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

Abstract

The present application provides methods of treating cancer using a combination of an inhibitor of A2A and / or A2B and an inhibitor of PD-1 and / or PD-L1.

Description

[Technical field]

[0001] Disclosed herein are combination therapies comprising an inhibitor of A2A / A2B and an inhibitor of PD-1 / PD-L1, and methods of using same to treat disorders such as cancer. [Background technology]

[0002] Some cancer patients have poor long-term prognosis and / or are resistant to one or more types of treatments commonly used in the art. Thus, there remains a need for effective cancer treatments with improved 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 comprising administering to the subject: (i) an inhibitor of A2A / A2B; and (ii) PD-1 / PD-L1 inhibitors.

[0004] Other features, objects, and advantages of the invention will become apparent from the description and the claims. [Brief description of the drawings]

[0005] [Figure 1] AC. Synergy of Compound 9 with (1A) pembrolizumab, (1B) antibody X, and (1C) compound Y in CHO-PD-L1 co-cultured with primary T cells (see Example 1). [Diagram 2] AD. Synergistic effect of Compound 9 or Compound 3A with Atezolizumab in PBMCs stimulated with CD3 antibody. [Diagram 3]A-C. Antitumor efficacy of Compound 9 and anti-PD1 (clone 29F.1A12 against murine PD-1) in preclinical CT26 and B16-F10 tumor models. (3A) Efficacy study of Compound 9 at 10 mg / kg BID in the CT26 syngeneic model as a single agent and in combination with an anti-PD1 antibody. (3B) Efficacy study of Compound 9 at 10 mg / kg BID in the CT-26 NSG xenograft model. (3C) Efficacy study of Compound 9 at 10 mg / kg BID in the B16 syngeneic model as a single agent and in combination with an anti-PD-L1 antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] The present application provides a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B; and (ii) PD-1 / PD-L1 inhibitors.

[0007] A2A / A2B inhibitors Adenosine is an extracellular signaling molecule that can regulate immune responses through many immune cell types. Adenosine was first recognized as a physiological regulator of coronary vascular tone by Drury and Szent-Gyorgyu (Sachdeva, S. and Gupta, M. Saudi Pharmaceutical Journal, 2013, 21, 245-253), but this was not recognized until 1970, when Sattin and Rall showed that adenosine regulates cell function through the occupancy of specific receptors on the cell surface (Sattin, A., and Rall, TW, 1970). Mol. Pharmacol. 6, 13-23; Hasko', G., at al., 2007, Pharmacol. Ther. 113, 264-275).

[0008] Adenosine plays an important role in various other physiological functions. It is involved in the synthesis of nucleic acids, and when linked to three phosphate groups, it forms ATP, an essential component of the cellular energy system. Adenosine can be generated by enzymatic degradation of extracellular ATP or can also be released from injured neurons and glial cells by crossing damaged cell membranes (Tautenhahn, M. et al. Neuropharmacology, 2012, 62, 1756-1766). Adenosine exerts various pharmacological effects in both the peripheral and central nervous systems through its action on specific receptors localized on the cell membrane (Matsumoto, T. et al. Pharmacol. Res., 2012, 65, 81-90). Alternative pathways for extracellular adenosine generation have been described. These include the generation of adenosine from nicotinamide dinucleotide (NAD) instead of ATP, through the coordinated action of CD38, CD203a, and CD73. CD73-independent production of adenosine can also occur through other phosphates such as alkaline phosphatase or prostate-specific phosphatase.

[0009] There are four known subtypes of adenosine receptors in humans, 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 to modulate the activity of adenylate cyclase, an enzyme responsible for increasing cyclic AMP (cAMP). Different receptors have different stimulatory and inhibitory effects on this enzyme. Increased intracellular concentrations of cAMP can suppress the activity of immune and inflammatory cells (Livingston, M. et al., Inflamm. Res., 2004, 53, 171-178).

[0010] A2A adenosine receptors can transmit signals in the periphery and in the CNS (central nervous system), with agonists being explored as anti-inflammatory drugs and antagonists being explored for neurodegenerative diseases (Carlsson, J. et al., J. Med. Chem., 2010, 53, 3748-3755). In most cell types, A2A subtypes inhibit intracellular calcium levels, whereas A2B enhances them. A2A receptors generally appear to inhibit inflammatory responses from immune cells (Borrmann, T. et al., J. Med. Chem., 2009, 52(13), 3994-4006).

[0011] 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). A2B receptors are structurally closely related to A2A receptors and can activate adenylate cyclase, but are functionally distinct. It has been hypothesized that this subtype may utilize signaling systems other than adenylate cyclase (Livingston, M. et al., Inflamm. Res., 2004, 53, 171-178). Among all adenosine receptors, A2B adenosine receptors are considered low affinity receptors that are believed to remain silent under physiological conditions and become activated as a result of 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 through activation of 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).

[0012] In the immune system, the involvement of adenosine signaling may be an important regulatory mechanism that protects 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).

[0013] In tumors, this pathway is hijacked by the tumor microenvironment, hindering the immune system's antitumor capabilities and promoting cancer progression. In the tumor microenvironment, adenosine is mainly generated from extracellular ATP by two ectonucleotidases, CD39 and CD73. Multiple cell types can generate 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 (MDSCs), endothelial cells, cancer-associated fibroblasts (CAFs), and mesenchymal stromal / stem cells (MSCs). Moreover, hypoxia and inflammation, conditions common to the tumor microenvironment, induce the expression of CD39 and CD73, leading to increased adenosine production. As a result, adenosine levels in solid tumors are elevated compared to normal physiological conditions.

[0014] A2A is primarily expressed on lymphocyte-derived cells, such as T effector cells, T regulatory cells, and natural killer (NK) cells. Blockade of the A2A receptor can prevent downstream immunosuppressive signals that temporarily inactivate T cells. A2B receptors are primarily expressed on monocyte-derived cells, such as dendritic cells, tumor-associated macrophages, myeloid-derived suppressor cells (MDSCs), and mesenchymal stromal / stem cells (MSCs). Blockade of the A2B receptor in preclinical models can suppress tumor growth, inhibit metastasis, and increase tumor antigen presentation.

[0015] Regarding the safety profile of ADORA2A / ADORA2B (A2A / A2B) blockade, A2A and A2B receptor knockout (KO) mice are all viable and fertile without growth abnormalities (Allard, B. et al. Current Opinion in Pharmacology, 2016, 29, 7-16). A2AKO mice showed increased levels of proinflammatory cytokines only upon challenge with lipopolysaccharide (LPS) and no evidence of inflammation at baseline (Antonioli, L. et al., Nature Reviews Cancer, 2013, 13, 842-857). A2B KO mice showed normal platelet, red blood cell, and white blood cell counts, but increased inflammation at baseline, including TNF-α and IL-6) (Antonioli, L. et al., Nature Reviews Cancer, 2013, 13, 842-857). After LPS treatment, further increases in the production of TNF-α and IL-6 were detected. A2B KO mice also showed increased vascular adhesion molecules that mediate inflammation and leukocyte adhesion / rolling; enhanced mast cell activation; increased susceptibility to IgE-mediated anaphylaxis; and increased vascular leakage and neutrophil influx under hypoxia (Antonioli, L. et al., Nature Reviews Cancer, 2013, 13, 842-857).

[0016] The adenosine pathway is an important 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 through two G protein-coupled receptors (GPCRs) known as 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 high levels of adenosine production observed in the tumor microenvironment, it has been reported that the antitumor capabilities of the immune system are suppressed, leading to cancer progression.

[0017] In some embodiments, the inhibitor of A2A / A2B is a compound selected from Table 1, or a pharma- ceutically acceptable salt thereof.

[0018] Table 1: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0019] In some embodiments, the inhibitor of A2A / A2B is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof, Cy 1 is phenyl substituted by 1 or 2 substituents independently selected from halo and CN; Cy 2 is a 5- to 6-membered heteroaryl or a 4- to 7-membered heterocycloalkyl, 2 Each of the 5- to 6-membered heteroaryl and 4- to 7-membered heterocycloalkyl groups is 1-3 Alkyl, C 1-3 Alkoxy, NH2, NH(C 1-3 Alkyl) and N(C 1-3 each optionally substituted with 1, 2, or 3 groups independently selected from alkyl; R 2 is 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 wherein 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- to 7-membered heterocycloalkyl)-C 1-3 Alkyl- is selected from 1, 2, or 3 independently selected R C optionally substituted with substituents; R a2 is one or two independently selected R C (5-7 membered heteroaryl)-C optionally substituted with 1-3 alkyl-; Each R C Halo, C 1-6Alkyl, C6 aryl, 5- to 7-membered heteroaryl, (4- to 7-membered heterocycloalkyl)-C 1-3 Alkyl-, OR a4 , and N.R. c4 R d4 are independently selected from; Each R a4 , R c4 , and R d4 H and C 1-6 alkyl.

[0020] In some embodiments of the compounds of Formula (I), Cy 2 is pyrimidinyl.

[0021] In some embodiments of the compounds of Formula (I), R 2 is 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.

[0022] In some embodiments, the compound of formula (I), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (Compound 1, see Table 1).

[0023] In some embodiments, the compound of formula (I), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 2, see Table 1).

[0024] In some embodiments, the compound of formula (I), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 3A, see Table 1).

[0025] In some embodiments, the compound of formula (I), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 3B, see Table 1).

[0026] In some embodiments, the compound of formula (I), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 4, see Table 1).

[0027] In some embodiments, the compound of formula (I), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 21A, see Table 1).

[0028] In some embodiments, the compound of formula (I), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 21B, see Table 1).

[0029] In some embodiments, the inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; 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, or a pharma- ceutically acceptable salt thereof. The synthesis and characterization of compounds of formula (I) can be found in WO2019 / 168847 and US 62 / 891,685, both of which are incorporated by reference in their entireties.

[0030] In some embodiments, the inhibitor of A2A / A2B is a compound of formula (II) [ka] or a pharma- ceutically acceptable salt thereof, R 2 is selected from H and CN; Cy 1 is phenyl substituted by 1 or 2 substituents independently selected from halo and CN; L is for C 1-3 alkylene, wherein the alkylene optionally has one, two, or three independently selected R 8D substituted with a substituent; Cy 4 is selected from phenyl, cyclohexyl, pyridyl, pyrrolidinonyl, and imidazolyl, where phenyl, cyclohexyl, pyridyl, pyrrolidinonyl, and imidazolyl are each independently selected from R 8D and R 8 optionally substituted with 1, 2, or 3 substituents independently selected from Each R 8 Independently, Halo, 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 1-3 Alkyl, and (4-7 membered heterocycloalkyl)-C 1-3 alkyl, wherein 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, C3-7 Cycloalkyl-C 1-3 Alkyl, (5-6 membered heteroaryl)-C 1-3 Alkyl, and (4-7 membered heterocycloalkyl)-C 1-3 Each alkyl is selected from 1, 2, or 3 independently selected R 8A optionally substituted with substituents; Each R 8A Independently, Halo, C 1-6 Alkyl, 5-6 membered heteroaryl, 4-7 membered heterocycloalkyl, CN, OR a81 , and N.R. c81 R d81 wherein R 8A C 1-3 Alkyl, 5- to 6-membered heteroaryl, and 4- to 7-membered heterocycloalkyl each have one, two, or three independently selected R 8B optionally substituted with substituents; Each R a81 , R c81 , and R d81 are independent, H, C 1-6 alkyl, and 4- to 7-membered heterocycloalkyl, a81 , R c81 , and R d81 C 1-6 Alkyl and 4- to 7-membered heterocycloalkyl each have one, two, or three independently selected R 8B optionally substituted with substituents; Each R 8B But halo and C 1-3 alkyl; and Each R 8D OH, CN, halo, C 1-6 Alkyl, and C 1-6 haloalkyl.

[0031] In some embodiments, the compound of formula (II), or a pharma- ceutically acceptable salt thereof, is 3-(5-amino-2-(hydroxy(phenyl)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile, or a pharma- ceutically acceptable salt thereof (compound 5, see Table 1).

[0032] In some embodiments, the compound of formula (II), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 6, see Table 1).

[0033] In some embodiments, the compound of formula (II), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 7, see Table 1).

[0034] In some embodiments, the compound of formula (II), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 8, see Table 1).

[0035] The synthesis and characterization of compounds of formula (II) can be found in WO2019 / 222677, which is incorporated herein by reference in its entirety.

[0036] In some embodiments, the inhibitor of A2A / A2B is a compound of formula (III) [ka] or a pharma- ceutically acceptable salt thereof, Cy 1 is phenyl substituted by 1 or 2 substituents independently selected from halo and CN; R 2 is selected from 5- to 6-membered heteroaryl and 4- to 7-membered heterocycloalkyl, wherein R 2 Each of the 5- to 6-membered heteroaryl and 4- to 7-membered heterocycloalkyl groups is optionally selected from 1, 2, or 3 independently selected R 2A substituted with a substituent; Each R 2A But, D, Halo, C 1-6 Alkyl, and C 1-6 independently selected from haloalkyl; R 4 is phenyl-C 1-3 Alkyl-, C 3-7 Cycloalkyl-C 1-3 Alkyl-, (5-6 membered heteroaryl)-C 1-3 Alkyl- and (4- to 7-membered heterocycloalkyl)-C 1-3 alkyl, wherein 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- to 7-membered heterocycloalkyl)-C 1-3 Alkyl- is selected from 1, 2, or 3 independently selected R 4A optionally substituted with substituents; Each R 4A But, Halo, C 1-6 Alkyl, C 1-6 Haloalkyl, CN, OR a41 , and N.R. c41 R d41 are independently selected from; and Each R a41 , R c41 , and R d41 But H and C 1-6 alkyl.

[0037] In some embodiments, the compound of formula (III), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 9, see Table 1).

[0038] In some embodiments, the compound of formula (III), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 10, see Table 1).

[0039] In some embodiments, the compound of formula (III), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 11, see Table 1).

[0040] In some embodiments, the compound of formula (III), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 12, see Table 1).

[0041] The synthesis and characterization of compounds of formula (III) can be found in PCT / US2019 / 040496, which is incorporated herein by reference in its entirety.

[0042] In some embodiments, the inhibitor of A2A / A2B is a compound of formula (IV) [ka] or a pharma- ceutically acceptable salt thereof, Cy 1 is phenyl substituted by 1 or 2 substituents independently selected from halo and CN; Cy 2 is selected from 5- to 6-membered heteroaryl and 4- to 7-membered heterocycloalkyl, wherein Cy 2 Each of the 5- to 6-membered heteroaryl and 4- to 7-membered heterocycloalkyl groups is selected from 1, 2, or 3 independently selected R 6 optionally substituted with substituents; Each R 6 But, Halo, C 1-6 Alkyl, and C 1-6 haloalkyl; R 2 is phenyl-C 1-3 Alkyl- or (5-6 membered heteroaryl)-C 1-3 alkyl-, wherein R 2 Phenyl-C 1-3 Alkyl- and (5-6 membered heteroaryl)-C 1-3 Each alkyl- is selected from 1, 2, or 3 independently selected R 2A optionally substituted with substituents; Each R 2A Independently, Halo, C 1-6 Alkyl, and C 1-6 haloalkyl.

[0043] In some embodiments, the compound of formula (IV), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 13, see Table 1).

[0044] In some embodiments, the compound of formula (IV), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 14, see Table 1).

[0045] In some embodiments, the compound of formula (IV), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 15, see Table 1).

[0046] In some embodiments, the compound of formula (IV), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 16, see Table 1).

[0047] The synthesis and characterization of compounds of formula (IV) can be found in US 62 / 798,180, which is incorporated herein by reference in its entirety.

[0048] In some embodiments, the inhibitor of A2A / A2B is a compound of formula (V) [ka] or a pharma- ceutically acceptable salt thereof, R 2 H, D, Halo, C 1-6 Alkyl, and C 1-6 haloalkyl; R 3 H and C 1-6 alkyl, R 4H and C 1-6 alkyl, R 5 H, halo, CN, C 1-6 alkyl, R 6 is phenyl, C 3-7 cycloalkyl, 5- to 7-membered heteroaryl, and 4- to 7-membered heterocycloalkyl; 6 Phenyl, C 3-7 Cycloalkyl, 5- to 7-membered heteroaryl, and 4- to 7-membered heterocycloalkyl are each selected from one, two, or three independently selected R A optionally substituted with substituents; Each R A is (5-10 membered heteroaryl)-C 1-3 Alkyl- and (4-10 membered heterocycloalkyl)-C 1-3 alkyl-, wherein R A (5-10 membered heteroaryl)-C 1-3 Alkyl- and (4-10 membered heterocycloalkyl)-C 1-3 Each alkyl- is selected from one or two independently selected R B optionally substituted with substituents; Each R B Halo, C 1-6 Alkyl, and C(O)R b26 are independently selected from; Each R b26 But H and C 1-3 alkyl, b26 C 1-3 Alkyl is one or two independently selected R C optionally substituted with substituents; Each R C But, Halo, C 1-6 Alkyl, CN, OR a36 , and N.R. c36 R d36 are independently selected from Each R a36 , R c36 , and R d36 H and C 1-6 alkyl.

[0049] In some embodiments, the compound of formula (V), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 17, see Table 1).

[0050] In some embodiments, the compound of formula (V), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 18, see Table 1).

[0051] In some embodiments, the compound of formula (V), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 19, see Table 1).

[0052] In some embodiments, the compound of formula (V), or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof (compound 20, see Table 1).

[0053] The synthesis and characterization of compounds of formula (V) can be found in US-2019-0337957, which is incorporated herein by reference in its entirety.

[0054] PD-1 / PD-L1 inhibitors The immune system plays a key role in controlling and eradicating diseases such as cancer. However, cancer cells often develop strategies to evade or suppress the immune system to promote their growth. One such mechanism is to alter the expression of costimulatory and co-inhibitory molecules expressed in immune cells (Postow et al, J. Clinical Oncology 2015, 1-9). Blocking the signaling of inhibitory immune checkpoints such as PD-1 has proven to be a promising and effective treatment.

[0055] 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 No. 2007 / 0202100; U.S. Patent Publication No. 2008 / 0202200). Nos. 311117; 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. WO01 / 14557).

[0056] 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 at lower levels on 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).

[0057] The extracellular region of PD-1 is composed of a single immunoglobulin (Ig) V domain with 23% identity to the equivalent domain of CTLA-4 (Martin-Orozco, N. et al. (2007) Semin. Cancer Biol. 17(4): 288-298). The extracellular IgV domain continues to 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 switch motif, suggesting that PD-1 negatively regulates TCR signaling. (Ishida, Y. et al. (1992) EMBO J. 11: 3887-2895; Blank, C. et al. (2006) Immunol. Immunother. 56(5): 739-745).

[0058] PD-1 mediates inhibition 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. Patent Nos. 6,803,192; 7,794,710; U.S. Patent Application Publication Nos. 2005 / 0059051; 2009 / 0055944; 2009 / 0274666; 2009 / 0313687; PCT Publication Nos. WO01 / 39722; WO02 / 086083).

[0059] The amino acid sequence of human PD-1 protein (Genbank accession number NP_005009) is MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 1).

[0060] 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), which differ in their expression patterns. PD-L1 protein is upregulated in macrophages and dendritic cells in response to lipopolysaccharide and GM-CSF treatment, and in T cells and B cells upon 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 predict prognosis 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). Ligation of PD-1 and its ligands PD-L1 and PD-L2 on T cells transmits signals that inhibit the production of IL-2 and IFN-γ, as well as cell proliferation induced upon 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 recruitment of 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 phosphorylation of the PKC-θ activation loop.This is required for activation of the NF-κB and AP1 pathways and production of cytokines such as 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).

[0061] 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 develop lupus-like glomerulonephritis and dilated cardiomyopathy (Nishimura et al, Immunity 1999, 11:141-151; Nishimura et al, Science 2001, 291:319-322). Using an LCMV model of chronic infection, PD-1 / PD-L1 interactions have been shown to inhibit the activation, expansion, and acquisition of effector functions of virus-specific CD8 T cells (Barber et al., Nature 2006, 439, 682-7). Taken together, these data support the development of therapeutic approaches that block PD-1-mediated inhibitory signaling cascades to augment or "rescue" T cell responses. Thus, new methods are needed to block PD-1 / PD-L1 protein / protein interactions and thereby treat cancers of interest.

[0062] In some embodiments, the PD-1 / PD-L1 inhibitor is nivolumab (OPDIVO®, BMS-936558, MDX1106, or MK-34775), pembrolizumab (KEYTRUDA®, MK-3475, SCH-900475, lambrolizumab, CAS Registry Number 1374853-91-4), atezolizumab (Tecentriq®, CAS Registry Number 1374853-91-4), or rituximab (Tecentriq®, CAS Registry Number 1374853-91-4). Reg No. 1380723-44-3), durvalumab, avelumab (Bavencio®), 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-1 210 (HR-301210, SHR1210, or SHR-1210), BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and U.S. Patent Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, and 8,217,149, or publication numbers WO03042402, WO2008 / 156712, WO 2010 / 089411, WO2010 / 036959, WO2011 / 066342, WO2011 / 159877, WO2011 / 082400, WO2011 / 161699, WO2017 / 0700 89, WO2017 / 087777, WO2017 / 106634, WO2017 / 112730, WO2017 / 192961, WO2017 / 205464, WO2017 / 222976, WO2018 / 013789, WO2018 / 04478, WO2018 / 119236, WO2018 / 119266, WO2018 / 119221, WO2018 / 119286, WO2018 / 119263, WO2018 / 119224, WO2019 / 191707, and WO2019 / 217821, and any combination thereof. The disclosures of each of the patents, applications, and patent application publications set forth herein are hereby incorporated by reference in their entirety.

[0063] In some embodiments, the PD-1 / PD-L1 inhibitor is selected from compounds such as those disclosed in WO2018 / 119266. (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 pharma- ceutically acceptable salt thereof; (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 pharma- ceutically acceptable salt thereof; (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 pharma- ceutically acceptable salt thereof; (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 pharma- ceutically acceptable salt thereof; (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 pharma- ceutically acceptable salt thereof; (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 pharma- ceutically acceptable salt thereof; and 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 pharma- ceutically acceptable salt thereof.

[0064] 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 pharma- ceutical acceptable salt thereof.

[0065] (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 pharma- ceutically acceptable salt thereof, is also referred to herein as compound Y. The synthesis and characterization of compound Y is disclosed in WO2018 / 119266, which is incorporated herein by reference in its entirety.

[0066] In some embodiments, the inhibitor of PD-1 / PD-L1 is selected from: (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; (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; (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; (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; (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 (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.

[0067] In some embodiments, the PD-1 / PD-L1 inhibitor is selected from compounds such as those disclosed in WO2018 / 119224. (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 pharma- ceutically acceptable salt thereof; (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 pharma- ceutically acceptable salt thereof; (S)—N-(2-chloro-3'-(5-(2-hydroxypropyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide)-2'-methylbiphenyl-3-yl)-5-isopropyl-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide, or a pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; trans-4-(2-(2-((2,2'-dichloro-3'-(5-(2-hydroxyethyl)-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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; and 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 pharma- ceutically acceptable salt thereof.

[0068] In some embodiments, the PD-1 / PD-L1 inhibitor is selected from compounds such as those disclosed in WO2019 / 191707. (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 pharma- ceutically acceptable salt thereof; (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 pharma- ceutically acceptable salt thereof; (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 pharma- ceutically acceptable salt thereof; (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 pharma- ceutically acceptable salt thereof; (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 pharma- ceutically acceptable salt thereof; and (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 pharma- ceutically acceptable salt thereof.

[0069] In some embodiments, the PD-1 / PD-L1 inhibitor is selected from compounds such as those disclosed in WO2019 / 217821. 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; (R)-4-(2-(2-((2,2'-dichloro-3'-(5-(2-hydroxypropyl)-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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; and 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 pharma- ceutically acceptable salt thereof.

[0070] In some embodiments, the PD-1 / PD-L1 inhibitor is pembrolizumab.

[0071] In some embodiments, the PD-1 / PD-L1 inhibitor is nivolumab.

[0072] In some embodiments, the PD-1 / PD-L1 inhibitor is atezolizumab.

[0073] In some embodiments, the inhibitor of PD-1 / PD-L1 is antibody X. As used herein, antibody X is a humanized IgG4 monoclonal antibody that binds to human PD-1. See hPD-1mAb7(1.2) of WO2017019846, which is incorporated herein by reference in its entirety. The amino acid sequences of the mature antibody X heavy and light chains are set forth below. Complementarity determining regions (CDRs) 1, 2, and 3 of the variable heavy (VH) and variable light (VL) domains are shown in order from the N-terminus to the C-terminus of the mature VL and VH sequences, both underlined and bold. An antibody consisting of the mature heavy chain (SEQ ID NO:2) and mature light chain (SEQ ID NO:3) listed below is referred to as antibody X.

[0074] Mature antibody X heavy chain (HC) [ka]

[0075] Mature antibody X light chain (LC) [ka]

[0076] The variable heavy (VH) domain of antibody X has the following amino acid sequence: [ka]

[0077] The variable light (VL) domain of antibody X has the following amino acid sequence: [ka]

[0078] The amino acid sequences of the VH CDRs of antibody X are listed below. VH CDR1: SYWMN (SEQ ID NO: 6), VH CDR2: VIHPSDSETWLDQKFKD (SEQ ID NO: 7), VH CDR3: EHYGTSPFAY (SEQ ID NO: 8)

[0079] The amino acid sequences of the VL CDRs of Antibody X are listed below: VL CDR1: RASESVDNYGMSFMNW (SEQ ID NO: 9), VL CDR2: AASNQGS (SEQ ID NO: 10), and VL CDR3: QQSKEVPYT (sequence number 11).

[0080] As used herein, "QD" is taken to mean a dose administered to a subject once a day. "QOD" is taken to mean a dose administered to a subject once every other day. "QW" is taken to mean a dose administered to a subject once a week. "Q2W" is taken to mean a dose administered to a subject once every two weeks. "Q3W" is taken to mean a dose administered to a subject once every three weeks. "Q4W" is taken to mean a dose administered to a subject once every four weeks.

[0081] As used herein, "about" when referring to a measurable value, such as an amount, dosage, temporal duration, etc., is meant to encompass a variation of ±10%. In certain embodiments, "about" can include a variation of ±5%, ±1%, or ±0.1% from the specified value, and any variation therebetween, such variations being appropriate for carrying out the disclosed methods.

[0082] In some embodiments, the compound disclosed herein is the (S)-enantiomer of one of the aforementioned compounds, or a pharma- ceutically acceptable salt thereof. In some embodiments, the compound is the (R)-enantiomer of one of the aforementioned compounds, or a pharma- ceutically acceptable salt thereof.

[0083] It is further understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0084] The term "n-membered", where n is an integer, typically refers to the number of ring-forming atoms at the moiety where n is the number of ring-forming atoms. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.

[0085] As used herein, the phrase "optionally substituted" means unsubstituted or substituted. The substituents are independently selected, and the substitution may occur at any chemically accessible position. As used herein, the term "substituted" means that a hydrogen atom is removed and replaced by a substituent. A single divalent substituent, for example, oxo, can replace two hydrogen atoms. It should be understood that substitution at a given atom is limited by the valence of the atom.

[0086] As used herein, the phrase "each 'variable' is independently selected from" means substantially the same as "at each occurrence, 'variable' is selected from."

[0087] Through definition, "C n~m " denotes a range inclusive of the endpoints, where n and m are integers and indicate the number of carbons. Examples include C 1-3 , C 1-4 , C 1-6 etc.

[0088] As used herein, the term "C" used alone or in combination with other terms means n-mThe term "alkyl" refers to a saturated hydrocarbon group having n to m carbons, which may be linear 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 homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like. In some embodiments, an alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0089] As used herein, the term "C" used alone or in combination with other terms means n~m The term "alkoxy" refers to a group of formula -O-alkyl, where the alkyl group has n to m carbons. Exemplary alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), butoxy (e.g., n-butoxy and tert-butoxy), and the like.

[0090] 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 (e.g., having 2, 3, or 4 fused rings). n~m The term "aryl" refers to an aryl group having n to m ring carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, an aryl group has 5 to 10 carbon atoms. In some embodiments, an aryl group is phenyl or naphthyl. In some embodiments, an aryl is phenyl (i.e., C6 aryl).

[0091] 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. In some embodiments, halo is F. In some embodiments, halo is Cl.

[0092] As used herein, the term "C" used alone or in combination with other terms means n~m The term "haloalkyl" refers to an alkyl group having 1 to 2s+1 halogen atoms (which may be the same or different), where "s" is the number of carbon atoms in the alkyl group, and 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. Exemplary haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCl3, CHCl2, C2Cl5, and the like.

[0093] As used herein, "cycloalkyl" refers to non-aromatic cyclic hydrocarbons, including cyclized alkyl and alkenyl groups. Cycloalkyl groups can include monocyclic or polycyclic (e.g., having two fused rings) groups, spirocyclic rings, and bridged rings (e.g., bridged bicycloalkyl groups). The ring-forming carbon atoms of a cycloalkyl group can be optionally substituted by oxo or sulfido (e.g., C(O) or C(S)). Also included in the definition of cycloalkyl are moieties having one or more aromatic rings fused to (i.e., having a bond in common with) a cycloalkyl ring, e.g., benzo or thienyl derivatives such as cyclopentane, cyclohexane, etc. Cycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbons (i.e., C 3-10 In some embodiments, the cycloalkyl may have the formula: 3-10In some embodiments, the cycloalkyl is a monocyclic or bicyclic cycloalkyl of the formula: 3-7 In some embodiments, the cycloalkyl is C 4-7 In some embodiments, the cycloalkyl is a monocyclic cycloalkyl of the formula: 4-10 It is a spirocyclic or bridged cycloalkyl (e.g., a bridged bicycloalkyl group). Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, cubane, adamantane, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, bicyclo[2.2.2]octanyl, spiro[3.3]heptanyl, and the like. In some embodiments, the cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0094] As used herein, "heteroaryl" refers to a monocyclic or polycyclic (e.g., having two 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 can 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 membered monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from N, O, S, and B. In some embodiments, the heteroaryl is a 5-6 membered monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from N, O, and S. In some embodiments, the heteroaryl group contains 3-10, 4-10, 5-10, 5-7, 3-7, or 5-6 ring-forming atoms. In some embodiments, the heteroaryl group has 1-4 ring-forming heteroatoms, 1-3 ring-forming heteroatoms, 1-2 ring-forming heteroatoms, or 1 ring-forming heteroatom. When the heteroaryl group contains more than one heteroatom ring member, the heteroatoms may be the same or different.Examples of heteroaryl groups include thienyl (or thiophenyl), furyl (or furanyl), 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-azaborine, pyridinyl, pyrimidinyl, pyrazinyl, pyrid ... These include, but are not limited to, diphenyl, azolyl, triazolyl, thiadiazolyl, quinolinyl, isoquinolinyl, indolyl, benzothiophenyl, benzofuranyl, benzisoxazolyl, imidazo[1,2-b]thiazolyl, purinyl, triazinyl, 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.

[0095] As used herein, "heterocycloalkyl" refers to a monocyclic or polycyclic heterocycle having at least one non-aromatic ring (saturated or partially unsaturated ring), in which one or more of the ring-forming carbon atoms of the heterocycloalkyl are replaced by a heteroatom selected from N, O, S, and B, and the ring-forming carbon atoms and heteroatoms of the heterocycloalkyl group may be optionally substituted by one or more oxo or sulfide (e.g., C(O), S(O), C(S), or S(O)2, etc.). When a ring-forming carbon atom or heteroatom of a heterocycloalkyl group is optionally substituted by one or more oxo or sulfide, the O or S of said group is additional to the number of ring-forming atoms specified herein (e.g., 1-methyl-6-oxo-1,6-dihydropyridazin-3-yl is a 6-membered heterocycloalkyl group in which a ring-forming carbon atom is substituted by an oxo group, and the 6-membered heterocycloalkyl group is further substituted by a methyl group). Heterocycloalkyl groups include monocyclic and polycyclic (e.g., having two fused rings) systems. Heterocycloalkyls include monocyclic and polycyclic 3-10 membered, 4-10 membered, 5-10 membered, 4-7 membered, 5-7 membered, or 5-6 membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles and bridged rings (e.g., 5-10 membered bridged biheterocycloalkyl rings having one or more ring-forming carbon atoms replaced by heteroatoms independently selected from N, O, S, and B). Heterocycloalkyl groups can be bonded through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, heterocycloalkyl groups contain 0-3 double bonds. In some embodiments, heterocycloalkyl groups contain 0-2 double bonds.

[0096] Also included within the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused to (i.e., having a bond in common with) a non-aromatic heterocyclic ring, e.g., a benzo or thienyl derivative, such as piperidine, morpholine, azepine, etc. Heterocycloalkyl groups that contain fused aromatic rings can be bonded through any ring-forming atom, including a ring-forming atom of the fused aromatic ring.

[0097] In some embodiments, the heterocycloalkyl group contains 3-10 ring-forming atoms, 4-10 ring-forming atoms, 3-7 ring-forming atoms, or 5-6 ring-forming atoms. In some embodiments, the heterocycloalkyl group has 1-4 heteroatoms, 1-3 heteroatoms, 1-2 heteroatoms, or 1 heteroatom. In some embodiments, the 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, the 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, the heterocycloalkyl is a monocyclic or bicyclic 5-10 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, the heterocycloalkyl is a monocyclic 5-6 membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S and having one or more oxidized ring members.

[0098] Exemplary heterocycloalkyl groups include pyrrolidin-2-one (or 2-oxopyrrolidinyl), 1,3-isoxazolidin-2-one, pyranyl, tetrahydropyran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, and azepanyl. , 1,2,3,4-Tetrahydroisoquinoline, benzazapene, azabicyclo[3.1.0]hexanyl, diazabicyclo[3.1.0]hexanyl, oxobicyclo[2.1.1]hexanyl, azabicyclo[2.2.1]heptanyl, diazabicyclo[2.2.1]heptanyl, azabicyclo[3.1.1]heptanyl, diazabicyclo[3.1.1]heptanyl, azabicyclo[3.2.1]octanyl, diazabicyclo[3.2.1]octanyl, oxobicyclo [2.2.2]octanyl, azabicyclo[2.2.2]octanyl, azaadamantanyl, diazaadamantanyl, oxo-adamantanyl, azaspiro[3.3]heptanyl, diazaspiro[3.3]heptanyl, oxo-azaspiro[3.3]heptanyl, azaspiro[3.4]octanyl, diazaspiro[3.4]octanyl, oxo-azaspiro[3.4]octanyl, azaspiro[2.5]octanyl, diazaspiro[2.5]octanyl, azaspiro[4.4 ]nonanyl, diazaspiro[4.4]nonanyl, oxo-azaspiro[4.4]nonanyl, azaspiro[4.5]decanyl, diazaspiro[4.5]decanyl, diazaspiro[4.4]nonanyl, oxo-diazaspiro[4.4]nonanyl, oxo-dihydropyridazinyl, oxo-2,6-diazaspiro[3.4]octanyl, oxohexahydropyrrolo[1,2a]pyrazinyl, 3-oxopiperazinyl, oxo-pyrrolidinyl, oxo-pyrazinyl, and the like. For example, heterocycloalkyl groups include the following groups (with or without N-methyl substitution): [ka]

[0099] As used herein, "C o~p Cycloalkyl-C n~m "Alkyl-" refers to a group of the formula cycloalkyl-alkylene- where the cycloalkyl has from o to p carbon atoms and the alkylene linking group has from n to m carbon atoms.

[0100] As used herein, "C o~p Aryl-C n~m "Alkyl-" refers to a group of the formula aryl-alkylene- where the aryl has from op carbon atoms and the alkylene linking group has from n to m carbon atoms.

[0101] As used herein, "heteroaryl-C n~m "Alkyl-" refers to a group of the formula heteroaryl-alkylene-, where the alkylene linking group has n to m carbon atoms.

[0102] As used herein, "heterocycloalkyl-C n-m "Alkyl-" refers to a group of the formula heterocycloalkyl-alkylene-, where the alkylene linking group has n to m carbon atoms.

[0103] In certain places, the definitions or embodiments refer to specific rings (e.g., azetidine ring, pyridine ring, etc.). Unless otherwise specified, these rings can be attached to any ring member, as long as the valence of the atom is not exceeded. For example, the azetidine ring can be attached at any position of the ring, whereas the pyridin-3-yl ring is attached at the 3-position.

[0104] As used herein, the term "oxo" refers to an oxygen atom (i.e., =O) as a divalent substituent which when attached to carbon forms a carbonyl group (e.g., C=O or C(O)), or to a nitrogen or sulfur heteroatom forms a nitroso, sulfinyl, or sulfonyl group.

[0105] As used herein, the term "independently selected from" means that each occurrence of a variable or substituent is independently selected at each occurrence from the applicable list.

[0106] The compounds described herein may be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise specified. Compounds of the present disclosure containing asymmetrically substituted atoms may be isolated 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 stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like, may also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms. In some embodiments, the compounds have the (R) configuration. In some embodiments, the compounds have the (S)-configuration. The formulas provided herein (e.g., formulas (I), (II), etc.) include stereoisomers of the compounds.

[0107] The resolution of racemic mixtures of compounds can be carried out by any of a number of methods well known in the art. An exemplary method includes fractional recrystallization using chiral resolving acids that are optically active salt-forming organic acids. Suitable resolving agents for fractional recrystallization are, for example, optically active acids such as D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or various optically active camphorsulfonic acids such as β-camphorsulfonic acid. Other suitable resolving agents for fractional crystallization include stereoisomerically pure forms of α-methylbenzylamine (e.g., S and R forms, or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

[0108] Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by one skilled in the art.

[0109] The compounds described herein also include tautomeric forms. Tautomeric forms are obtained by the exchange of adjacent double and single bonds with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy more than one position of a heterocyclic ring system, such as 1H-imidazole and 3H-imidazole, 1H-1,2,4-triazole, 2H-1,2,4-triazole, and 4H-1,2,4-triazole, 1H-isoindole and 2H-isoindole, 2-hydroxypyridine and 2-pyridone, and 1H-pyrazole and 2H-pyrazole. Tautomeric forms may be in equilibrium or may be sterically locked into one form by appropriate substitution.

[0110] All compounds and their pharma- ceutically acceptable salts may be found together with other substances such as water and solvents (eg, hydrates and solvates) or may be isolated.

[0111] In some embodiments, preparation of compounds may involve the addition of acids or bases, for example to affect catalysis of a desired reaction or the formation of salt forms, such as acid addition salts.

[0112] In some embodiments, the compounds described herein or salts thereof are substantially isolated. "Substantially isolated" means that the compounds are at least partially or substantially separated from the environment in which they were formed or detected. Partial separation can include, for example, compositions enriched in the compounds described herein. Substantial separation can include compositions containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compounds described herein or salts thereof. Methods for isolating compounds and their salts are routine in the art.

[0113] The term "compound," as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. Compounds herein that are identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

[0114] The phrase "pharmacologically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals, within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0115] The present application also includes pharma- ceutically acceptable salts of the compounds described herein. As used herein, "pharma- ceutically acceptable salts" refers to derivatives of the disclosed compounds, in which the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharma- ceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharma- ceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharma- ceutically acceptable salts of the present disclosure can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two, generally with non-aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, isopropanol, or butanol) or acetonitrile (ACN) being preferred. Lists of suitable salts can be 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.

[0116] The compounds described herein, including their salts, can be prepared using known organic synthesis techniques, or can be synthesized according to any of a number of possible synthetic routes.

[0117] The reactions for preparing the compounds described herein can be carried out in a suitable solvent that can be easily selected by those skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out, which can range from the freezing temperature of the solvent to the boiling temperature of the solvent. A given reaction can be carried out in one solvent or a mixture of two or more solvents. Depending on the particular reaction step, a suitable solvent for a particular reaction step can be selected by those skilled in the art.

[0118] The preparation of the compounds described herein may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of suitable protecting groups, can be easily determined by those skilled in the art. The chemistry of protecting groups can be found, for example, in TW Greene, and PG M Huts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley & Sons, Inc., New York (1999), which is incorporated herein by reference in its entirety.

[0119] The reaction can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H or 13C), spectroscopic means such as infrared spectroscopy, spectrophotometry (e.g., UV-Vis), mass spectrometry, or chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LCMS), or thin layer chromatography (TLC). Compounds can be purified by those skilled in the art by a variety of methods, 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, which is incorporated herein by reference in its entirety) and normal phase silica chromatography.

[0120] The compounds described herein can modulate the activity of one or more of various GPCRs, including, for example, A2A / A2B. The term "modulate" is meant to refer to the ability to increase or decrease the activity of one or more members of the A2A / A2B family. Thus, the compounds described herein can be used in methods to modulate 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 act as inhibitors of one or both of A2A and A2B. In further embodiments, the compounds described herein can be used to modulate the activity of A2A / A2B in an individual in need of receptor modulation by administering a modulating amount of a compound described herein or a pharma- ceutically acceptable salt thereof. In some embodiments, the modulation is inhibition.

[0121] Given that the growth and survival of cancer cells are affected by multiple signaling pathways, the present invention is useful for treating disease states characterized by drug-resistant mutants. Furthermore, different GPCR inhibitors that show different preferences in the GPCRs that they regulate activity can be used in combination. This approach is highly efficient in treating disease states by targeting multiple signaling pathways, reducing the possibility of drug resistance occurring in cells and reducing the toxicity of disease treatment.

[0122] The GPCRs that the present compounds bind to and / or modulate (eg, inhibit) include any member of the A2A / A2B family.

[0123] In some embodiments, two or more compounds described herein are used to inhibit the activity of one GPCR (eg, A2A).

[0124] In some embodiments, two or more compounds described herein are used to inhibit two or more GPCRs, such as at least two GPCRs (eg, A2A and A2B).

[0125] 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).

[0126] The A2A / A2B inhibitors described herein can be selective. By "selective" it is meant that the compound binds to or inhibits the 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., over A2B). In some embodiments, the compounds described herein are selective inhibitors of A2B (e.g., over A2A). In some embodiments, the selectivity can be at least about 2-fold, 5-fold, 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about 1000-fold. Selectivity can be measured by methods routine in the art. In some embodiments, selectivity can be tested by biochemical affinity for each GPCR. In some embodiments, the selectivity of the compounds of the present disclosure can be determined by cellular assays related to specific A2A / A2B GPCR activity.

[0127] As used herein, the term "contacting" refers to bringing the indicated moieties together in an in vitro system or 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, such as a human, having A2A / A2B, as well as introducing a compound described herein into a sample, including, for example, a cell preparation or purified preparation that contains A2A / A2B.

[0128] As used herein, the terms "individual" or "patient", used interchangeably, refer to any animal, including a mammal, preferably a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, or primate, most preferably a human.

[0129] As used herein, the phrase "therapeutically effective amount" refers to that amount of an active compound or pharmaceutical agent that elicits the biological or medicinal response that a researcher, veterinarian, physician, or other clinician is looking for in a tissue, system, animal, individual, or human.

[0130] As used herein, the term "treating" or "treatment" refers to one or more of: (1) preventing a disease, e.g., preventing a disease, condition, or disorder in an individual who may be predisposed to the disease, condition, or disorder but has not yet experienced or exhibited the pathology or symptoms of the disease; (2) inhibiting a disease, e.g., inhibiting a disease, condition, or disorder in an individual who is experiencing or exhibiting the pathology or symptoms of the disease, condition, or disorder (i.e., arresting further progression of the pathology and / or symptoms); and (3) ameliorating a disease, e.g., ameliorating a disease, condition, or disorder in an individual who is experiencing or exhibiting the pathology or symptoms of the disease, condition, or disorder (i.e., reversing the pathology and / or symptoms), e.g., reducing the severity of the disease. In some embodiments, the term "treating" or "treatment" refers to inhibiting or ameliorating a disease.

[0131] Dosage and Route of Administration In some embodiments, the A2A / A2B inhibitor or a pharma- ceutically acceptable salt thereof is administered to the subject at a dose of about 0.1 mg to about 1000 mg on a free base basis. In some embodiments, the A2A / A2B inhibitor or a pharma- ceutically acceptable salt thereof is administered to the subject at a dose of about 1 mg to about 500 mg on a free base basis. In some embodiments, the A2A / A2B inhibitor or a pharma- ceutically acceptable salt thereof is administered to the subject at a dose of about 5 mg to about 250 mg on a free base basis. In some embodiments, the A2A / A2B inhibitor or a pharma- ceutically acceptable salt thereof is administered to the subject at a dose of about 10 mg to about 100 mg on a free base basis.

[0132] In some embodiments, the inhibitor of A2A / A2B, or a pharma- ceutically acceptable salt thereof, is administered at 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 , approx. 155 mg, approx. 160 mg, approx. 165 mg, approx. 170 mg, approx. 175 mg, approx. 180 mg, approx. 185 mg, approx. 190 mg, approx. 195 mg, approx. 200 mg, approx. g, approx. 245 mg, approx. 250 mg, approx. 255 mg, approx. 260 mg, approx. 265 mg, approx. 270 mg, approx. 275 mg, approx. 280 mg, approx. mg, approx. 335 mg, approx. 340 mg, approx. 345 mg, approx. 350 mg, approx. 355 mg, approx. 360 mg, approx. 365 mg, approx. 370 mg, approx. 375 mg, approx. 0 mg, approx. 425 mg, approx. 430 mg, approx. 435 mg, approx. 440 mg, approx. 445 mg, approx. 450 mg, approx. 455 mg, approx. 460 mg, approx. 465 mg, approx. 10mg, approx. 515mg, approx. 520mg, approx. 525mg, approx. 530mg, approx. 535mg, approx. 540mg, approx. 545mg, approx. 550mg, approx. 555mg, approx. 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 77 0mg, 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 845mg, about 850mg, The subject is administered a dose selected from about 855 mg, about 860 mg, about 865 mg, about 870 mg, about 875 mg, about 880 mg, about 885 mg, about 890 mg, about 895 mg, about 900 mg, about 905 mg, about 910 mg, about 915 mg, about 920 mg, about 925 mg, about 930 mg, about 935 mg, about 940 mg, about 945 mg, about 950 mg, about 955 mg, about 960 mg, about 965 mg, about 970 mg, about 975 mg, about 980 mg, about 985 mg, about 990 mg, about 995 mg, and about 1000 mg. In some embodiments, the A2A / A2B inhibitor, or a pharma- ceutically acceptable salt thereof, is administered to a subject at a dose ranging from about 0.1 mg to about 500 mg, or any dose value therebetween, on a free base basis. In some embodiments, the A2A / A2B inhibitor, or a pharma- ceutically acceptable salt thereof, is administered to a subject at a dose ranging from about 1 mg to about 100 mg, or any dose value therebetween, on a free base basis.

[0133] In some embodiments, the A2A / A2B inhibitor, or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof, is administered to the subject once a day. In some embodiments, the A2A / A2B inhibitor, or a pharma- ceutically acceptable salt thereof, is administered to the subject every other day. In some embodiments, the A2A / A2B inhibitor, or a pharma- ceutically acceptable salt thereof, is administered to the subject once a week.

[0134] In some embodiments, each of the doses is administered as a single, once-daily dose.In some embodiments, each of the doses is administered as a single, once-daily oral dose.

[0135] In some embodiments, the PD-1 / PD-L1 inhibitor, or a pharma- ceutically acceptable salt thereof, is administered to the subject at a dose of about 0.1 mg to about 1000 mg of the free base. In some embodiments, the PD-1 / PD-L1 inhibitor, or a pharma- ceutically acceptable salt thereof, is administered to the subject at a dose of about 1 mg to about 500 mg of the free base. In some embodiments, the PD-1 / PD-L1 inhibitor, or a pharma- ceutically acceptable salt thereof, is administered to the subject at a dose of about 5 mg to about 250 mg of the free base. In some embodiments, the PD-1 / PD-L1 inhibitor, or a pharma- ceutically acceptable salt thereof, is administered to the subject at a dose of about 10 mg to about 100 mg of the free base.

[0136] In some embodiments, the inhibitor of PD-1 / PD-L1, or a pharma- ceutically acceptable salt thereof, is administered at a dose of 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 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 500 mg, about 550 mg, about 600 mg, about 650 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 415 mg, approx. 420 mg, approx. 425 mg, approx. 430 mg, approx. 435 mg, approx. 440 mg, approx. 445 mg, approx. 450 mg, approx. 455 mg, approx. 460 mg, approx. 5mg, approx. 510mg, approx. 515mg, approx. 520mg, approx. 525mg, approx. 530mg, approx. 535mg, approx. 540mg, approx. 545mg, approx. 550mg, approx. 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,Approx. 685 mg, approx. 690 mg, approx. 695 mg, approx. 700 mg, approx. 705 mg, approx. 710 mg, approx. 715 mg, approx. 720 mg, approx. 725 mg, approx. 730 mg, approx. g, approx. 770 mg, approx. 775 mg, approx. 780 mg, approx. 785 mg, approx. 790 mg, approx. 795 mg, approx. 800 mg, approx. 805 mg, approx. 810 mg, approx. In some embodiments, the compound is administered to a subject at a dose selected from about 855 mg, about 860 mg, about 865 mg, about 870 mg, about 875 mg, about 880 mg, about 885 mg, about 890 mg, about 895 mg, about 900 mg, about 905 mg, about 910 mg, about 915 mg, about 920 mg, about 925 mg, about 930 mg, about 935 mg, about 940 mg, about 945 mg, about 950 mg, about 955 mg, about 960 mg, about 965 mg, about 970 mg, about 975 mg, about 980 mg, about 985 mg, about 990 mg, about 995 mg, and about 1000 mg. In some embodiments, the PD-1 / PD-L1 inhibitor, or a pharma- ceutically acceptable salt thereof, is administered to a subject at a dose ranging from about 0.1 mg to about 500 mg, on a free base basis, or any dose value therebetween. In some embodiments, the PD-1 / PD-L1 inhibitor, or a pharma- ceutically acceptable salt thereof, is administered to a subject at a dose ranging from about 1 mg to about 100 mg, on a free base basis, or any dose value therebetween.

[0137] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dosage of about 1 mg / kg to about 10 mg / kg. In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dosage 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 the subject at a dosage of about 200 mg to about 1000 mg. In some embodiments, the inhibitor of PD-1 / PD-L1 is administered to a subject at a dosage 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.

[0138] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject once daily, every other day, once weekly, or any time interval in between. In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject once daily. 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 weekly.

[0139] In some embodiments, each of the doses is administered as a single, once-daily dose.In some embodiments, each of the doses is administered as a single, once-daily oral dose.

[0140] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject every 2 weeks, every 3 weeks, or every 4 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.

[0141] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dosage of 1 mg / kg Q2W.

[0142] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dosage of 3 mg / kg Q2W.

[0143] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dosage of 3 mg / kg Q4W.

[0144] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dosage of 10 mg / kg Q2W.

[0145] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to the subject at a dosage of 10 mg / kg Q4W.

[0146] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to a subject at a dosage of 200 mg Q3W.

[0147] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to a subject at a dosage of 250 mg Q3W.

[0148] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to a subject at a dosage of 375 mg Q3W.

[0149] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to a subject at a dosage of 500 mg Q4W.

[0150] In some embodiments, the PD-1 / PD-L1 inhibitor is administered to a subject at a dosage of 750 mg Q4W.

[0151] In some embodiments, the PD-1 / PD-L1 inhibitor is antibody X. In some embodiments, antibody X is administered to the subject at a dosage of about 250 mg to about 850 mg. In some embodiments, antibody X is administered to the subject at a dosage of about 375 mg to about 850 mg. In some embodiments, antibody X is administered to the subject at a dosage of about 450 mg to about 850 mg. In some embodiments, antibody X is administered to the subject at a dosage of about 500 mg to about 750 mg. In some embodiments, antibody X is administered to the subject at a dosage of about 500 mg. In some embodiments, antibody X is administered to the subject at a dosage of about 750 mg. In some embodiments, antibody X is administered to the subject every 4 weeks. In some embodiments, antibody X is administered to the subject by intravenous administration.

[0152] In some embodiments, antibody X is administered to a subject at a dosage of 1 mg / kg Q2W.

[0153] In some embodiments, antibody X is administered to a subject at a dosage of 3 mg / kg Q2W.

[0154] In some embodiments, antibody X is administered to a subject at a dosage of 3 mg / kg Q4W.

[0155] In some embodiments, antibody X is administered to a subject at a dosage of 10 mg / kg Q2W.

[0156] In some embodiments, antibody X is administered to a subject at a dosage of 10 mg / kg Q4W.

[0157] In some embodiments, antibody X is administered to a subject at a dosage of 200 mg Q3W.

[0158] In some embodiments, antibody X is administered to a subject at a dosage of 250 mg Q3W.

[0159] In some embodiments, antibody X is administered to a subject at a dosage of 375 mg Q3W.

[0160] In some embodiments, antibody X is administered to a subject at a dosage of 500 mg Q4W.

[0161] In some embodiments, antibody X is administered to a subject at a dosage of 750 mg Q4W.

[0162] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) an inhibitor of PD-1 / PD-L1, which is antibody X;

[0163] In some embodiments, the A2A / A2B inhibitor is administered to a subject at a dose of about 0.1 mg to about 500 mg on a free base basis, and the A2A / A2B inhibitor is administered once daily or every other day.

[0164] In some embodiments, antibody X is administered to a subject at a dosage of about 100 mg to about 1000 mg Q4W.

[0165] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) an inhibitor of PD-1 / PD-L1, which is antibody X; wherein the A2A / A2B inhibitor is administered to the subject at a dosage of about 0.1 mg to about 500 mg on a free base basis, wherein the A2A / A2B inhibitor is administered once daily or every other day; and Antibody X is administered to a subject at a dosage of about 100 mg to about 1000 mg Q4W.

[0166] In some embodiments, antibody X is administered to a subject at a dose of about 375 mg Q4W. In some embodiments, antibody X is administered to a subject at a dose of about 500 mg Q4W. In some embodiments, antibody X is administered to a subject at a dose of about 750 mg Q4W.

[0167] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is pembrolizumab;

[0168] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is pembrolizumab;

[0169] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is atezolizumab;

[0170] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is atezolizumab;

[0171] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) An inhibitor of PD-1 / PD-L1 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 pharma- ceutically acceptable salt thereof (Compound Y).

[0172] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) An inhibitor of PD-1 / PD-L1 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 pharma- ceutically acceptable salt thereof (Compound Y).

[0173] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) an inhibitor of PD-1 / PD-L1, which is antibody X;

[0174] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) an inhibitor of PD-1 / PD-L1, which is antibody X;

[0175] In some embodiments, the A2A / A2B inhibitor is administered to a subject at a dose of about 0.1 mg to about 500 mg on a free base basis, and the A2A / A2B inhibitor is administered once daily or every other day.

[0176] In some embodiments, antibody X is administered to a subject at a dosage of about 100 mg to about 1000 mg Q4W.

[0177] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) an inhibitor of PD-1 / PD-L1, which is antibody X; wherein the A2A / A2B inhibitor is administered to the subject at a dosage of about 0.1 mg to about 500 mg on a free base basis, wherein the A2A / A2B inhibitor is administered once daily or every other day; and Antibody X is administered to a subject at a dosage of about 100 mg to about 1000 mg Q4W.

[0178] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) an inhibitor of PD-1 / PD-L1, which is antibody X; wherein the A2A / A2B inhibitor is administered to the subject at a dosage of about 0.1 mg to about 500 mg on a free base basis, wherein the A2A / A2B inhibitor is administered once daily or every other day; and Antibody X is administered to a subject at a dosage of about 100 mg to about 1000 mg Q4W.

[0179] In some embodiments, antibody X is administered to the subject at a dose of about 375 mg Q4W. In some embodiments, antibody X is administered to the subject at a dose of about 500 mg Q4W. In some embodiments, antibody X is administered to the subject at a dose of about 750 mg Q4W.

[0180] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is pembrolizumab;

[0181] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is pembrolizumab;

[0182] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is pembrolizumab;

[0183] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is pembrolizumab;

[0184] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is atezolizumab;

[0185] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is atezolizumab;

[0186] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is atezolizumab;

[0187] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor, which is atezolizumab;

[0188] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) An inhibitor of PD-1 / PD-L1 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 pharma- ceutically acceptable salt thereof (Compound Y).

[0189] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) An inhibitor of PD-1 / PD-L1 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 pharma- ceutically acceptable salt thereof (Compound Y).

[0190] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) An inhibitor of PD-1 / PD-L1 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 pharma- ceutically acceptable salt thereof (Compound Y).

[0191] In some embodiments, provided herein is a method of treating a cancer 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 Merkel cell carcinoma in a subject, comprising administering to the subject: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) An inhibitor of PD-1 / PD-L1 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 pharma- ceutically acceptable salt thereof (Compound Y).

[0192] In some embodiments, the A2A / A2B inhibitor and the PD-1 / PD-L1 inhibitor are administered simultaneously.

[0193] In some embodiments, the A2A / A2B inhibitor and the PD-1 / PD-L1 inhibitor are administered sequentially.

[0194] When the PD-1 / PD-L1 inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof, the PD-1 / PD-L1 inhibitor can be administered to a subject, e.g., a subject in need thereof, e.g., a human subject, by various methods. The methods and dosages discussed herein are applicable to all anti-PD-1 antibodies or antigen-binding fragments thereof, e.g., antibody X. In many applications, the route of administration is one of intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP), or intramuscular injection. Intra-articular delivery can also be used. Other methods of parenteral administration can also be used. Examples of such modes include intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, and epidural and intrasternal injection. In some cases, administration can be oral.

[0195] The route and / or mode of administration of the antibody or antigen-binding fragment thereof can also be tailored to individual cases, e.g., by monitoring the subject, e.g., using tomography imaging, e.g., to visualize the tumor.

[0196] The antibody or antigen-binding fragment thereof may be administered as a fixed dose or in mg / kg patient weight doses. The dose may also be selected to reduce or avoid production of antibodies against the anti-PD-1 antibody. The dosing regimen is adjusted to provide a desired response (e.g., therapeutic response) or combined therapeutic effect. In general, the dose of the anti-PD-1 antibody (and optionally the second agent) may be used to provide the subject with a bioavailable amount of the agent. For example, doses ranging from 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 may be administered. Other doses may also be used. In certain embodiments, a subject in need of treatment is administered an anti-PD-1 antibody at 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.

[0197] The composition may contain 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.

[0198] Dosage unit form or "fixed dose" or "uniform dose" as used herein refers to physically discrete units suitable as a unitary dosage for treating a subject, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with the necessary pharmaceutical carrier, and optionally in association with other agents. Single or multiple doses may be administered. Alternatively, or in addition, the antibody may be administered via continuous infusion. Exemplary fixed doses include 375 mg, 500 mg, and 750 mg.

[0199] Doses of the anti-PD-1 antibody or antigen-binding fragment thereof can be administered, for example, at least two doses, three doses, five doses, ten doses, or more, for example, once or twice daily, or about 1-4 times per week, or preferably once weekly, every other week (every 2 weeks), every 3 weeks, or once monthly, at periodic intervals over a period sufficient to encompass, for example, about 1-12 weeks, preferably 2-8 weeks, more preferably about 3-7 weeks, and even more preferably about 4, 5, or 6 weeks (course of treatment). Factors that can affect the dosage and timing required to effectively treat a subject include, for example, the severity of the disease or disorder, the formulation, the route of delivery, previous treatments, the general health and / or age of the subject, and other diseases present. Furthermore, treatment of a subject with a therapeutically effective amount of a compound can include a single treatment or, preferably, includes a series of treatments.

[0200] A pharmaceutical composition may include 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 two or more agents are used. The "therapeutically effective amount" of an agent may also vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the compound to elicit a desired response in an individual, such as improvement of at least one disorder parameter, or improvement of at least one disorder symptom. A therapeutically effective amount is also one in which any toxic or adverse effects of the composition are outweighed by the therapeutically beneficial effects.

[0201] Pharmaceutical preparations When used as a pharmaceutical, the compounds of the present disclosure can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art and can be administered by various routes depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including transdermal, epidermal, ocular, and mucosal delivery, including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, or injection or infusion, or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose or can be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration can 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.

[0202] The present disclosure also includes pharmaceutical compositions that contain the compounds of the present disclosure or pharma- ceutically acceptable salts thereof as active ingredients in combination with one or more pharma- ceutically acceptable carriers (excipients). In some embodiments, the compositions are suitable for topical administration. In making the compositions, the active ingredients are typically mixed with excipients, diluted by excipients, or enclosed in such carriers, for example, in the form of capsules, sachets, paper or other containers. When the excipient functions as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredients. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or liquid media), for example, ointments containing up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0203] When preparing formulation, active compound can be milled to provide suitable particle size before being combined with other components.If active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh.If active compound is substantially water-soluble, its particle size can be adjusted by milling to provide substantially uniform distribution in formulation, for example about 40 mesh.

[0204] The compounds of the present disclosure can be milled using known milling procedures, such as wet milling, to obtain a particle size suitable for tableting and other formulation types.Finely divided (nanoparticulate) preparations of the compounds of the present disclosure can be prepared by processes known in the art, see, for example, the disclosure of International Application No. WO2002 / 000196.

[0205] 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 further include lubricants such as talc, magnesium stearate, and mineral oil, wetting agents, emulsifying and suspending agents, preservatives such as methylbenzoate and propylhydroxybenzoate, sweeteners, and flavoring agents.The composition of the present invention can be formulated to provide quick, sustained, or delayed release of active ingredient after administration to a patient by using procedures known in the art.

[0206] The compositions can be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[0207] To prepare solid compositions such as tablets, the main active ingredient is mixed with pharmaceutical excipients to form a solid preformulation composition containing a homogenous mixture of the compound of the present invention.When these preformulation compositions are referred to as homogenous, the active ingredient is typically uniformly 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 unit dosage forms.

[0208] The tablet or pill of the present disclosure can be coated or otherwise compounded to provide a dosage form that provides the advantage of long-term action.For example, the tablet or pill can include an inner dose and an outer dose component, the latter being in the form of an envelope on the former.The two components can be separated by an enteric layer that resists disintegration in the stomach and allows the inner component to pass intact into the duodenum or to be delayed in release.A variety of materials can be utilized as such enteric layer or coating, including a number of polymeric acids, and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0209] Liquid forms into which the compounds and compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and emulsions flavored with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0210] Compositions for inhalation or insufflation include liquids and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by oral or nasal respiratory route for local or systemic effect. Compositions may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered orally or nasally from a device that delivers the formulation in an appropriate manner.

[0211] Topical formulations may contain one or more conventional carriers. In some embodiments, ointments may contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white petrolatum (petrolatum), and the like. The carrier composition of creams may be based on water in combination with glycerol and one or more other components, for example, glycerol monostearate, PEG-glycerol monostearate, and cetylstearyl alcohol. Gels may be formulated using isopropyl alcohol and water, suitably in combination with other components, such as, for example, glycerol, hydroxyethylcellulose, and the like. In some embodiments, topical formulations include at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2, or at least about 5% by weight of a compound of the present disclosure. Topical formulations may be suitably packaged in 100 g tubes, optionally accompanied by instructions for treating a selected indication, such as, for example, psoriasis or other skin conditions.

[0212] The amount of compound or composition administered to a patient will vary depending on what is being administered, the purpose of administration, such as prophylaxis or treatment, the condition of the patient, the method of administration, etc. In therapeutic applications, the compositions 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 physician based on factors such as the severity of the disease, the age, weight, and general condition of the patient.

[0213] The compositions administered to a patient may be in the form of pharmaceutical compositions described above. These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. Aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparations being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations will typically be 3-11, more preferably 5-9, most preferably 7-8. It will be understood that the use of certain of the aforementioned excipients, carriers, or stabilizers will result in the formulation of pharmaceutical salts.

[0214] The therapeutic amount of a compound of the present disclosure can vary according to, for example, the particular application for which the treatment is being performed, the method of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the present disclosure in a pharmaceutical composition can vary depending on a number of factors, including the dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the present disclosure can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w / v of the compound for parenteral administration.

[0215] The compositions of the invention can further comprise one or more additional pharmaceutical agents, such as chemotherapeutic agents, steroids, anti-inflammatory compounds, or immunosuppressants, examples of which are listed herein.

[0216] In certain embodiments, anti-PD-1 antibodies can be prepared with carriers that protect the compound from rapid release, such as sustained release formulations, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed. Marcel Dekker, Inc., New York (1978).

[0217] Solid Tumors and Cancer Examples of cancers treatable using the therapeutic methods and regimens of the present disclosure include, but are not limited to, bone cancer, splenic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrium cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, ureteral cancer, urethral cancer, urachal cancer, gastric cancer, Cancers of the present disclosure include, but are not limited to, chronic or acute leukemias, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphomas, bladder cancer, kidney or urethral cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of the above cancers. The methods of the present disclosure are also useful for treating metastatic cancers, particularly metastatic cancers that express PD-L1.

[0218] In some embodiments, cancers treatable by the methods of the present disclosure include melanoma (e.g., metastatic malignant melanoma), renal carcinoma (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), head and neck squamous cell carcinoma, urothelial carcinoma (e.g., bladder), and microsatellite instability high cancer (MSIhigh). Additionally, the present disclosure includes refractory or recurrent malignancies whose growth may be inhibited using the methods of the present disclosure.

[0219] In some embodiments, cancers treatable 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, renal cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.), hematological cancers (e.g., lymphoma, 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), lymphomas such as Hodgkin's lymphoma or multiple myeloma, leukemia), and combinations of said cancers.

[0220] In some embodiments, cancers treatable using the methods of the present disclosure include, but are not limited to, cholangiocarcinoma, 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, pancreatic islet cell carcinoma, oral cavity cancer (oral cancer), mouth cancer, pharyngeal cancer, laryngeal cancer, lip cancer, mesothelioma, cervical cancer, nasal cavity cancer, ocular cancer, ocular melanoma, pelvic cancer, rectal cancer, renal cell carcinoma, salivary gland cancer, paranasal sinus cancer, spinal cancer, tongue cancer, tubular cancer, urethral cancer, and ureteral cancer.

[0221] In some embodiments, the disease or disorder is 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, kidney cancer, gastric cancer, or sarcoma. In some embodiments, the cancer is selected from acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, multiple myelogenous leukemia, essential thrombocythemia, chronic myelogenous leukemia, myelofibrosis, primary myelofibrosis, post-polycythemia / essential thrombocythemia myelofibrosis, post-essential thrombocythemia myelofibrosis, and post-polycythemia myelofibrosis. 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.

[0222] 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 metastases), 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, bile duct cancer, oral cancer, non-melanoma skin cancer, and Merkel cell carcinoma.

[0223] In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).

[0224] In some embodiments, the colon cancer is colorectal cancer (CRC).

[0225] 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, color cancer, endometrial cancer, bladder cancer, skin cancer, uterine cancer, renal cancer, gastric cancer, or sarcoma. In some embodiments, the sarcoma is Askin tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant nerve sheath tumor, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystosarcoma 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, or undifferentiated pleomorphic sarcoma.

[0226] In some embodiments, the cancer is mesothelioma or adenocarcinoma. In some embodiments, the disease or disorder is mesothelioma. In some embodiments, the cancer is adenocarcinoma.

[0227] MDSCs (myeloid-derived suppressor cells) are a heterogeneous group of immune cells from the myeloid lineage (a family of cells originating from bone marrow stem cells). MDSCs are strongly expanded in pathological situations, such as chronic infections and cancer, as a result of altered hematopoiesis. MDSCs are distinct from other myeloid cell types, which have potent immunosuppressive activity rather than immunostimulatory properties. Like other myeloid cells, MDSCs interact with other types of immune cells, including T cells, dendritic cells, macrophages, and natural killer cells, to control their function. In some embodiments, for example, the compounds described herein may be used in methods related to cancer tissues (e.g., tumors) with high infiltration of MDSCs, including solid tumors with high basal levels of infiltration of macrophages and / or MDSCs. In some embodiments, the combination therapy described herein may be used in methods related to cancer tissues (e.g., tumors) with tumors or tumor-infiltrating lymphocytes (TILs) expressing PD-1 or PD-L1.

[0228] In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (e.g., colon cancer), melanoma, ovarian cancer, bladder cancer, renal cell carcinoma, liver cancer, or hepatocellular carcinoma.

[0229] 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 Merkel cell carcinoma.

[0230] In some embodiments, the cancer is selected from a cancer selected from melanoma, endometrial cancer, lung cancer, kidney cancer, bladder cancer, breast cancer, pancreatic cancer, and colon cancer.

[0231] In some embodiments, the cancer is selected from endometrial cancer, anal cancer, and cholangiocarcinoma.

[0232] In some embodiments, the cancer is a tumor that exhibits high adenosine levels in the tumor microenvironment. These tumors may be enriched by gene expression signatures or by high expression levels of CD73 and / or other alkaline phosphatases, including tissue non-specific alkaline phosphatases (i.e., TNAP and PAP).

[0233] In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is endometrial cancer (e.g., endometiral adenocarcinoma). In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is small cell lung cancer or non-small cell lung cancer. In some embodiments, the cancer is kidney renal clear cell carcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma. In some embodiments, the cancer is a sarcoma. In some embodiments, the sarcoma is Askin tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant nerve sheath tumor, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystosarcoma 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, or undifferentiated pleomorphic sarcoma.

[0234] Labeled Compounds and Assay Methods 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 have been replaced, i.e. substituted, with an atom having an atomic mass or mass number different from that normally found in nature (i.e. naturally occurring). Suitable radionuclides that may be incorporated into the compounds of the present disclosure include: 2 H (also written as D for deuterium), 3 H (also written as T for tritium), 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 For example, one or more hydrogen atoms in the compounds of the present disclosure can be replaced by a deuterium atom (e.g., one or more hydrogen atoms of the alkyl group of the compounds of the present disclosure can be optionally replaced by a deuterium atom, such as -CH3 being replaced by -CD3).

[0235] One or more constituent atoms of the compounds presented herein can be replaced or substituted with natural or non-natural abundance isotopes of the atoms. In some embodiments, the compounds contain at least one deuterium atom. In some embodiments, the compounds contain two or more deuterium atoms. In some embodiments, the compounds contain 1-2, 1-3, 1-4, 1-5, or 1-6 deuterium atoms. In some embodiments, all hydrogen atoms in the compounds can be replaced or substituted with deuterium atoms.

[0236] In some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms bonded to carbon atoms of any Formula A substituent are each optionally replaced by a deuterium atom.

[0237] Synthetic methods for incorporating isotopes into organic compounds are well known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, NY, Appleton-Century-Crofts, 1971; The Renaissance of H / D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in a variety of studies, such as NMR spectroscopy, metabolic experiments, and / or assays.

[0238] Substitution with heavier isotopes, such as deuterium, can provide certain therapeutic advantages, such as increased in vivo half-life or reduced dosage requirements, due to greater metabolic stability, and therefore may be preferred in some cases (see, e.g., A. Kerekes et.al. J. Med. Chem. 2011, 54, 201-210; R. Xu et.al. J. Label Compd. Label Compd. Radiopharm. 2015, 58, 308-312). In particular, substitution at one or more metabolic sites may provide one or more therapeutic advantages.

[0239] The radionuclide that is incorporated into the radiolabeled compound will depend on the particular application of that radiolabeled compound. For example, for in vitro A2A / A2B labeling and competition assays: 3 H, 14 C. 82 Br, 125 I, 131 I, or 35 Compounds incorporating S may be useful. For radioimaging applications, 11 C. 18 F,125 I, 123 I, 124 I, 131 I, 75 Br, 76 Br, or 77 Br may be useful.

[0240] A "radiolabel" or "labeled compound" is understood to be a compound that incorporates at least one radionuclide. In some embodiments, the radionuclide is 3 H, 14 C. 125 I, 35 S, and 82 Br.

[0241] The present disclosure can further include synthetic methods for incorporating radioisotopes into the disclosed compounds. Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and one of ordinary skill in the art will readily recognize methods applicable to the disclosed compounds.

[0242] Methods for producing antibodies 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, such as transformed cell lines (e.g., CHO, 293E, COS). In addition, antibodies (e.g., scFv') can be expressed in yeast cells, such as Pichia (e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hanseula, or Saccharomyces. To produce the 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 the recombinant expression vector, transfect the host cell, select for transformants, culture the host cell, and recover the antibody.

[0243] If the antibody is expressed in bacterial cells (eg, E. coli), the expression vector should possess characteristics that allow for the amplification of the vector within the bacterial cell. Furthermore, when E. coli such as JM109, DH5α, HB101, or XL1-Blue is used as a host, the vector must have a promoter, such as the lacZ promoter (Ward et al., 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or a T7 promoter that can allow efficient expression in E. coli. Examples of such vectors include, for example, M13 series vectors, pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21, which expresses T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. For production into the periplasm of E. coli, the pelB signal sequence (Lei et al., 2002) is used. al., J. Bacteriol., 169:4379 (1987) may be used as a signal sequence for antibody secretion. For bacterial expression, the calcium chloride method or electroporation may be used to introduce the expression vector into bacterial cells.

[0244] When the antibody is expressed in animal cells such as CHO, COS, and NIH3T3 cells, the expression vector contains a promoter necessary for expression in these cells, such as the SV40 promoter (Mulligan et al. Nature, 277:108 (1979)), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or the CMV promoter. In addition to the nucleic acid sequence encoding the immunoglobulin or a domain thereof, additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origin of replication) and selection marker genes, can be carried in the recombinant expression vector. The selection marker gene can facilitate selection of host cells into which the vector has been introduced (see, for example, U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, the selectable marker gene usually confers resistance to drugs such as G418, hygromycin, methotrexate, etc. to a host cell into which the vector has been introduced. Examples of vectors having selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0245] In one embodiment, the antibody is produced in a mammalian cell. Exemplary mammalian host cells for expressing the antibody include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, e.g., used with a DHFR selection marker 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 derived from transgenic animals, e.g., transgenic mammals.

[0246] In an exemplary system for antibody expression, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain of an antibody that binds to an anti-PD-1 antibody (e.g., antibody X) is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to enhancer / promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus, etc., such as the CMV enhancer / AdMLP promoter regulatory element or the SV40 enhancer / AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for the selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformed host cells are cultured to allow expression of the antibody heavy and light chains, and the antibody is recovered from the culture medium.

[0247] Antibodies are also produced by genetically modified animals. For example, U.S. Patent No. 5,849,992 describes a method for expressing antibodies in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and a nucleic acid that encodes the antibody of interest and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes the antibody of interest secreted therein. The antibody can be purified from the milk or used directly for several applications. Animals that include one or more of the nucleic acids described herein are also provided.

[0248] The antibody of the present disclosure can be isolated from the inside or outside (such as the medium) of a host cell and purified as a substantially pure and homogeneous antibody. Isolation and purification methods commonly used for antibody purification may be used for antibody isolation and purification, and are not limited to any particular method. The antibody can be isolated 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. Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et at., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be performed using liquid phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include protein A columns and protein G columns. Examples of columns that use Protein A columns include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The present disclosure also includes antibodies that are highly purified using these purification methods.

[0249] Antibodies such as Antibody X can be made, for example, by preparing and expressing a synthetic gene encoding the recited amino acid sequence, or by mutating a human germline gene to provide a gene encoding the recited amino acid sequence. Additionally, this and other anti-PD-1 antibodies can be obtained, for example, using one or more of the following methods.

[0250] Humanized antibodies can be generated by replacing sequences of Fv variable regions that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, SL, Science, 229:1202-1207 (1985), Oi et al., BioTechniques, 4:214 (1986), as well as US5,585,089, US5,693,761, US5,693,762, US5,859,205, and US6,407,213. These methods include isolating, manipulating, and expressing nucleic acid sequences that encode all or part of an immunoglobulin Fv variable region from at least one of the heavy or light chains. Sources of such nucleic acids are well known to those skilled in the art and can be obtained, for example, from hybridomas producing antibodies against a given target, from germline immunoglobulin genes, or from synthetic constructs, as described above. The recombinant DNA encoding the humanized antibody can then be cloned into an appropriate expression vector.

[0251] 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. Bio. 227:799-817 (1992); and Tomlinson et al., EMBO J., 14:4628-4638 (1995). The V BASE 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, for example, as a source of human sequences for framework regions and CDRs. Consensus human framework regions can also be used, for example, as described in U.S. Patent No. 6,300,064.

[0252] Other methods for humanizing antibodies can also be used. For example, other methods can account for the three-dimensional structure of the antibody, the framework positions three-dimensionally adjacent to the binding determinants, and the immunogenic peptide sequence. See, for example, WO90 / 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. Yet another method is called "humaneering" and is described, for example, in US 2005-008625.

[0253] The antibody can comprise a human Fc region, e.g., a wild-type Fc region or an Fc region that comprises one or more modifications. In one embodiment, the constant region is modified to modify the properties of the antibody (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). For example, a human IgG1 constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237 (based on Kabat numbering). The antibody can have mutations in the CH2 region of the heavy chain that reduce or modify effector function, e.g., Fc receptor binding and complement activation. For example, the antibody can have mutations as described in U.S. Patent 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 (e.g., Angal et al. (1993) Mol. Immunol. 30:105-08), as disclosed in the art. See also, e.g., US2005 / 0037000.

[0254] 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 (e.g., biologically active antibody fragment or minibody) of the anti-PD-1 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) that contain a single variable chain, such as VH or VL, or a biologically active fragment 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 Davieset al.(1996) Protein Eng., 9(6):531-7. Like whole antibodies, sdAbs can selectively bind to specific antigens. With a molecular weight of only 12-15 kDa, sdAbs are much smaller than typical antibodies, even smaller than Fab fragments and single chain variable fragments.

[0255] Provided herein are compositions comprising an anti-PD-1 antibody, or antigen-binding fragment thereof, and a mixture of one or more acidic variants, e.g., the amount of acidic variant(s) 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 antigen-binding fragment thereof, comprising at least one deamidation site, wherein the pH of the composition is about 5.0 to about 6.5, e.g., such that at least about 90% of the 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, such as 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.

[0256] An "acidic variant" is a variant of a polypeptide of interest that is more acidic (e.g., as determined by cation exchange chromatography) than the polypeptide of interest. An example of an acidic variant is a deamidated variant.

[0257] "Deamidated" variants of a polypeptide molecule are those in which one or more asparagine residue(s) of the original polypeptide have been converted to aspartic acid, i.e., the neutral amide side chain has been converted to a residue having overall acidic properties.

[0258] As used herein, the term "mixture" refers to a composition comprising an anti-PD-1 antibody or antigen-binding fragment thereof and refers to the presence of both the desired antibody or antigen-binding fragment thereof that binds to human PD-1 or human PD-L1, and one or more acidic variants thereof. The acidic variants may include primarily deamidated antibodies that bind to human PD-1 or human PD-L1, along with minor amounts of other acidic variant(s).

[0259] In certain embodiments, the binding affinity (K D ), on-rate (K D On) and / or off rate (K D Off) is similar to that of a wild-type antibody, for example, with a difference of less than about 5-fold, 2-fold, 1-fold (100%), 50%, 30%, 20%, 10%, 5%, 3%, 2% or 1%.

[0260] antibody fragment Antibody fragments (e.g., Fab, Fab', F(ab')2, Facb, and Fv) can be prepared by proteolysis of intact antibodies. For example, antibody fragments can be obtained by treating whole antibodies with enzymes such as papain, pepsin, or plasmin. Papain digestion of whole antibodies produces F(ab)2 or Fab fragments, pepsin digestion of whole antibodies produces F(ab')2 or Fab', and plasmin digestion of whole antibodies produces Facb fragments.

[0261] Alternatively, antibody fragments can be produced recombinantly: for example, a nucleic acid encoding the 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, allowing the facile production of large amounts of these fragments. 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 approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having increased in vivo half-lives are described in U.S. Pat. No. 5,869,046.

[0262] Mini Body Antibody minibodies that bind to human PD-1 or human PD-L1 include diabodies, single chain (scFv), and single chain (Fv)2 (sc(Fv)2).

[0263] A "diabody" is a bivalent minibody constructed by gene fusion (see, for example, Holliger, P. et al. Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), EP404,097, WO93 / 11161). A diabody is a dimer consisting of two polypeptide chains. The VL and VH domains of each polypeptide chain of a diabody are linked by a linker. The number of amino acid residues constituting the linker can be 2 to 12 residues (e.g., 3 to 10 residues, or 5 or about 5 residues). The linker of the polypeptides in a diabody is typically too short to allow the VL and VH to bind to each other. Thus, the VL and VH encoded by the same polypeptide chain cannot form a single chain variable region fragment, but instead form a dimer with different single chain variable region fragments. As a result, the diabody has two antigen binding sites.

[0264] scFv is a single-chain polypeptide antibody obtained by linking 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 Monoclonal Antibodies" Vol. 113, Ed Resenburg and Moore, Springer Verlag, New York, pp. 269-315, (1994)). The order of VH and VL to be linked 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 L chain V region in ScFv can be derived from any antibody that binds to human PD-1 or human PD-L1, or an antigen-binding fragment thereof, as described herein.

[0265] sc(Fv)2 is a minibody in which two VHs and two VLs are linked 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 connecting scFvs with a linker. In the sc(FV)2 of the present invention, the two VHs and two VLs are preferably arranged in the order of VH, VL, VH, and VL ([VH] linker [VL] linker [VH] linker [VL]) starting from the N-terminus of the single-chain polypeptide, but the order of the two VHs and two VLs is not limited to the above arrangement, and they can be arranged in any order.

[0266] bispecific antibody A bispecific antibody is an antibody that has binding specificities for at least two different epitopes. An exemplary bispecific antibody may bind to two different epitopes of the PD-1 protein. Other such antibodies may combine a PD-1 binding site with a 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, diabody bispecific antibodies).

[0267] Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where these two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). According to a different approach, antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host cell. This provides greater flexibility in adjusting the ratio of the three polypeptide fragments. However, if the expression of at least two polypeptide chains in equal ratios results in high yields, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector.

[0268] According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. A preferred interface is H3 The method comprises at least a portion of a domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other undesired end products such as homodimers.

[0269] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies can be made using any convenient cross-linking method.

[0270] "Diabody" technology provides an alternative mechanism for the production of bispecific antibody fragments. These fragments contain a VH connected to a VL by a linker that is too short to allow pairing between the two domains on the same chain. Thus, 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.

[0271] Multivalent antibodies A multivalent antibody may be internalized (and / or catabolized) more quickly than a bivalent antibody by a cell expressing the antigen to which it binds. The antibodies provided herein may be multivalent antibodies (e.g., tetravalent antibodies) having three or more antigen binding sites, which may be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody. The multivalent antibody may comprise a dimerization domain and three or more antigen binding sites. Exemplary dimerization domains include (or consist of) an Fc region or a hinge region. The multivalent antibody may comprise (or consist of) from three to about eight (e.g., four) antigen binding sites. The multivalent antibody optionally comprises at least one polypeptide chain (e.g., at least two polypeptide chains), where the polypeptide chain(s) comprise two or more variable domains. For example, the polypeptide chain(s) may comprise VD1-(X1) n -VD2-(X2) n -Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or peptide spacers, and n is 0 or 1.

[0272] Conjugated antibodies The antibodies disclosed herein can be coupled to polymers (e.g., polyethylene glycol (PEG), polyethyleneimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers), hyaluronic acid, radioactive materials (e.g., 90 Y, 131 I) They may be conjugated antibodies that are coupled to a variety of molecules including fluorescent substances, luminescent substances, haptens, enzymes, metal chelates, drugs, and macromolecular substances such as toxins (e.g., calcheamicin, Pseudomonas exotoxin A, ricin (e.g., deglycosylated ricin A chain)).

[0273] In one embodiment, to improve the cytotoxicity of anti-PD-1 antibodies and therefore their therapeutic efficacy, the antibodies are conjugated with highly toxic substances, including radioisotopes and cytotoxic agents. These conjugates can selectively deliver a toxic load to the target site (i.e., cells expressing the antigen recognized by the antibody), while cells not recognized by the antibody are spared. To minimize toxicity, the conjugates are generally engineered based on molecules with short serum half-lives (hence the use of mouse sequences, and IgG3 or IgG4 isotypes).

[0274] In certain embodiments, the anti-PD-1 antibody, or antigen-binding fragment thereof, is modified with a moiety that improves its stabilization and / or retention in the circulation, e.g., at least 1.5, 2, 5, 10, or 50-fold, e.g., in blood, serum, or other tissues. For example, the antibody, or antigen-binding fragment thereof, that binds human PD-1 or human PD-L1 can be associated (e.g., conjugated) with a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or polyethylene oxide. Suitable polymers vary substantially by weight. Polymers having molecular number-average weights 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 antibody, or antigen-binding fragment thereof, that binds human PD-1 or human PD-L1 can be conjugated to a water-soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinyl alcohol or polyvinylpyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Further useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene, polymethacrylates, carbomers, and branched or unbranched polysaccharides.

[0275] The above-mentioned conjugated antibody can be prepared by chemically modifying the antibody or its low molecular weight form described herein.Methods for modifying antibodies are well known in the art (e.g., US5057313 and US5156840).

[0276] kit The present disclosure also includes pharmaceutical kits, useful, for example, in the treatment or prevention of A2A / A2B-related diseases or disorders, which include one or more containers housing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure. As will be apparent to one of skill in the art, such kits can optionally further include one or more of a variety of conventional pharmaceutical kit components, such as, for example, a container with one or more pharma- ceutically acceptable carriers, additional containers, etc. Instructions, either as an insert or label, indicating the amounts of components to be administered, administration guidelines, and / or guidelines for mixing the components, can also be included in the kit.

[0277] 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 manner. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to produce essentially the same results. It is understood that certain features of the present invention that are described in the context of separate embodiments for clarity can also be provided in a single embodiment. Conversely, various features of the present invention that are described in the context of a single embodiment for brevity can also be provided separately or in any suitable subcombination.

[0278] In addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Each reference cited in this application, including all patents, patent applications, and patent publications, is hereby incorporated by reference in its entirety. EXAMPLES

[0279] Example 1: In vitro CHO-PD-L1 co-culture assay 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 an adenosine mimetic, 5'-N-ethylcarboxamidoadenosine (NECA) reagent, was used to activate adenosine signaling. Under these conditions, compound 9 is able to restore T cell function with anti-PD1 reagents.

[0280] Anti-PD1 reagents tested in this system include (A) pembrolizumab, (B) antibody X, and (C) compound Y under the treatment of 2 μM NECA, as shown in FIG. 1.

[0281] protocol On day 0, 10,000 CHOPDL1+ cells were plated in a plate 96 tissue culture flat bottom plate in 100ul of CHO medium without antibiotics. On day 1, T cells were thawed and resuspended in T cell medium at 1x106 cells / ml. Medium was removed from the CHOPDL1+ cell plate and 130ul of T cell medium was added. 198ul of T cell medium was added to the compound plate at 2ul, or -1:100 dilution, then resuspended. 20ul of compound from the compound plate was added to the CHO cell plate at a final dilution of compound at 1:1000. 50ul of T cells (50,000 cells) were added to the plate containing CHO cells for a total volume of 200ul and incubated at 37°C for 72 hours. After 3 days of culture, supernatants were collected for hIFNg and hIL2 cytokine assays performed using ProCartaplex 2-plex kits (Life Technologies catalog number PPX-02) for hIFNg and hIL2 (manufacturer's protocol). Cytokine assays using ProCartaplex kits are performed on a Flex Map3D Luminex multiplex platform.

[0282] Example 2: In vitro mixed lineage reaction assay In another in vitro assay, the mixed lineage reaction assay (MLR), PBMCs from healthy donors were stimulated with CD3 antibody and treated with atezolizumab, compound 9, or compound 3A in the presence of 10 μM of the adenosine mimetic reagent, NECA (Figures 2A-2D).

[0283] protocol On day 0, 10,000 PBMCs from a healthy donor were co-cultured with 10,000 γ-irradiated PBMCs from another healthy donor. Cells were plated in 200ul RPMI-1640 medium supplemented with 10% FBS in 96-well tissue culture round bottom plates and treated with or without 10μM NECA, 5ng / ml CD3 antibody (clone OKT3), and the indicated concentrations of compounds / antibodies. Cells were incubated at 37°C for 4 days. IFN-γ in the supernatant of each well was measured with a HTRF kit (Cisbio, 62HIFNGPEH) and the fluorescent signal was detected on day 4 with a Pherastar FS plate reader (BMG Labtech).

[0284] When compound 9 was combined with an anti-PD-L1 antibody (i.e., atezolizumab), it was able to significantly increase IFNγ production (Figures 2A-2B). Also, when compound 3A was combined with an anti-PD-L1 antibody (i.e., atezolizumab), it was able to significantly increase IFNγ production (Figures 2C-2D).

[0285] Example 3: In vivo efficacy study in mouse synergy model Compound 9 was evaluated for inhibition of tumor growth in two different models. The CT-26 murine colon carcinoma has been demonstrated to have high levels of adenosine in the tumor microenvironment, reflecting the adenosine-rich tumors selected for clinical investigation (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 52% tumor growth inhibition (TGI) compared to vehicle control and further demonstrated additivity in combination with an anti-PD-1 antibody (77% TGI vs. vehicle) (Figure 3A). In contrast, no single-agent efficacy was observed when the same regimen was applied to the model when hosted in NSG mice, which lack the T and NK cells where compound 9 is believed to exert most of its therapeutic action (Figure 3B).

[0286] Compound 9 was further evaluated in the B16 melanoma model, an immunologically hypothermic model, for its ability to subvert immune checkpoint resistance. Both compound 9 and anti-PD-L1 had modest but non-significant single-agent activity, but when combined, acted synergistically to result in 54% tumor growth inhibition (Figure 3C). These data suggest that compound 9 can alter the microenvironment of adenosine-rich tumors and cooperate with other immuno-oncology drugs to promote effective anti-tumor responses.

[0287] Example A: Activity of A2A / A2B Inhibitors I. A2A Tag-lite® HTRF Assay The assay was performed in black low volume 384-well polystyrene plates (Greiner 784076-25) in a final volume of 10 μL. Test compounds were first serially diluted in DMSO, then 100 nl was added to the wells of the plate before adding other reaction components. The final concentration of DMSO was 1%. Tag-lite® adenosine A2A labeled cells (CisBio C1TT1A2A) were diluted 1:5 in Tag-lite buffer (CisBio LABMED) and spun at 1200 g for 5 min. The pellet was resuspended in Tag-lite buffer at 10.4 times the volume of the initial cell suspension, and adenosine A2A receptor red antagonist fluorescent ligand (CisBio L0058RED) was added at a final concentration of 12.5 nM. 10ul of the cell-ligand mixture was added to the assay wells and incubated at room temperature for 45 minutes before being read on a PHERAstarFS plate reader (BMG Labtech) equipped with an HTRF337 / 620 / 665 optical module. Percent binding of fluorescent ligand was calculated, with 100nM A2A antagonist ZM241385 (Tocris1036) control replacing 100% ligand and 1% DMSO replacing 0%. Data of % binding versus log inhibitor concentration were fitted to a one-site competitive binding model (GraphPad Prism version 7.02) (ligand constant = 12.5nM and ligand Kd = 1.85nM). The K values ​​of the examples obtained by this method were i The data are shown in Table 2.

[0288] II. Adenosine A2B Receptor Cyclic AMP GS Assay Stably transfected HEK-293 cells expressing human adenosine A2B receptors (Perkin Elmer) were maintained in MEM medium with 10% FBS and 100 μg / ml Geneticin (Life Technologies). Geneticin was removed from the cultures 18-24 hours prior to the assay. Intracellular cAMP accumulation was measured using cisbio's cAMP-GS Dynamic Kit, which utilizes FRET (fluorescence resonance energy transfer) technology. Appropriate concentrations of compounds of the present disclosure are mixed with 10,000 cells / well in white 96-well half-area plates (Perkin Elmer) with gentle shaking for 30 minutes at room temperature (RT). 12 nM of the agonist NECA (R&D Technologies) was added to each well with gentle shaking for 60 minutes at room temperature. Detection reagents d2-labeled cAMP (receptor) and anti-cAMP cryptate (donor) were added to each well with gentle shaking for 60 minutes at room temperature. Plates were read on a Pherastar (BMG Labtech) and the fluorescence ratio 665 / 620 was calculated and the EC was calculated by curve fitting the percent of control versus the logarithm of compound concentration using GraphPad Prism. 50 The EC of the example obtained by this method was determined. 50 The data are shown in Table 2.

[0289] Table 2.A 2A _Ki data (Example A(I)) and A 2B _cAMP_EC 50 The data (Example A(II)) is provided below. [Table 2]

[0290] Example A1: 3-(5-amino-2-(pyridin-2-ylmethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (compound 1) [ka] Step 1: 3-(2-amino-6-chloropyrimidin-4-yl)benzonitrile [ka] A mixture of 4,6-dichloropyrimidin-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 then heated and stirred at 60° C. for 2 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 eluted with 8% EtOAc in dichloromethane to give the desired product. 11 LC-MS (M+H) calculated for H8ClN4 + :231.0, actual value:231.0.

[0291] Step 2: 2-(pyridin-2-yl)acetohydrazide [ka] 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) at room temperature. The mixture was heated at 85° C. for 4 h, stirred, and then cooled to room temperature. Upon standing, a white solid formed, which was collected by filtration and used in the next step without further purification. C 10 LC-MS calculated for NO (M+H) + : 152.1, actual value: 152.0.

[0292] Step 3: 3-(5-amino-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile [ka] 2-(Pyridin-2-yl)acetohydrazide (2.62 g, 17.34 mmol) was added to a solution of 3-(2-amino-6-chloropyrimidin-4-yl)benzonitrile (4.00 g, 17.34 mmol) in ethanol (35 mL) at room temperature. After heating and stirring under reflux for 2 h, the reaction mixture was cooled to room temperature and concentrated. The resulting residue was taken up in N,O-bis(trimethylsilyl)acetamide (20 mL) and stirred at 120° C. for 7 h. The mixture was then cooled to room temperature, poured onto ice, and stirred at room temperature for 1 h. The resulting solid was collected by filtration and taken up in 20 mL of 1N HCl solution. The resulting mixture was stirred at room temperature for 1 h, filtered, and the aqueous layer was neutralized by the addition of saturated NaHCO3 solution. The resulting solid was collected by filtration and dried to give the desired product. C 18 H 14 LC-MS calculated for N7 (M+H) + :328.1;Actual value 328.1.

[0293] Step 4: 3-(5-amino-8-bromo-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile [ka] 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) at -30 °C was added NBS (1.09 g, 6.11 mmol) in small portions. The reaction mixture was slowly warmed to 0 °C to obtain a homogeneous solution. After stirring at 0 °C for 1 h, the reaction mixture was diluted with saturated NaHCO3 and the resulting precipitate was collected by filtration. The solid was then purified by flash chromatography on a silica gel column eluting with 0-10% MeOH in DCM to give the desired product. C 18 H 13 LC-MS (M+H) calculated for BrN7 + :406.0;Actual value 406.0.

[0294] Step 5: 3-(5-amino-2-(pyridin-2-ylmethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile Pd(Ph3P)4 (284 mg, 0.246 mmol) was added to a mixture of 4-(tributylstannyl)pyrimidine (1090 mg, 2.95 mmol), 3-(5-amino-8-bromo-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (1000 mg, 2.46 mmol), and copper(I) chloride (244 mg, 2.46 mmol) in 1,4-dioxane (12 mL). The reaction mixture was purged with N2 and stirred at 80 °C for 7 h. The resulting mixture was cooled to room temperature, concentrated, diluted with DCM (50 mL), and washed with saturated NH4OH solution. The organic layer was dried over Na2SO4, concentrated, and purified by preparative LC-MS (pH 2, acetonitrile / water with TFA) to give the product as a TFA salt. C 22 H 16 LC-MS calculated for N9 (M+H) + :406.2;Actual value 406.2. 1 H NMR(500MHz,DMSO)δ 8.95(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).

[0295] 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) [ka] Step 1: 2-(2,6-difluorophenyl)-2-hydroxyacetate methyl [ka] 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) at 0° C. The mixture was stirred at room temperature 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 give the crude product, which was used in the next step without further purification. C 11 H 12 F2NO3(M+H+MeCN) + LC-MS calculated for: m / z = 244.1; found 244.2.

[0296] 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 This compound was prepared using a procedure similar to that described for Example A1, substituting 2-(2,6-difluorophenyl)-2-hydroxymethyl acetate 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) eluted with an isocratic mobile phase of 25% MeOH in CO2 at a flow rate of 80 mL / min. Peak 1 was isolated and purified by preparative LCMS (pH=2, MeCN / water with TFA) to give the desired product as a TFA salt. 23 H 15 LC-MS calculated for F2N8O (M+H) + : m / z = 457.1; measured value 457.1. 1H NMR(500MHz,DMSO)δ 8.94(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).

[0297] Example A3: Synthesis of 3-(5-amino-2-((5-(pyrimidin-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-(pyrimidin-2-yl)-1H-tetrazol-1-yl)methyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (compound 3B) [ka] Step 1: 3-(5-amino-2-(hydroxymethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile [ka] 2-Hydroxyacetohydrazide (2.34 g, 26.01 mmol) was added to a solution of 3-(2-amino-6-chloropyrimidin-4-yl)benzonitrile (4.00 g, 17.34 mmol) (Example A1, step 1) in ethanol (35 mL) at room temperature. After heating and stirring under reflux for 2 hours, the reaction mixture was cooled to room temperature and concentrated. The resulting residue was taken up in N,O-bis(trimethylsilyl)acetamide (20 mL) and stirred at 120° C. for 7 hours. The mixture was then cooled to room temperature, poured onto ice, and stirred at room temperature for 1 hour. The resulting solid was collected by filtration and taken up in 20 mL of 1N HCl solution. The resulting mixture was stirred at room temperature for 1 hour, filtered, and the aqueous layer was neutralized by the addition of saturated NaHCO3 solution. The resulting solid was collected by filtration and dried to give the desired product. C 13 H 11 LC-MS calculated for NO (M+H) + :267.1;Actual value 267.1.

[0298] Step 2: 3-(5-amino-8-bromo-2-(hydroxymethyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile [ka] 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) at -30°C was added NBS (0.67 g, 3.76 mmol) in small portions. The reaction mixture was slowly warmed to 0°C to give a homogeneous solution. After stirring at 0°C for 1 h, the reaction mixture was diluted with saturated NaHCO3 solution and the desired product was collected by filtration and dried. 13 H 10 LC-MS (M+H) calculated for BrNO + :345.0;Actual value 345.0.

[0299] Step 3: 3-(5-amino-2-(hydroxymethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile [ka] 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 room temperature, concentrated, and purified by flash column chromatography eluting with 0% to 10% methanol in DCM to give the product. C 17 H 13 LC-MS calculated for NO (M+H) + :345.1;Actual value 345.1.

[0300] Step 4: 3-(5-amino-2-(chloromethyl)-8-(pyrimidin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile [ka] 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 room temperature. The reaction mixture was stirred at room temperature for 5 h, concentrated and purified by flash chromatography eluting with 0% to 5% methanol in DCM to give the product. 17 H 12LC-MS (M+H) calculated for ClN8 + :363.1;Actual value 363.1.

[0301] Step 5: 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) 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 room temperature, diluted with methanol (4 mL) and purified by preparative LC-MS (pH 2, acetonitrile / water with TFA) to give the product as a TFA salt. 23 H 16 N 13 LC-MS (M+H) calculated for + :474.2;Actual value 474.2.

[0302] Compound 3A: 1 H NMR(500MHz,DMSO)δ 8.99(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=7.7,1.1Hz,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).

[0303] 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) [ka] Step 1: 6-Chloro-N 2 ,N 2 -Bis(4-methoxybenzyl)pyrimidine-2,4-diamine [ka] To a solution of 2,6-dichloropyrimidin-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 room temperature, diluted with water (100 mL), and extracted with EtOAc (100 mL). The organic layer was washed with water, dried over anhydrous sodium sulfate, and concentrated to give the crude product, which was used in the next step without further purification. 20 H 22 LC-MS (M+H) calculated for ClNO2 + : m / z = 385.1; measured value 385.1.

[0304] Step 2: 7-Chloro-N 5 ,N 5 -Bis(4-methoxybenzyl)-[1,2,4]triazolo[1,5-c]pyrimidine-2,5-diamine [ka] O-Ethyl carbonyl isothiocyanate (3.1 mL, 26 mmol) was dissolved in 6-chloro-N 2 ,N 2To a solution of N,N-bis(4-methoxybenzyl)pyrimidine-2,4-diamine (1.0 g, 2.6 mmol) at room temperature in 1,4-dioxane (5.0 mL) was added. The reaction mixture was then stirred at 90° C. overnight, cooled to room temperature, and filtered. 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 hours, cooled to room temperature, and concentrated. The resulting material was taken up in EtOAc, washed with water, dried over anhydrous sodium sulfate, and concentrated. The crude material was purified by silica column chromatography eluting with 0% to 50% EtOAc / hexane to give the product. 21 H 22 LC-MS (M+H) calculated for ClNO2 + :425.1;Actual value:425.2.

[0305] Step 3: 3-(2-amino-5-(bis(4-methoxybenzyl)amino)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile [ka] Chloro(2-dicyclohexylphosphino-2',4',6'-tri-i-propyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (330 mg, 0.42 mmol) in 1,4-dioxane (8.8 mL) and water (1.8 mL) (3-cyanophenyl)boronic acid (460 mg, 3.2 mmol), 7-chloro-N 5 , N 5 To a mixture of 1,2,4-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) was added. The mixture was purged with N2 and stirred at 95 °C overnight. The reaction mixture was then cooled to room temperature, concentrated, and purified by silica gel column chromatography eluting with 0% to 50% EtOAc in DCM to give the desired product.28 H 26 LC-MS calculated for N7O2 (M+H) + :492.2;Actual value 492.2.

[0306] Step 4: 3-(2-amino-5-(bis(4-methoxybenzyl)amino)-8-bromo-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile [ka] 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 added NBS (120 mg, 0.66 mmol) slowly at 0° C. The reaction mixture was then stirred at room temperature for 30 min before adding water (10 mL). The resulting solid was collected by filtration and dried to give the desired product. 28 H 25 LC-MS (M+H) calculated for BrNO2 + : m / z = 570.1; measured value 570.2.

[0307] 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 [ka] 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 for 30 min in a microwave reactor. The reaction mixture was then cooled to room temperature, 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 give the desired product. C 32 H 28 LC-MS calculated for N9O2 (M+H) + : m / z = 570.2; measured value 570.3.

[0308] 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 [ka] To a mixture of copper(II) bromide (91 mg, 0.407 mmol) and tert-butylnitrile (0.054 ml, 0.407 mmol) in acetonitrile (3 mL) at 50 °C under nitrogen was added 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) dropwise. The mixture was stirred at 50 °C for 2 h. After cooling to room temperature, 1N aqueous NH4OH solution (20 mL) was added and the mixture was extracted three times with CHCl2 (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 give the desired product. 32 H 26 LC-MS (M+H) calculated for BrNO + : m / z = 633.1; measured value 633.2.

[0309] 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 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 and stirred at 110° C. under nitrogen overnight. The reaction mixture was then cooled to room temperature, concentrated and TFA (1.0 mL) was added. The resulting mixture was then stirred at 110° C. for 30 min, cooled to room temperature, diluted with acetonitrile, filtered and purified by preparative LC-MS (pH 2, acetonitrile / water with TFA) to give the desired product as a TFA salt. 23 H 18 LC-MS calculated for NO (M+H)+ : m / z = 436.2; measured value 436.2. 1 H NMR(600MHz,DMSO)δ 8.97(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=5.4,1.4Hz,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).

[0310] Example A5: Synthesis of 3-(5-amino-2-(hydroxy(phenyl)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile (Compound 5) [ka] Step 1: 3-(2-amino-6-chloropyrimidin-4-yl)benzonitrile [ka] A mixture of 4,6-dichloropyrimidin-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 then heated at 60° C. for 2 days. After cooling to room temperature (RT), the mixture was concentrated, diluted with water and extracted with dichloromethane (DCM, 3×30 mL). The combined organic layers were dried over MgSO4, filtered and concentrated. The crude residue was purified by flash chromatography on a silica gel column with 8% ethyl acetate (EtOAc) in dichloromethane to give the desired product. 11 LC-MS (M+H) calculated for H8ClN4 + :231.0, actual value:231.0.

[0311] Step 2: 3-(5-amino-2-(hydroxy(phenyl)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)benzonitrile A solution of 3-(2-amino-6-chloropyrimidin-4-yl)benzonitrile (100 mg, 0.434 mmol) and 2-hydroxy-2-phenylacetohydrazide (108 mg, 0.650 mmol) in ethanol (2 ml) was heated and stirred at 95° C. for 3 h. After cooling to RT, the reaction mixture was concentrated to dryness, taken up in N,O-bis(trimethylsilyl)acetamide (1 mL) and stirred at 120° C. for 7 h. The resulting mixture was cooled to RT, poured onto ice and stirred for 1 h. The resulting suspension was extracted three times with DCM. The combined organic layers were dried over MgSO4, filtered and concentrated. The crude residue was dissolved in methanol (MeOH) and purified by preparative LC-MS (pH 2, acetonitrile / water with TFA) to give the product as a TFA salt. C 19 H 15 LC-MS calculated for NO (M+H) + : m / z = 343.1; measured value 343.1.

[0312] 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) [ka] Step 1: 3-(2-amino-6-chloropyrimidin-4-yl)-2-fluorobenzonitrile [ka] To a solution of 3-bromo-2-fluorobenzonitrile (18.3 g, 91 mmol) in THF (60 mL) cooled to 0° C. was added i-PrMgClLiCl complex (70.4 mL, 91 mmol) in THF (1.3 M) over 20 min. The mixture was stirred at 0° C. for 50 min, then zinc chloride (48.1 mL, 91 mmol) in 2-MeTHF (1.9 M) was added at 0° C. The reaction was stirred at room temperature for 25 min, at which point 4,6-dichloropyrimidin-2-amine (10 g, 61.0 mmol) was added in one portion. The solution was stirred for 10 min. Tetrakis(triphenylphosphine)palladium (1.41 g, 1.22 mmol) was added to the mixture and the reaction was stirred at room temperature for 16 h. Upon completion, 2,4,6-trimercaptotriazine silica gel (2 g) was added to the reaction solution. The mixture was stirred for 1 hour and then 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 give 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 carried forward without further purification. 11 LC-MS (M+H) calculated for H8ClNO + : m / z = 249.0; measured value 249.0.

[0313] Step 2: 2-(2,6-difluorophenyl)-2-hydroxyacetate methyl [ka] 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) at 0° C. The mixture was stirred at room temperature 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 give the crude product, which was used in the next step without further purification. C 11 H 12 F2NO3(M+H+MeCN) +LC-MS calculated for: m / z = 244.1; found 244.2.

[0314] Step 3: 2-(2,6-difluorophenyl)-2-hydroxyacetylhydrazide [ka] 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. LC-MS (M+H) calculated for C8H9F2N2O2 + :203.1;Actual value:203.2.

[0315] Step 4: 3-(5-amino-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile The title compound was prepared using a procedure similar to that described in step 2 of Example A5, substituting 3-(2-amino-6-chloropyrimidin-4-yl)-2-fluorobenzonitrile for 3-(2-amino-6-chloropyrimidin-4-yl)benzonitrile and 2-(2,6-difluorophenyl)-2-hydroxyacetohydrazide for 2-hydroxy-2-phenylacetohydrazide. 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 peak 1 and peak 2 were 3.8 and 5.3 min, respectively. After concentration, peak 2 was purified by preparative LCMS (pH=2, MeCN / water with TFA) to give the desired product as a TFA salt. C 19 H 17 LC-MS calculated for NO (M+H) + : m / z = 397.1; measured value 397.1.

[0316] Example A7: Synthesis of 5-amino-7-(3-cyano-2-fluorophenyl)-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazole[1,5-c]pyrimidine-8-carbonitrile (compound 7) [ka] Step 1: 3-(5-amino-8-bromo-2-((2,6-difluorofluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile [ka] This compound was prepared using a procedure similar to that described for Example A1, step 4, substituting 3-(5-amino-2-((2,6-difluorofluorophenyl)(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 LC-MS (M+H) calculated for BrF3N6O + :475.0;Actual value 475.0.

[0317] 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 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 room temperature, the reaction was diluted with saturated NaHCO3 and the organics were extracted with EtOAc (3x). The combined organics were dried over MgSO4 and concentrated. The two enantiomers were separated using Phenomenex HPLC with an isocratic mobile phase of 60% EtOH in hexanes at a flow rate of 20 mL / min. The compounds were separated by chiral HPLC using a Lux Cellulose-4 column (21.2×250 mm, 5 μm particle size). The retention times of peak 1 and peak 2 were 4.9 and 7.2 min, respectively. After concentration, peak 1 was purified by preparative LC-MS (pH 2, acetonitrile / water with TFA) to give the desired product as the TFA salt. 20 H 11 LC-MS calculated for F3N7O (M+H) + :422.1;Actual value 422.1.

[0318] 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) [ka] Step 1: Methyl 2-(2-fluoro-6-vinylphenyl)acetate [ka] A mixture of 2-(2-bromo-6-fluorophenyl)methyl acetate (6.0 g, 24 mmol), potassium phosphate, tribasic (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. 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 then added, and the reaction mixture was purged with N2 and stirred at 80 °C for 16 h. The reaction mixture was then cooled to room temperature, concentrated, and extracted with EtOAc (x3). The combined organic layers were dried over MgSO4, concentrated, and purified by column chromatography (0 to 50% EtOAc in DCM). C 11 H 12 LC-MS calculated for FO2 (M+H) + :195.1;Actual value 195.1.

[0319] Step 2: 2-(2-fluoro-6-vinylphenyl)-2-hydroxyacetate methyl [ka] 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-dimethyltetrahydrofuran-4H-4a, 7-methanobenzo[c][1,2]oxazileno[2,3-b]isothiazole 3,3-dioxide (4.7 g, 20.6 mmol) was added dropwise in THF (0.5 M). After stirring at -78 °C for 30 min, the reaction mixture was warmed to 0 °C and stirred for 1 h. The reaction was quenched with saturated NH4Cl solution. The aqueous layer was extracted with DCM (3x). The combined organics were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by column chromatography eluting with 0-50% ethyl acetate / hexanes to give the desired product. 11 H 11LCMS calculated for FO3Na (M+Na) + :233.1;Actual value 233.1.

[0320] Step 3: 2-(2-fluoro-6-vinylphenyl)-2-hydroxyacetohydrazide [ka] This compound was prepared using a procedure similar to that described for Example A6, step 3, substituting 2-(2-fluoro-6-vinylphenyl)-2-hydroxyacetate for methyl 2-(2,6-difluorophenyl)-2-hydroxyacetate. 10 H 11 LC-MS calculated for FN2O2 (M+H) + : 211.1, actual value: 211.1.

[0321] Step 4: 3-(5-amino-2-((2-fluoro-6-vinylphenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile [ka] 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 LCMS calculated for F2NO (M+H) + :405.1;Actual value:405.1.

[0322] Step 5: 3-(5-amino-2-((2-fluoro-6-formylphenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-fluorobenzonitrile [ka] Osmium tetroxide in water (4% w / w, 0.36 mL, 0.12 mmol) 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 room temperature 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 (x3). The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. The crude material was purified by column chromatography eluting with 0-100% ethyl acetate / hexane to give the desired product. C 20 H 13 LC-MS (M+H) calculated for F2N6O2 + :407.1;Actual value 407.1.

[0323] 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 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 Lux Cellulose-4 column (21.2×250 mm, 5 μm particle size) eluted with an isocratic mobile phase of 45% EtOH in hexane at a flow rate of 20 mL / min. The retention times of peak 1 and peak 2 were 14.9 min and 17.5 min, respectively. After concentration, peak 2 was further separated by chiral HPLC using a Phenomenex Lux Cellulose-1 column (21.2×250 mm, 5 μm particle size) eluted with an isocratic mobile phase of 30% EtOH in hexane at a flow rate of 20 mL / min. The retention times of peak 1 and peak 2 were 11.0 min and 15.5 min, respectively. After concentration, peak 1 was purified by preparative LCMS (pH=2, MeCN / water with TFA) to give the desired product as a TFA salt. C 25 H 23 LC-MS (M+H) calculated for F2N8O2 + :505.2;Actual value 505.2.

[0324] 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) [ka] Step 1: 3-bromo-1-(2-(3-cyanophenyl)-2-oxoethyl)-1H-1,2,4-triazole-5-carboxylate methyl [ka] To methyl 3-bromo-1H-1,2,4-triazole-5-carboxylate (5.0 g, 24.3 mmol), 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 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 give the desired product as a white solid (5.2 g, 61%). C 13 H 10 LC-MS (M+H) calculated for BrNO + : m / z = 349.0; measured value 349.0.

[0325] Step 2: 3-(2-bromo-8-oxo-7,8-dihydro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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 give the product (8.4 g, 88%). C 12 LC-MS calculated for H7BrNO (M+H) + : m / z = 316.0; measured value 316.0.

[0326] Step 3: 3-(2-bromo-8-chloro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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) were 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 give the product (8.8 g, 99%). 12 LC-MS (M+H) calculated for H6BrClN5 + : m / z = 333.9; measured value 334.0.

[0327] Step 4: 3-(8-(bis(4-methoxybenzyl)amino)-2-bromo-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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 give the product (14.1 g, 94%). 28 H 24 LC-MS (M+H) calculated for BrNO2 + : m / z = 555.1; measured value 555.1.

[0328] Step 5: 3-(8-(bis(4-methoxybenzyl)amino)-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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, after which 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.

[0329] 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) were 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 in the previous step was added slowly via syringe. The reaction mixture was then stirred overnight at 60 °C, 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 by flash chromatography to give the product (0.11 g, 71%). 34 H 30 LC-MS calculated for N7O2 (M+H) + : m / z = 568.2; measured value 568.3.

[0330] Step 6: 3-(8-amino-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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 as a white solid (TFA salt) (57 mg, 90%). 18 H 14 LC-MS calculated for N7 (M+H) + : m / z = 328.1; measured value 328.1.

[0331] Step 7: 3-(8-amino-5-bromo-2-(pyridin-2-ylmethyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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 give the crude product, which was used in the next step without further purification. 18 H 13 LC-MS (M+H) calculated for BrN7 + :406.0;Actual value 406.0.

[0332] 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 A mixture of 6-chloro-2-methylpyridazin-3(2H)-one (30 mg, 0.21 mmol), bis(pinacolato)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 with TFA) to give the product as a TFA salt. 23 H 18 LC-MS calculated for NO (M+H) + : m / z = 436.2; measured value 436.2.

[0333] 1 H 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).

[0334] 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) [ka] Step 1: 3-bromo-1-(2-(3-cyanophenyl)-2-oxoethyl)-1H-1,2,4-triazole-5-carboxylate methyl [ka] To methyl 3-bromo-1H-1,2,4-triazole-5-carboxylate (5.0 g, 24.3 mmol), 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 give the desired product as a white solid (5.2 g, 61%). C 13 H 10 LC-MS (M+H) calculated for BrNO + : m / z = 349.0; measured value 349.0.

[0335] Step 2: 3-(2-bromo-8-oxo-7,8-dihydro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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 mixture was diluted with water. The resulting precipitate was collected by filtration, washed with water and dried under vacuum to give the product (8.4 g, 88%). C 12 LC-MS calculated for H7BrNO (M+H) + : m / z = 316.0; measured value 316.0.

[0336] Step 3: 3-(2-bromo-8-chloro-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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 give the product (8.8 g, 99%). 12 LC-MS (M+H) calculated for H6BrClN5 + : m / z = 336.0; measured value 336.0.

[0337] Step 4: 3-(8-(bis(4-methoxybenzyl)amino)-2-bromo-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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 give the product (14.1 g, 94%). 28 H 24 LC-MS (M+H) calculated for BrNO2 + : m / z = 555.1; Measured value: 555.1.

[0338] Step 5: 3-(8-(bis(4-methoxybenzyl)amino)-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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), potassium phosphate tribasic (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 h. The reaction mixture was cooled to room temperature and most of the 1,4-dioxane was removed. The resulting precipitate was collected by filtration and dried to give the crude product (9.1 g), which was used directly in the next step. 30 H 27 LC-MS calculated for N6O2 (M+H) + : m / z = 503.2; measured value 503.1.

[0339] Step 6: 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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 was added 1-bromopyrrolidine-2,5-dione (254 mg, 1.43 mmol) at 0° C. The resulting mixture was stirred for 4 h and directly purified on a silica gel column to give the desired product (780 mg, 94%). 30 H 26 LC-MS (M+H) calculated for BrNO2 +: m / z = 581.1; measured value 581.2.

[0340] 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 [ka] 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) were 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 on a silica gel column to give the desired product (176 mg, 67%). C 34 H 29 LC-MS calculated for N8O2 (M+H) + : m / z = 581.2; measured value 581.1.

[0341] 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 [ka] 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 on a silica gel column to give the desired product (130 mg, 74%). C 33 H 27 LC-MS calculated for N8O3 (M+H) + : m / z = 583.2; measured value 583.2.

[0342] 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 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.

[0343] 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) was added the freshly prepared Grignard reagent from the previous step at -10 °C. 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 in vacuo. 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 adding aqueous NaHCO3 solution.

[0344] The crude material was directly purified by silica gel column to give the desired product (60 mg, 64%) as a racemic mixture, which was then separated by chiral HPLC using a chiral column (Phenomenex Lux 5um Cellulose-4, 21.2x250mm) and 75% EtOH in hexane (20mL / min) solvent system.

[0345] Peak 2 was isolated and further purified by preparative LC / MS (pH=2, acetonitrile / water with TFA) to give the desired product as a TFA salt. 23 H 15 LC-MS calculated for F2N8O (M+H) + : m / z = 457.1; Measured value: 457.0.

[0346] 1 H 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).

[0347] 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) [ka] Step 1: 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-vinyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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 give the crude product, which was used in the next step without further purification. 30 H 26 LC-MS (M+H) calculated for BrNO2 + : m / z = 581.1; Measured value: 581.1.

[0348] Step 2: 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-formyl-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)benzonitrile [ka] 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 on a silica gel column to give the desired product. 29 H 24 LC-MS calculated for N6O3Br (M+H) + : m / z = 583.1; measured value 583.1.

[0349] 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 [ka] 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.

[0350] 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) was added the freshly prepared Grignard reagent from the previous step at -10 °C. 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 give the desired product as a racemic mixture. C 35 H 28 LC-MS (M+H) calculated for N6O3BrF2 + : m / z = 697.1; measured value 697.1.

[0351] 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 [ka] 3-(8-(bis(4-methoxybenzyl)amino)-5-bromo-2-((2,6-difluorophenyl)(hydroxy)methyl)-[1,2,4]triazolo[1,5 A mixture of 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) was purged with N2 and heated at 95 °C for 7 h. The mixture was concentrated and purified by flash chromatography to give the desired product as a colorless oil. 39 H 32 N7O4F2(M+H) + LCMS calculated for: 700.2; found 700.2.

[0352] 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 [ka] 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 room temperature. The resulting mixture was stirred for 4 h, concentrated, and used in the next step without further purification. C 39 H 31 LC-MS (M+H) calculated for N7O3ClF2 + : m / z = 718.2; measured value 718.2.

[0353] 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 To 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 (40 mg, 0.084 mmol) in 1 mL of DMSO was added ammonia solution (1 mL). The mixture was heated at 100° C. for 10 h under 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 basified by adding aqueous NaHCO3 solution. The crude material was directly purified by silica gel column to give the desired product as a racemic mixture. The products were then separated by chiral HPLC using a chiral column (AM-1) and a 45% EtOH in hexanes (20 mL / min) solvent system. Peak 1 was isolated and further purified by preparative LCMS (pH=2, acetonitrile / water with TFA) to give the desired product as a TFA salt. 23 H 17 LC-MS calculated for F2N8O (M+H) + : m / z = 459.1; measured value 459.0.

[0354] 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) [ka] 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-tetramethylphenyl)-2-(2,6-difluorophenyl)-2-hydroxyethyl)benzonitrile in dioxane (3.0 mL) and water (0.60 mL). To a solution of 2-(2-(2-phenyl-1,3,2-dioxaborolan-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) was added potassium phosphate tribasic (0.472 g, 2.23 mmol). The resulting 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) and heated to 80° C. for 20 min. The reaction mixture was then cooled to room temperature, concentrated, and basified by the addition of aqueous NaHCO3 solution. The crude material was directly purified by silica gel column to give the desired product (257 mg, 72%) as a racemic mixture.

[0355] The products were then separated by chiral HPLC using a chiral column (Phenomenex Lux 5um Cellulose-2, 21.1 x 250 mm) and a 35% EtOH in hexane (20 mL / min) solvent system. Peak 2 was isolated and further purified using preparative LC / MS (pH = 2, acetonitrile / water with TFA) to give the desired product as the TFA salt. 26 H 20 LC-MS calculated for F2N7O (M+H) + : m / z = 484.2; measured value 484.2. 1H 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).

[0356] 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) [ka] Step 1. 4,6-Dichloro-3H-[1,2,3]triazolo[4,5-c]pyridine [ka] 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) at 0 °C. The solution was stirred for 30 min. Water (20 mL) was added and the white precipitate was filtered, washed with water and dried to give the desired product. LC-MS calculated for C5H3Cl2N4: 189.0 (M+H). + ;Measured value: 189.0 (M+H) + .

[0357] Step 2. 6-Chloro-N-(2,4-dimethoxybenzyl)-3H-[1,2,3]triazolo[4,5-c]pyridin-4-amine [ka] 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 for ClNO: 320.1 (M+H) + ;Measured value: 320.3 (M+H) + .

[0358] Step 3. 6-Chloro-N-(2,4-dimethoxybenzyl)-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-4-amine [ka] 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) was added diisopropyl azodicarboxylate (0.647 mL, 3.28 mmol) at 0° C. The resulting mixture was stirred at 0° C. for 1 h. Direct purification on a silica gel column afforded the desired product (375 mg, 33.4%). C 20 H 20 LC-MS calculated for ClNO: 411.1 (M+H) + ;Measured value: 411.2 (M+H) + .

[0359] Step 4. 3-(4-((2,4-dimethoxybenzyl)amino)-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile [ka] 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. under microwave irradiation for 90 min. 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 give the desired product (300 mg, 68.9%). 27 H 24 LC-MS calculated for N7O2: 478.2 (M+H) + ;Measured value: 478.3 (M+H) + .

[0360] Step 5. 3-(4-amino-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile [ka] 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, 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 give the desired product (175 mg, 85%). C 18 H 14 LC-MS calculated for N7: 328.1 (M+H) + ;Measured value: 328.2 (M+H) + .

[0361] Step 6. 3-(4-amino-7-bromo-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile [ka] 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 layer was separated, dried over Na2SO4, filtered, and evaporated under reduced pressure. The resulting residue was purified on a silica gel column to give the desired product (135 mg, 62.2%). C 18 H 13 LC-MS calculated for BrN7: 406.0 (M+H) + and 408.0 (M+H) + ;Measured value: 406.1 (M+H) + and 408.2 (M+H) + .

[0362] 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 [ka] 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, then heated and stirred at 90° C. for 2 h. The reaction was diluted with methanol and purified by preparative LCMS (pH=2) to give the desired product. 22 H 16 LC-MS calculated for N9: 406.2 (M+H) + ;Measured value: 406.2 (M+H) + .

[0363] 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) [ka] 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 [ka] 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) was added diisopropyl azodicarboxylate (739 μl, 3.75 mmol) at 0° C. 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%). C 20 H 19 LC-MS calculated for ClFN6O2: 429.1 (M+H) + ;Measured value: 429.3 (M+H) + .

[0364] 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 [ka] Cesium carbonate (658 mg, 2.019 mmol) in 1,4-dioxane (10.0 mL) and water (1.0 mL) 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). The resulting mixture was sparged 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 under microwave irradiation at 120° C. for 1.5 h. 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 give the desired product (357 mg, 71%). 27 H 23 LC-MS calculated for FN7O2: 496.2 (M+H) + ; Measured value: 496.3 (M+H) + .

[0365] Step 3. 3-(4-amino-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)benzonitrile [ka] 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, 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 give the desired product (213 mg, 61%). C 18 H 13 LC-MS m / z calculated for FN7: 346.1 (M+H) + ;Measured value: 346.3 (M+H) + .

[0366] 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 [ka] 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 for BrFN7: 424.0 (M+H) + and 426.0 (M+H) + ;Measured value: 424.3 (M+H) + and 426.3 (M+H) + .

[0367] 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 [ka] 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, then heated and stirred at 90° C. for 2 h. The reaction was diluted with methanol and purified by preparative LCMS (pH=2) to give the desired product. 22 H 15 LC-MS calculated for FN9: 424.1 (M+H) + ;Measured value: 424.3 (M+H) + . 1 H NMR(500MHz,DMSO-d6)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).

[0368] 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) [ka] Cesium carbonate (46.1 mg, 0.141 mmol) in 1,4-dioxane (2 mL) and water (0.2 mL) 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). The resulting mixture was sparged with N2 for 2 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 by preparative HPLC afforded the desired product. 23 H 16 LC-MS calculated for FN8: 423.1 (M+H) + ;Measured value: 423.3 (M+H) + .

[0369] 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) [ka] Step 1. 3-(4-amino-7-bromo-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)-2-fluorobenzonitrile [ka] This compound was prepared following a procedure similar to that described for Example A13, steps 1-6, substituting (3-cyano-2-fluorophenyl)boronic acid for (3-cyanophenyl)boronic acid in step 4. 18 H 12LC-MS calculated for BrFN7: 424.0 (M+H) + and 426.0 (M+H) + ;Measured value: 424.3 (M+H) + and 426.3 (M+H) + .

[0370] 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; [ka] This compound was prepared according to a procedure similar to that of Example A15, using (1-methyl-1H-pyrazol-5-yl)boronic acid instead of pyridin-4-ylboronic acid, and using 3-(4-amino-7-bromo-2-((3-fluoropyridin-2-yl)methyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)-2-fluorobenzonitrile instead of 3-(4-amino-7-bromo-2-(pyridin-2-ylmethyl)-2H-[1,2,3]triazolo[4,5-c]pyridin-6-yl)-2-fluorobenzonitrile. 22 H 17 LC-MS calculated for FN9: 426.2 (M+H) + ;Measured value: 426.3 (M+H) + .

[0371] 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) [ka] Step 1: Ethyl 3-(pentylamino)-1H-pyrrole-2-carboxylate [ka] Ethyl 3-amino-1H-pyrrole-2-carboxylate (5 g, 32.4 mmol), pentanal (3.79 ml, 35.7 mmol), and sodium cyanoborohydride (2.038 g, 32.4 mmol) were mixed in methanol (64.9 ml) at room temperature overnight. The reaction mixture was concentrated under reduced pressure. The crude residue was purified by flash chromatography (0 to 100% EtOAc in hexanes) to give the desired product (4.4 g, 61%). 12 H 21 LCMS calculated for N2O2 (M+H): 225.2. Found: 225.1

[0372] Step 2: 3-(3-(ethoxycarbonyl)-1-pentylthioureido)-1H-pyrrole-2-carboxylate ethyl [ka] A vial 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 phases 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. 16 H 26 LCMS calculated for N3O4S (M+H): 356.2. Found: 356.1.

[0373] Step 3: 1-Pentyl-2-thioxo-2,3-dihydro-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one [ka] A microwave vial was charged with 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 degrees Celsius for 10 minutes. 1M HCl solution was added to bring the reaction mixture to neutral pH and the solid product was filtered and dried (3.1 g, 64%). 11 H 16 LCMS calculated for N3OS (M+H): 238.1. Found: 238.1.

[0374] Step 4: 2-Hydrazono-1-pentyl-2,3-dihydro-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one [ka] A vial was charged with 1-pentyl-2-thioxo-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 degrees Celsius overnight. The solid formed was filtered and washed with water to give the desired product (2.2 g, 70%). 11 H 18 LCMS calculated for NO (M+H): 236.1. Found: 236.1.

[0375] Step 5: 3-Methyl-9-pentyl-6,9-dihydro-5H-pyrrolo[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one [ka] A vial 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 degrees Celsius for 3 hours. The suspension was filtered, washed with hexane, and dried (4.0 g, 76%). 13 H 18 LCMS calculated for NO (M+H): 260.1. Found: 260.2.

[0376] 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 [ka] A vial 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 h. 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). C 19 H 22 LCMS calculated for N5O3S (M+H): 400.1. Found: 400.1.

[0377] 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 [ka] A vial 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), dry THF (30 mL) and the mixture was cooled to -78°C. A solution of lithium diisopropylamide (1M 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 dry THF (3 ml) was added dropwise to the reaction mixture and the reaction mixture was maintained at -78°C for an additional 1.5 hours. The reaction mixture was quenched with saturated NH4Cl solution (30 mL) and diluted with dichloromethane (30 mL). The layers were separated and the aqueous layer was extracted with DCM (3x30ml). The combined organic fractions were dried over MgSO4, filtered and concentrated. The crude residue was purified by automated flash chromatography (0-100% EtOAc in DCM) to give the desired product (0.84g, 70%). 19 H 21 LCMS calculated for BrN5O3S (M+H): 478.1. Found: 478.1.

[0378] Step 8: 3-chloro-5-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)pyridine [ka] A vial 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 degrees Celsius 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 organic extracts were dried over MgSO4, filtered, and concentrated. Purification by automated flash chromatography (0-100% EtOAc in DCM) afforded the product (0.548 g, 67%). C 15 H 20 LCMS calculated for BClN3O2 (M+H): 320.1, 322.1. Found: 320.1, 322.1

[0379] 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 A vial 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-triazole- 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 potassium phosphate tribasic (0.016 g, 0.074 mmol) were added. 1,4-Dioxane (0.35 ml) and water (0.07 ml) were added and the reaction mixture was sparged with nitrogen gas for 5 minutes and then stirred at 90° C. for 2 hours. The reaction mixture was then cooled to room temperature and sodium hydroxide (10 mg) 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 (5 mL). The final material was purified by preparative HPLC (pH 2, acetonitrile / water with TFA) to give the product as a TFA salt (2 mg, 21%). 22 H 24 LC-MS (M+H) calculated for ClNO + :451.2;actual value 453.2.

[0380] 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) [ka] This compound was prepared using a procedure similar to that described in Example A17, using 3-(bromomethyl)-5-methylpyridine instead of 3-(bromomethyl)-5-chloropyridine hydrobromide in step 8. 23 H 27LC-MS calculated for NO (M+H): 431.2. Found: 431.3.

[0381] 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) [ka] This compound was prepared using a procedure similar to that described in Example A17, using 6-(bromomethyl)thieno[3,2-b]pyridine instead of 3-(bromomethyl)-5-chloropyridine hydrobromide in step 8. 24 H 25 LC-MS calculated for N8OS (M+H): 473.2. Found: 473.3.

[0382] 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) [ka] 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 [ka] 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 reaction mixture was cooled and diluted with ethyl acetate, then washed with water, dried, and concentrated. The product was purified by column chromatography eluting with hexane / EtOAc (up to 60% EtOAc) to give the product. C 24 H 35 BN3O4(M+H) + LCMS calculated for m / z 440.3; found 440.3.

[0383] 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 [ka] TBAF (1.0M in THF) (2.0ml, 2.0mmol) 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 (0.360g, 0.753mmol) in THF (4.0ml), then the reaction was stirred at 50°C for 1 hour. The solvent was removed and the product was purified by column chromatography eluting with CHCl / MeOH (up to 10% MeOH). 13 H 17 LC-MS (M+H) calculated for BrNO + : m / z = 338.1; measured value 338.1.

[0384] 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 [ka] 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), tert-butyl 6-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)-3,4-dihydroisoquinol A mixture of benzene-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 replaced with N2 three times. The reaction mixture was then stirred at 105°C for 2 h, cooled to room temperature, diluted with ethyl acetate, washed with water, dried and concentrated. The product was purified by column eluting with CHCl / MeOH (up to 10% MeOH). 31 H 39 LC-MS calculated for N8O3 (M+H) + : m / z = 571.3; Measured value: 571.5.

[0385] 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 [ka] TFA (0.5 ml, 6.49 mmol) was added to 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 CHCl (0.5 ml) and the reaction was then stirred at room temperature for 30 minutes. The solvent was then removed to give the crude product as the TFA salt. 26 H 31 LC-MS calculated for NO (M+H) + : m / z = 471.3; measured value 471.2.

[0386] 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 Dimethylglycinoyl 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 CHCl (0.8 ml) and stirred at room temperature for 30 min. The solvent was removed and the mixture was diluted with acetonitrile / water and purified by preparative HPLC (pH 2, acetonitrile / water with TFA) to give the desired compound as its TFA salt. 30 H 38 LC-MS calculated for N9O2 (M+H) + : m / z = 556.3; measured value 556.3.

[0387] 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) [ka] This compound was prepared using a procedure similar to that described for Example A3, substituting 5-(1H-pyrazol-1-yl)-1H-tetrazole for 2-(1H-tetrazol-5-yl)pyridine. Compound 21A was purified by preparative LC-MS (pH 2, acetonitrile / water with TFA) to give the product as a TFA salt. 21 H 15 N 14 LC-MS (M+H) calculated for + :463.2;Actual value 463.2.

[0388] In addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Each reference cited in this application, including all patents, patent applications, and publications, is hereby incorporated by reference in its entirety. The present application also includes the following aspects. [Aspect 1] 1. A method of treating cancer in a subject, comprising: (i) an inhibitor of A2A / A2B; and (ii) PD-1 / PD-L1 inhibitors to said subject. [Aspect 2] The A2A / A2B inhibitor is a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, Cy 1 is phenyl substituted by 1 or 2 substituents independently selected from halo and CN; Cy 2 is a 5- to 6-membered heteroaryl or a 4- to 7-membered heterocycloalkyl, 2 Each of the 5- to 6-membered heteroaryl and 4- to 7-membered heterocycloalkyl groups is 1-3 Alkyl, C 1-3 Alkoxy, NH 2 , NH(C 1-3 Alkyl) and N(C 1-3 Alkyl) 2 each optionally substituted with 1, 2, or 3 groups independently selected from R 2 is 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 wherein 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- to 7-membered heterocycloalkyl)-C 1-3 Alkyl- is selected from 1, 2, or 3 independently selected R C optionally substituted with substituents; R a2 is one or two independently selected R C (5-7 membered heteroaryl)-C optionally substituted with 1-3 alkyl-; Each R C Halo, C 1-6 Alkyl, C 6 Aryl, 5- to 7-membered heteroaryl, (4- to 7-membered heterocycloalkyl)-C 1-3 Alkyl-, OR a4 , and N.R. c4 R d4 are independently selected from; Each R a4、R c4 , and R d4 H and C 1-6 The method of embodiment 1, wherein said alkyl is independently selected from alkyl. [Aspect 3] The A2A / A2B inhibitor 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; and 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 pharma- ceutically acceptable salt thereof 3. The method of embodiment 1 or 2, selected from: [Aspect 4] The A2A / A2B inhibitor is a compound of formula (II):

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Claims

(i) an inhibitor of A2A / A2B; and (ii) PD-1 / PD-L1 inhibitor A pharmaceutical combination for treating cancer in a subject, comprising: The A2A / A2B inhibitor has the formula (III): 【Chemistry 1】 [In the formula, Cy 1 is phenyl substituted by 1 or 2 substituents independently selected from halo and CN; R 2 is selected from 5-6 membered heteroaryl and 4-7 membered heterocycloalkyl, wherein said 5-6 membered heteroaryl and 4-7 membered heterocycloalkyl of R 2 are each optionally substituted with 1, 2, or 3 independently selected R 2A substituents; each R 2A is independently selected from D, halo, C 1-6 alkyl, and C 1-6 haloalkyl; R 4 is selected from phenyl-C 1-3 alkyl-, C 3-7 cycloalkyl-C 1-3 alkyl-, (5- to 6-membered heteroaryl)-C 1-3 alkyl-, and (4- to 7-membered heterocycloalkyl)-C 1-3 alkyl, wherein the phenyl-C 1-3 alkyl-, C 3-7 cycloalkyl-C 1-3 alkyl-, (5- to 6-membered heteroaryl)-C 1-3 alkyl-, and (4- to 7-membered heterocycloalkyl)-C 1-3 alkyl- of said R 4 are each optionally substituted with 1, 2, or 3 independently selected R 4A substituents; each R 4A is independently selected from halo, C 1-6 alkyl, C 1-6 haloalkyl, CN, OR a41 , and NR c41 R d41 ; and Each R a41 , R c41 , and R d41 is independently selected from H and C 1-6 alkyl. or a pharma- ceutically acceptable salt thereof; and, The PD-1 / PD-L1 inhibitor, (a) atezolizumab; or (b) an antibody X, said antibody X being an antibody or an antigen-binding fragment thereof, the antibody comprises a variable heavy (VH) domain comprising a VH complementarity determining region (CDR) 1, a VH CDR2, and a 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, The antibody further comprises a variable light (VL) domain comprising a VL CDR1, a VL CDR2, and a 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); The antibody X, The combination drug.

2. 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; 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 pharma- ceutically acceptable salt thereof; and 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 pharma- ceutically acceptable salt thereof Selected from: The pharmaceutical combination according to claim 1.

3. 2. The pharmaceutical combination of claim 1, 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 pharma- ceutically acceptable salt thereof.

4. The combination pharmaceutical of any one of claims 1 to 3, wherein the PD-1 / PD-L1 inhibitor is atezolizumab.

5. The pharmaceutical combination according to any one of claims 1 to 3, wherein the PD-1 / PD-L1 inhibitor is antibody X.

6. The pharmaceutical combination according to claim 5, wherein antibody X is a humanized antibody.

7. The method of claim 1, 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 pharma- ceutically acceptable salt thereof; and The PD-1 / PD-L1 inhibitor is antibody X. The pharmaceutical combination according to claim 1.

8. The pharmaceutical combination according to any one of claims 1 to 7, wherein the A2A / A2B inhibitor is administered to the subject in a dose of about 0.1 mg to about 1000 mg on a free base basis.

9. The pharmaceutical combination according to any one of claims 1 to 8, wherein the A2A / A2B inhibitor is administered to the subject once daily, every other day, or once weekly.

10. The pharmaceutical combination according to any one of claims 1 to 9, wherein the A2A / A2B inhibitor and the PD-1 / PD-L1 inhibitor are administered simultaneously.

11. The pharmaceutical combination according to any one of claims 1 to 9, wherein the A2A / A2B inhibitor and the PD-1 / PD-L1 inhibitor are administered sequentially.

12. The combination pharmaceutical according to any one of claims 1 to 11, wherein the cancer is selected from bladder cancer, breast cancer, colon cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, and prostate cancer.

13. The combination pharmaceutical of claim 12, wherein the cancer is head and neck cancer.

14. The combination pharmaceutical of claim 13, wherein the head and neck cancer is head and neck squamous cell carcinoma.

15. The combination pharmaceutical of claim 12, wherein the cancer is lung cancer.

16. The combination pharmaceutical of claim 15, wherein the lung cancer is non-small cell lung cancer (NSCLC).

17. The combination pharmaceutical of claim 12, wherein the cancer is ovarian cancer.

18. The combination pharmaceutical of claim 12, wherein the cancer is prostate cancer.

19. The combination pharmaceutical of claim 18, wherein the prostate cancer is metastatic castration-resistant prostate cancer.

20. The combination pharmaceutical of claim 12, wherein the cancer is breast cancer.

21. The combination pharmaceutical of claim 12, wherein the cancer is bladder cancer.

22. The combination pharmaceutical of claim 12, wherein the cancer is colorectal cancer.

23. The combination pharmaceutical of claim 12, wherein the cancer is pancreatic cancer.

24. 1. A combination pharmaceutical for use in treating a cancer selected from bladder cancer, breast cancer, colon cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, and prostate cancer in a subject, the combination pharmaceutical comprising: (i) an inhibitor of A2A / A2B 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 pharma- ceutically acceptable salt thereof; and (ii) a PD-1 / PD-L1 inhibitor that is antibody X where the A2A / A2B inhibitor is administered to the subject at a dose of about 0.1 mg to about 500 mg on a free base basis, the A2A / A2B inhibitor is administered once daily or once every other day; and said antibody X is administered to said subject at a dose of about 100 mg to about 1000 mg Q4W, The antibody X is an antibody or an antigen-binding fragment thereof, the antibody comprises a variable heavy (VH) domain comprising a VH complementarity determining region (CDR) 1, a VH CDR2, and a 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, The antibody further comprises a variable light (VL) domain comprising a VL CDR1, a VL CDR2, and a 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); The combination drug.

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