Example A18: Synthesis of 3-methyl-7-(1-((5-methylpyridin-3-yl)methyl)-1H-pyrazol-4-yl)-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (Compound 18)

[0752]

[0753] This compound was prepared using a procedure similar to that described in Example A17, using 3-(bromomethyl)-5-methylpyridine in place of 3-(bromomethyl)-5-chloropyridine hydrobromide in step 8. 23 H 27 LCMS Calcd for N8O (M+H): 431.2. Found: 431.3.

[0754] Example A19: Synthesis of 3-methyl-9-pentyl-7-(1-(thieno[3,2-b]pyridin-6-ylmethyl)-1H-pyrazol-4-yl)-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (Compound 19)

[0755]

[0756] This compound was prepared using a procedure similar to that described in Example A17, using 6-(bromomethyl)thieno[3,2-b]pyridine in place of 3-(bromomethyl)-5-chloropyridine hydrobromide in step 8. 24 H 25 LCMS Calcd. for N8OS (M+H): 473.2. Found: 473.3.

[0757] Example A20: 7-(1-((2-(2-(dimethylamino)acetyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazol-4-yl)-3-methyl-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (Compound 20)

[0758]

[0759] Step 1: tert-Butyl 6-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate

[0760]

[0761] A flask was charged with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (.5 g, 2.58 mmol), tert-butyl 6-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (0.339 g, 1.288 mmol), triphenylphosphine (0.743 g, 2.83 mmol) and THF (12 ml). The solution was cooled to 0 °C and DIAD (0.601 ml, 3.09 mmol) was added dropwise. The reaction mixture was stirred at room temperature overnight. The mixture was diluted with ethyl acetate and washed with water, dried and concentrated. The product was purified by column chromatography eluting with hexane / EtOAc (maximum 60% EtOAc) to provide the product. For C 24 H 35 BN3O4(M+H) +LCMS calculated value: m / z = 440.3; found value: 440.3.

[0762] Step 2: 7-Bromo-3-methyl-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one

[0763]

[0764] TBAF (1.0 M in THF) (2.0 ml, 2.0 mmol) was added to a solution of 7-bromo-3-methyl-9-pentyl-6-(phenylsulfonyl)-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (0.360 g, 0.753 mmol) in THF (4.0 ml), and the reaction was stirred at 50° C. for 1 h. The solvent was removed and the product was purified by column chromatography, eluting with CH2Cl2 / MeOH (max 10% MeOH). For C 13 H 17 BrN5O(M+H) + LCMS calculated value: m / z = 338.1; found value: 338.1.

[0765] Step 3: tert-Butyl 6-((4-(3-methyl-5-oxo-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-7-yl)-1H-pyrazol-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate

[0766]

[0767] 7-Bromo-3-methyl-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (from Example A20, step 2) (0.040 g, 0.118 mmol), 6-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)-3,4-dihydro A mixture of tert-butyl isoquinoline-2(1H)-carboxylate (0.062 g, 0.142 mmol), dichloro[1,1'-bis(dicyclohexylphosphino)ferrocene]palladium(II) dichloromethane adduct (Pd-127) (8.94 mg, 0.012 mmol) and cesium fluoride (0.090 g, 0.591 mmol) in t-BuOH (1.5 ml) / water (0.6 ml) was evacuated and replenished with N2 3 times. The reaction was then stirred at 105°C for 2 h, cooled to rt, diluted with ethyl acetate, washed with water, dried and concentrated. The product was purified by column eluting with CH2Cl2 / MeOH (maximum 10% MeOH). For C 31 H 39 N8O3(M+H) + LCMS calculated value: m / z = 571.3; found value: 571.5.

[0768] Step 4: 3-methyl-9-pentyl-7-(1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazol-4-yl)-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one

[0769]

[0770] TFA (0.5 ml, 6.49 mmol) was added to a solution of tert-butyl 6-((4-(3-methyl-5-oxo-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-7-yl)-1H-pyrazol-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (50.0 mg, 0.088 mmol) in CH2Cl2 (0.5 ml), and the reaction was stirred at room temperature for 30 min. The solvent was then removed to provide the crude product as a TFA salt. For C 26 H 31 N8O(M+H) + LCMS calculated value: m / z = 471.3; found value: 471.2.

[0771] Step 5: 7-(1-((2-(2-(dimethylamino)acetyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazol-4-yl)-3-methyl-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one

[0772] Dimethylglycyl chloride (3.10 mg, 0.026 mmol) was added to a solution of 3-methyl-9-pentyl-7-(1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazol-4-yl)-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (6.0 mg, 0.013 mmol) and triethylamine (8.89 μl, 0.064 mmol) in CH2Cl2 (0.8 ml) at room temperature and stirred for 30 min. The solvent was removed, the mixture was diluted with acetonitrile / water and purified by preparative HPLC (pH 2, acetonitrile / water containing TFA) to provide the desired compound as its TFA salt. For C 30 H 38 N9O2(M+H) + LC-MS calculated value: m / z = 556.3; found value: 556.3.

[0773] Example A21. 3-(2-((5-(1H-pyrazol-1-yl)-2H-tetrazol-2-yl)methyl)-5-amino-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (Compound 21A) and 3-(2-((5-(1H-pyrazol-1-yl)-1H-tetrazol-1-yl)methyl)-5-amino-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (Compound 21B)

[0774]

[0775] The mixture of title compounds was prepared using a procedure similar to that described for Example A3, substituting 5-(1H-pyrazol-1-yl)-1H-tetrazolyl for 2-(1H-tetrazolyl-5-yl)pyridine. Compound 21A was purified by preparative LC-MS (pH 2, acetonitrile / water containing TFA) to afford the product as a TFA salt. 21 H 15 N 14 (M+H) + LCMS calcd: 463.2; found: 463.2.

[0776] In addition to those modifications described herein, those skilled in the art will appreciate the various modifications of the present invention based on the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The full text of each reference cited in this application (including all patents, patent applications and publications) is incorporated herein by reference.

Claims

1. Use of an A2A / A2B inhibitor and a PD-1 / PD-L1 inhibitor in the preparation of a medicament for treating a cancer in a subject, wherein the cancer is selected from breast cancer, colorectal cancer, head and neck cancer, non-small cell lung cancer and prostate cancer, wherein the A2A / A2B inhibitor is 3-(8-amino-5-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile or a pharmaceutically acceptable salt thereof, and wherein the PD-1 / PD-L1 inhibitor is antibody X, wherein antibody X is an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) domain, wherein the variable heavy chain domain comprises VH CDR1, VH CDR2 and VH CDR3, wherein: The VH CDR1 comprises the amino acid sequence SYWMN (SEQ ID NO: 6); The VH CDR2 comprises the amino acid sequence VIHPSDSETWLDQKFKD (SEQ ID NO: 7); and The VH CDR3 comprises the amino acid sequence EHYGTSPFAY (SEQ ID NO: 8); and wherein the antibody comprises a variable light chain (VL) domain comprising VL CDR1, VL CDR2 and VL CDR3, wherein: The VL CDR1 comprises the amino acid sequence RASESVDNYGMSFMNW (SEQ ID NO: 9); The VL CDR2 comprises the amino acid sequence AASNQGS (SEQ ID NO: 10); and The VL CDR3 comprises the amino acid sequence QQSKEVPYT (SEQ ID NO: 11).

2. The use according to claim 1, wherein antibody X is a humanized antibody.

3. The use of claim 1 or 2, wherein the treatment comprises administering to the subject the A2A / A2B inhibitor at a dose of 0.1 mg to 1000 mg based on the free base.

4. The use of claim 1 or 2, wherein the treatment comprises administering the A2A / A2B inhibitor to the subject once a day, every other day, or once a week.

5. The use according to claim 1 or 2, wherein the treatment comprises administering the A2A / A2B inhibitor and the PD-1 / PD-L1 inhibitor simultaneously.

6. The use according to claim 1 or 2, wherein the treatment comprises sequential administration of the A2A / A2B inhibitor and the PD-1 / PD-L1 inhibitor.

7. The use according to claim 1, wherein the cancer is head and neck cancer.

8. The use according to claim 7, wherein the head and neck cancer is head and neck squamous cell carcinoma.

9. The use according to claim 1, wherein the cancer is non-small cell lung cancer.

10. The use according to claim 1, wherein the cancer is prostate cancer.

11. The use according to claim 10, wherein the prostate cancer is metastatic castration-resistant prostate cancer.

12. The use according to claim 1, wherein the cancer is breast cancer.

13. The use according to claim 1, wherein the cancer is colorectal cancer.

Images