Combination of DGK inhibitors and checkpoint antagonists

Abstract

A combination of a DGK inhibitor and a checkpoint antagonist is provided. [Solution] We provide inhibitors of diacylglycerol kinase (DGK) and methods for treating diseases (e.g., cancer and infectious diseases) that may benefit from stimulating the immune system, which methods are characterized by administering a DGK inhibitor in combination with an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4.

Description

[Technical Field]

[0001] (Related Applications) This application claims priority to U.S. Provisional Application No. 62 / 950,570, filed December 19, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Human cancers contain numerous genetic and epigenetic alterations, producing neoantigens that can potentially be recognized by the immune system (Sjoblom et al. (2006) Science 314:268-74). The adaptive immune system, comprised of T and B lymphocytes, has the potential to exert potent anticancer effects, possessing broad capabilities and high specificity to respond to diverse tumor antigens. Furthermore, this immune system possesses considerable plasticity and memory components. Harnessing all these properties of the adaptive immune system makes immunotherapy unique among all cancer treatment approaches. However, although endogenous immune responses to cancer have been observed in preclinical models and patients, these responses are ineffective, and established cancers are considered "self" and resistant to the immune system. This tolerance allows tumors to actively subvert antitumor immunity using multiple distinct mechanisms. These mechanisms include dysfunctional T cell signaling (Mizoguchi et al., (1992) Science 258:1795-98), suppressive regulatory cells (Facciabene et al., (2012) Cancer Res. 72:2162-71), and the utilization of endogenous "immune checkpoints," which act to reduce the strength of the adaptive immune response and protect normal tissues from collateral tumor damage, allowing them to escape immune destruction (Topalian et al., (2012) Curr. Opin. Immunol. 24:1-6; Mellman et al. (2011) Nature 480:480-489).

[0003] Diacylglycerol kinase (DGK) is a lipid kinase that converts diacylglycerol to phosphatidic acid, thereby mediating the termination of T cell function via the TCR signaling pathway. Therefore, DGK functions as an intracellular checkpoint, and inhibition of DGK is expected to enhance T cell signaling and activation. Supporting evidence is that knockout mice models of either DGKα or DGKζ exhibit a hyperreactive T cell phenotype and improved antitumor immune activity (Riese MJ et al., Journal of Biological Chemistry, (2011) 7: 5254-5265; Zha Y et al., Nature Immunology, (2006) 12:1343; Olenchock BA et al., (2006) 11: 1174-81). Furthermore, tumor-infiltrating lymphocytes isolated from human renal cell carcinoma patients have been observed to overexpress DGKα, which results in the inhibition of T cell function (Prinz, PU et al., J Immunology (2012) 12:5990-6000). Therefore, DGKα and DGKζ are considered targets for cancer immunotherapy (Riese MJ et al., Front Cell Dev Biol. (2016) 4: 108; Chen, SS et al., Front Cell Dev Biol. (2016) 4: 130; Avila-Flores, A. et al., Immunology and Cell Biology (2017) 95: 549-563; Noessner, E., Front Cell Dev Biol. (2017) 5: 16; Krishna, S., et al., Front Immunology (2013) 4:178; Jing, W. et al., Cancer Research (2017) 77: 5676-5686). Summary of the Invention

[0004] The present application provides a method for treating a disease or disorder, comprising administering to a subject an inhibitor of DGKα, DGKζ, or both DGKα and DGKζ (e.g., a compound of Formula (I) or (II), e.g., a compound selected from Compounds 1-34, or a pharmaceutically acceptable salt thereof) in combination with an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4. Examples of diseases or disorders that can be benefited by stimulating the immune system include cancer and infectious diseases. The present application also provides the use of an inhibitor of DGKα, DGKζ, or both DGKα and DGKζ (e.g., a compound of Formula (I) or (II), e.g., a compound selected from Compounds 1-34, or a pharmaceutically acceptable salt thereof) for the manufacture of a medicament for treating a disease or disorder that can be benefited by stimulating the immune system (e.g., cancer and infectious diseases). The inhibitor is administered in combination with an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4. The present application relates to the use of an inhibitor of DGKα, DGKζ, or both DGKα and DGKζ (e.g., a compound of formula (I) or (II), e.g., a compound selected from compounds 1-34, or a pharmaceutically acceptable salt thereof) for the manufacture of a medicament for treating a disease or disorder that can be benefited by stimulating the immune system (e.g., cancer and infectious diseases), wherein the inhibitor is administered in combination with an antagonist of PD1 / PD-L1 binding and an antagonist of CTLA4.

[0005] The present application also provides a use of an antagonist of PD1 / PD-L1 binding for the manufacture of a medicament for treating a disease or disorder (e.g., cancer and infectious diseases) that may benefit from stimulating the immune system, wherein the antagonist is administered in combination with an inhibitor of DGKα, DGKζ, or both DGKα and DGKζ (e.g., a compound of Formula (I) or (II), e.g., a compound selected from Compounds 1-34, or a pharmaceutically acceptable salt thereof) and / or a CTLA4 antagonist. The present application also provides a use of an antagonist of PD1 / PD-L1 binding for the manufacture of a medicament for treating a disease or disorder (e.g., cancer and infectious diseases) that may benefit from stimulating the immune system, wherein the antagonist is administered in combination with an inhibitor of DGKα, DGKζ, or both DGKα and DGKζ (e.g., a compound of Formula (I) or (II), e.g., a compound selected from Compounds 1-34, or a pharmaceutically acceptable salt thereof) and a CTLA4 antagonist.

[0006] The present application also provides a use of a CTLA4 antagonist for the manufacture of a medicament for treating a disease or disorder (e.g., cancer and infectious diseases) that may benefit from stimulating the immune system, wherein the antagonist is administered in combination with an inhibitor of DGKα, DGKζ, or both DGKα and DGKζ (e.g., a compound of Formula (I) or (II), e.g., a compound selected from Compounds 1-34, or a pharmaceutically acceptable salt thereof) and / or an antagonist of PD1 / PD-L1 binding. The present application also provides a use of a CTLA4 antagonist for the manufacture of a medicament for treating a disease or disorder (e.g., cancer and infectious diseases) that may benefit from stimulating the immune system, wherein the antagonist is administered in combination with an inhibitor of DGKα, DGKζ, or both DGKα and DGKζ (e.g., a compound of Formula (I) or (II), e.g., a compound selected from Compounds 1-34, or a pharmaceutically acceptable salt thereof) and an antagonist of PD1 / PD-L1 binding.

[0007] Exemplary compounds of formula I, and pharmaceutically acceptable salts thereof, described herein are described in PCT / US2019 / 039131, filed June 26, 2019, and PCT / US2019 / 039135, filed June 26, 2019, the contents of both of which are specifically incorporated herein by reference. For example, exemplary compounds of formula II, and pharmaceutically acceptable salts thereof, described herein are described in PCT / US2020 / 048070, filed August 27, 2020, the contents of which are specifically incorporated herein by reference.

[0008] These and other features of the novel method of treatment are described in greater detail below. [Brief explanation of the drawings]

[0009] [Figure 1A-1B] Figures 1A and 1B show that T cells incubated with increasing concentrations of DGKi and nivolumab (A) or ipilimumab (B) in an MLR assay secreted increased amounts of IFN-γ compared to the same assay in the absence of nivolumab or ipilimumab. [Figures 2A-H] Figures 2A-H show that tumor growth was suppressed by the combination of anti-PD-1 and anti-CTLA4 antibodies with DGKi compared to mice treated with anti-PD-1 and anti-CTLA4 antibodies alone. Figures 2A-H show the tumor size over time after transplantation of murine B16 melanoma cells into mice treated with vehicle alone (Figure 2A), anti-PD-1 antibody alone (Figure 2B), anti-PD-1 and anti-CTLA4 antibodies (Figure 2C), DGKi and anti-PD-1 antibody (Figure 2D), DGKi and anti-CTLA4 antibody (Figure 2E), DGKi alone (Figure 2F), and DGKi, anti-PD-1, and anti-CTLA4 antibodies (Figure 2G). Figure 2H shows the average tumor size after implantation of B16 cells in mice treated with (i) anti-PD-1 antibody and anti-CTLA4 antibody, (ii) DGKi and anti-PD1 antibody, (iii) DGKi and CTLA4 antibody, and (iv) DGKi, anti-PD1 antibody, and anti-CTLA4 antibody. [Figure 3A-I]Figures 3A-I show that combined treatment with a DGK inhibitor and anti-PD-1 and / or anti-CTLA4 antibodies improved the complete remission rate (Figure 3A), and the degree of response correlated with an increase in AH1+ CD8 T cells in the CT26 mouse model (Figure 3B). [Figure 4A-F] Figures 4A–F show that DGK inhibition lowers the antigen threshold required for TCR activation. Figures 4A–F show the amount of IL-2 secreted by OT1 CD8 T cells incubated with increasing amounts of antigen and one of the peptides OVA (A), A2 (B), Q4 (C), T4 (D), and Q4H7 (E), indicating that DGK inhibition reduces the concentration of tumor antigen required for T cell activation. Figure 4F shows the amount of IL-2 secreted in the presence of each peptide shown in Figures 4A–E as well as recombinant peptide (1000 ng / mL), indicating that DGK inhibition promotes T cell responses even to tumor antigens with low affinity. [Figure 5A-5B] Figures 5A and 5B show that inhibition of DGK activates human CTL effector function and enhances tumor cell killing. Figure 5A shows the amount of IFN-γ secreted by T cells incubated with peptides in the presence of increasing concentrations of DGKi. Figure 5B shows that after 3 days of tumor cell incubation, increasing amounts of the cognate peptide enhanced tumor cell killing. [Figures 6A-6B] Figures 6A and 6B show that DGKi can restore the reduced T cell effector function caused by reduced B2M levels. Figure 6A shows the amount of β2 microglobulin in CRISPR-KO of B2M in HCT116 cells. Figure 6B shows that DGKi increases the amount of IFN-γ. [Figure 7] Figure 7 shows the relationship between tumor volume and days after tumor cell implantation in the CT26 animal model, showing that mice treated with DGKi compound 16 and anti-PD-1 antibody in the presence or absence of a CD8-depleting antibody showed less tumor shrinkage in mice treated with the CD8-depleting antibody. [Figure 8]Figure 8 shows the relationship between tumor volume and days after tumor cell implantation in the CT26 animal model, showing that mice treated with DGKi compound 16 and anti-PD-1 antibody in the presence or absence of a CD4-depleting antibody showed greater tumor reduction in mice treated with the CD4-depleting antibody. [Figure 9] Figure 9 shows the relationship between tumor volume and days after tumor cell implantation in the CT26 animal model, showing that mice treated with DGKi compound 16 and anti-PD-1 antibody in the presence or absence of an NK cell-depleting antibody showed less tumor shrinkage in mice treated with the NK cell-depleting antibody. [Figure 10] Figure 10 shows that the combination of DGKi with either anti-PD-1 or anti-CTLA4 can induce complete tumor regression (CR) in the MC38 tumor model. Tumor volumes are shown for each animal group after treatment with vehicle alone (Figure 10A), DGKi (Figure 10B), anti-PD-1 (Figure 10C), anti-CTLA4 (Figure 10D), DGKi and anti-PD-1 (Figure 10E), or DGKi and anti-CTLA4 (Figure 10F). Treatment with DGKi, anti-PD-1, and anti-CTLA4 alone can slow tumor growth. The combination of DGKi and anti-PD-1 resulted in complete tumor regression in 100% of test animals, while the combination of DGKi and anti-CTLA4 led to complete tumor regression in 70% of test mice. [Figure 11]Figure 11 shows that adding DGKi to anti-PD-1 treatment can induce complete tumor regression (CR) in both the MC38 and CT26 animal models, and that cured animals in these groups build sufficient immune memory to reject re-implanted tumors. Tumor volumes are shown for each animal group after treatment with vehicle alone (Figures 11A and 11E), anti-PD-1 (Figures 11B and 11F), or anti-PD-1 and DGKi (Figures 11C and 11G). In the MC38 and CT26 models, respectively, DGKi showed complete tumor regression in 100% and 60% of cases, due to the potent effect of its combination with anti-PD-1. To assess immune memory in cured animals, mice were re-implanted with 10 times the number of tumor cells used in the initial implantation, and tumor volumes were measured and correlated with the number of days since implantation. All re-implanted animals in the MC38 (Figure 11D) and CT26 (Figure 11H) cohorts spontaneously rejected tumors, confirming that the combined DGKi and anti-PD-1 treatment confers long-lasting immune memory. [Figure 12] Figure 12 shows that treatment with anti-PD-1, anti-CTLA4, and DGKi triplet species can reduce tumor growth in the checkpoint inhibitor-refractory B16F10 tumor model. Tumor volumes for each animal group are shown after treatment with vehicle alone (Figure 12A), anti-PD-1 and anti-CTLA4 (Figure 12B), anti-PD-1 and DGKi (Figure 12C), anti-CTLA4 and DGKi (Figure 12D), or anti-PD-1, anti-CTLA4, and DGKi (Figure 12E). The mean tumor volume for each group is shown in Figure 12F. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present application provides a method for treating a proliferative disease (e.g., cancer) or a viral infection, or any disease, disorder, or condition that can be benefited by stimulating the immune system more generally, as well as any disease, disorder, or condition that can be prevented, ameliorated, or cured by inhibiting DGKα and / or DGKζ enzymatic activity, comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of DGKα and / or DGKζ or a pharmaceutically acceptable salt thereof and (i) an antagonist of PD1 / PD-L1 binding (e.g., an antagonist of human PD1 or human PD-L1) and / or (ii) an antagonist of human CTLA4.

[0011] (definition) The features and usefulness of the methods of treatment will be more readily understood by those skilled in the art upon reading the following detailed description. It is understood that, for clarity, certain features of the methods of treatment that are described before or after in the context of separate embodiments may be combined to form a single embodiment. Conversely, various features of the methods of treatment that are described for brevity in the context of a single embodiment may be combined to form subcombinations thereof. Any embodiments identified herein as exemplary or preferred are intended to be illustrative, not limiting.

[0012] Unless otherwise stated herein, references made in the singular may also include the plural, for example, "a" and "an" may refer to either "one" or "one or more."

[0013] As used herein, the phrase "compound and / or its pharmaceutically acceptable salt" refers to at least one compound, at least one salt of a compound, or a combination thereof. For example, the compound of formula (I) and / or its pharmaceutically acceptable salt includes the compound of formula (I); two compounds of formula (I); a pharmaceutically acceptable salt of the compound of formula (I); a compound of formula (I) and one or more pharmaceutically acceptable salts of the compound of formula (I); and two or more pharmaceutically acceptable salts of the compound of formula (I).

[0014] Unless otherwise specified, any atom with unsatisfied valences is assumed to include enough hydrogen atoms to satisfy the valences.

[0015] The definitions set forth herein supersede any definitions set forth in any patents, patent applications, and / or published patent applications incorporated herein by reference.

[0016] Listed below are definitions of various terms used in this specification. These definitions apply to the terms as they are used throughout the specification, either individually or as part of a larger group (unless limited in specific instances).

[0017] Throughout the specification, groups and substituents may be chosen by one skilled in the art to provide stable moieties and compounds.

[0018] According to the practice used in the art, [ka] is used in structural formulas herein to represent the bond that is the point of attachment of a moiety or substituent to the core or backbone structure.

[0019] As used herein, the terms "halo" and "halogen" refer to F, Cl, Br, and I.

[0020] The term "cyano" refers to the group --CN.

[0021] The term "amino" refers to the group --NH.sub.2.

[0022] The term "oxo" refers to the group =O.

[0023] The term "alkyl" as used herein refers to both branched and straight-chain saturated aliphatic hydrocarbon groups, for example, having 1 to 12 carbon atoms, 1 to 6 carbon atoms, and 1 to 4 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and i-propyl), butyl (e.g., n-butyl, i-butyl, sec-butyl, and t-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylpentyl, 2-ethylbutyl, 3-methylpentyl, and 4-methylpentyl. When a number appears as a subscript after the symbol "C," the subscript more specifically qualifies the number of carbon atoms that a particular group may contain. For example, "C 1-4 "Alkyl" means straight and branched chain alkyl groups having from 1 to 4 carbon atoms.

[0024] As used herein, the term "fluoroalkyl" is intended to encompass both branched and straight-chain saturated aliphatic hydrocarbon groups substituted with one or more fluorine atoms. For example, "C 1-4 The term "fluoroalkyl" is meant to include C, C, C, and C alkyl groups substituted with one or more fluorine atoms. Representative examples of fluoroalkyl groups include, but are not limited to, -CF and -CHCF.

[0025] The term "cyanoalkyl" includes both branched and straight-chain saturated alkyl groups substituted with one or more cyano groups. For example, "cyanoalkyl" includes -CHCN, -CHCHCN, and -C 1-4 Examples include cyanoalkyl.

[0026] The term "aminoalkyl" includes both branched and straight-chain saturated alkyl groups substituted with one or more amino groups. For example, "aminoalkyl" includes -CH2NH2, -CH2CH2NH2, and C 1-4 Aminoalkyl is an example.

[0027] The term "hydroxyalkyl" includes both branched and straight-chain saturated alkyl groups substituted with one or more hydroxyl groups. For example, "hydroxyalkyl" includes -CHOH, -CHCHOH, and C 1-4 Hydroxyalkyl is an example.

[0028] The term "alkenyl" refers to a straight or branched chain hydrocarbon radical containing 2 to 12 carbon atoms and at least one carbon-carbon double bond. Examples of such groups include ethenyl or allyl. For example, "C 2-6 "Alkenyl" refers to a straight or branched chain alkenyl group having 2 to 6 carbon atoms.

[0029] The term "alkynyl" refers to a straight or branched chain hydrocarbon radical containing 2 to 12 carbon atoms and at least one carbon-carbon triple bond. Examples of such groups include ethynyl. For example, "C 2-6 "Alkynyl" refers to a straight or branched chain alkynyl group having 2 to 6 carbon atoms.

[0030] As used herein, the term "cycloalkyl" refers to a group derived from a non-aromatic monocyclic hydrocarbon molecule or a non-aromatic polycyclic hydrocarbon molecule by removing one hydrogen atom from a saturated ring carbon atom. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. When a number appears as a subscript after the symbol "C," the subscript more specifically qualifies the number of carbon atoms that a particular cycloalkyl group may contain. For example, "C3-C6 cycloalkyl" refers to a cycloalkyl group having from 3 to 6 carbon atoms.

[0031] The term "alkoxy," as used herein, refers to an alkyl group attached to the parent molecular moiety through an oxygen atom, for example, a methoxy group (-OCH). 1-3 "Alkoxy" means an alkoxy group having 1 to 3 carbon atoms.

[0032] The terms "fluoroalkoxy" and "-O(fluoroalkyl)" refer to a fluoroalkyl group as defined above attached through an oxygen linkage (-O-). For example, "C 1-4 "Fluoroalkoxy" is intended to include C1, C2, C3, and C4 fluoroalkoxy groups.

[0033] The term "alcalenyl" refers to a saturated carbon chain having two points of attachment to a core or backbone structure. An alcalenyl group has the structure -(CH) n -, where n is an integer equal to or greater than 1. Examples of alcalenyl bonds include -CH2CH2-, -CH2CH2CH2-, and -(CH2) 2-4 - are some examples.

[0034] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, substances, compositions, and / or dosage forms that are, within the scope of ordinary medical judgment, suitable for contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, and that offer a reasonable benefit / risk ratio.

[0035] For example, compounds of Formula (I) can form pharmaceutically acceptable salts, which may be used in the methods described herein. Unless otherwise specified, reference to a compound is understood to include reference to one or more pharmaceutically acceptable salts thereof. The term "salt" refers to pharmaceutically acceptable acid and / or base salts formed with inorganic and / or organic acids and bases. Furthermore, the term "salt" can include zwitterions (internal salts), for example, when a compound of Formula (I) contains both a basic moiety (e.g., an amine or pyridine or imidazole ring) and an acidic moiety (e.g., a carboxylic acid). Pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salts are preferred, for example, acceptable metal salts and amine salts in which the cation does not significantly contribute to the toxicity or biological activity of the salt. However, other salts may also be useful, for example, in isolation or purification steps that may be used in manufacturing processes, and are therefore contemplated herein. For example, salts of compounds of formula (I) may be formed by reacting a compound of formula (I) with a certain amount of acid or base (e.g., 1 equivalent) and precipitating the salt in a solvent, for example, or by subsequent lyophilization of the aqueous solution.

[0036] Examples of acid addition salts include acetates (e.g., acetates prepared from acetic acid or trihaloacetic acids (e.g., trifluoroacetic acid)), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (prepared from hydrochloric acid), hydrobromides (prepared from hydrogen bromide), and the like. salts such as those described herein, tartrates, thiocyanates, toluenesulfonates (e.g., tosylates), undecanoates, and the like.

[0037] Examples of base salts include ammonium salts, alkali metal salts (e.g., sodium, lithium, and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), barium, zinc, and aluminum salts, organic base salts such as organic amines (e.g., trialkylamines (e.g., triethylamine), procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, N,N'-dibenzylethylene-diamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, dicyclohexylamine), or similar pharmaceutically acceptable amines, and amino acid salts (e.g., arginine, lysine), and the like. Basic nitrogen-containing groups may be quaternized with reagents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), long-chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and other groups. Preferred salts include monohydrochlorides, hydrogen sulfates, methanesulfonates, phosphates, or nitrates.

[0038] For example, the compound of formula (I) may be provided as an amorphous solid or a crystalline solid. For example, the compound of formula (I) may be provided as a solid by lyophilization.

[0039] Additionally, solvates (e.g., hydrates) of, for example, compounds of Formula (I) should also be considered for use in the methods described herein. The term "solvate" refers to a physical association of, for example, a compound of Formula (I) with one or more organic or inorganic solvent molecules. This physical association includes hydrogen bonding. In some cases, it is possible to isolate the solvate, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" includes both solution-phase and isolable solvates. Examples of solvates include hydrates, ethanolates, methanolates, isopropanolates, acetonitrile solvates, and ethyl acetate solvates. Methods of solvation are known in the art.

[0040] Various forms of prodrugs are well known in the art: a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., Ch 31, (Academic Press, 1996); b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds. Ch 5, pgs 113 - 191 (Harwood Academic Publishers, 1991); and d) Hydrolysis in Drug and Prodrug Metabolism, Bernard Testa and Joachim M. Mayer, (Wiley-VCH, 2003) is described in.

[0041] Additionally, for example, a compound of formula (I) may be prepared, isolated, and purified to obtain a composition containing 99% or greater of the compound of formula (I) ("substantially pure"), which is then used or formulated as described herein. Such "substantially pure" compounds of formula (I) are also contemplated herein.

[0042] By "stable compound" and "stable structure" is intended a compound that is sufficiently robust that it will not decompose upon isolation to a useful degree of purity from a reaction mixture, nor upon formulation into an efficacious therapeutic agent. As used herein, compound is intended to embody a stable compound.

[0043] The compounds described herein are intended to include all isotopes of atoms contained in the compounds of the present invention. Isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium (D) and tritium (T). Isotopes of carbon include 13 C and 14 C. Isotopically labeled compounds can generally be prepared by conventional techniques known to those skilled in the art, or by methods analogous to those described herein, substituting the appropriate isotopically labeled reagent for the unlabeled reagent otherwise used.

[0044] As used herein, "treatment" covers any medication or method of treating a disease in a human, including inhibiting the progression of the disease or one or more disease symptoms, reducing the progression of the disease or one or more disease symptoms, preventing its progression, partially or totally alleviating the disease or one or more disease symptoms, or preventing the recurrence of one or more symptoms of the disease.

[0045] The terms "subject" and "patient" are used interchangeably herein and refer to a human, unless otherwise specified.

[0046] "DGKα and / or DGKζ inhibitors" refer to "inhibitors of DGKα and / or DGKζ enzyme activity," and are both inhibitors of human DGKα and / or human DGKζ. For example, DGKα having the amino acid sequence shown in SEQ ID NO: 2 or DGKα having the amino acid sequence shown in SEQ ID NO: 2 without unnatural amino acids (e.g., His tag or specific N-terminal amino acids), and DGKζ having the amino acid sequence shown in SEQ ID NO: 4 or DGKζ having the amino acid sequence shown in SEQ ID NO: 4 without unnatural amino acids (e.g., His tag or specific N-terminal amino acids).

[0047] As used herein, target proteins such as DGK, PD-1, PD-L1, and CTLA4 refer to human target proteins unless otherwise specified. For example, "mouse DGK" refers to mouse DGK, as specifically indicated.

[0048] "PD1" is used synonymously with "PD-1."

[0049] "CTLA4" is used synonymously with "CTLA-4."

[0050] The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug effective in treating a disease or disorder in a subject, such as an amount that partially or completely alleviates one or more symptoms. In some embodiments, an effective amount refers to an amount, at the dosage and for the period necessary, to achieve the desired therapeutic or prophylactic result.

[0051] The term "cancer" as used herein refers to a type of cell that exhibits abnormal proliferation and growth. Cancer can be benign (also called a benign tumor), precancerous, or malignant. Cancer cells can be solid cancer cells or leukemic cancer cells. Examples of cancers to which the therapeutic methods herein are applicable include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of cancer include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous non-small cell lung cancer), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain tumor, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, stomach cancer, melanoma, and various types of head and neck cancer (including head and neck squamous cell carcinoma).

[0052] As used herein, the term "tumor growth" refers to the proliferation or growth of cells or cells comprising a cancer, increasing in size or extent to the corresponding cancer.

[0053] Administration "in combination with" one or more therapeutic agents includes simultaneous and sequential administration in any order.

[0054] (Treatment method) The present application provides a method for treating a proliferative disease (e.g., cancer) or a viral infection, or any disease, disorder, or condition that can be benefited by stimulating the immune system more generally, as well as any disease, disorder, or condition that can be prevented, ameliorated, or cured by inhibiting DGKα and / or DGKζ enzymatic activity, comprising administering to a subject in need thereof therapeutically effective amounts of (i) an inhibitor of DGKα and / or DGKζ and (ii) an antagonist of PD1 / PD-L1 binding (e.g., an antagonist of human PD1 or human PD-L1) and / or an antagonist of human CTLA4. The present application provides a method for treating a proliferative disease (e.g., cancer) or a viral infection, or a disease, disorder, or condition that can be benefited by stimulating the immune system more generally, as well as any disease, disorder, or condition that can be prevented, ameliorated, or cured by inhibiting DGKα and / or DGKζ enzymatic activity, comprising administering to a subject in need thereof therapeutically effective amounts of (i) an inhibitor of DGKα and / or DGKζ and (ii) an antagonist of PD1 / PD-L1 binding (e.g., an antagonist of human PD1 or human PD-L1).The present application provides a method for treating a proliferative disease (e.g., cancer) or a viral infection, or a disease, disorder, or condition that can be benefited by stimulating the immune system more generally, as well as any disease, disorder, or condition that can be prevented, ameliorated, or cured by inhibiting DGKα and / or DGKζ enzymatic activity, comprising administering to a subject in need thereof therapeutically effective amounts of (i) an inhibitor of DGKα and / or DGKζ and an antagonist of human CTLA4. In some embodiments, treatment of a proliferative disease (e.g., cancer) or a viral infection, or more generally a disease, disorder, or condition that benefits from stimulating the immune system, and any disease, disorder, or condition that can be prevented, ameliorated, or cured by inhibiting DGKα and / or DGKζ enzymatic activity, comprises administering to a subject in need thereof therapeutically effective amounts of (i) an inhibitor of DGKα and / or DGKζ and (ii) an antagonist of PD1 / PD-L1 binding (e.g., an antagonist of human PD1 or human PD-L1) and an antagonist of human CTLA4.

[0055] (i) an inhibitor of DGKα and / or DGKζ and (ii) an antagonist of PD1 / PD-L1 binding (e.g., an antagonist of human PD1 or human PD-L1) and / or an antagonist of human CTLA4 can be administered simultaneously or sequentially. For example, in some embodiments, a method of treating cancer or a disease that can be treated by promoting an immune response includes first administering to a subject in need thereof an inhibitor of DGKα and / or DGKζ, and then later (e.g., 6 hours, 12 hours, 24 hours, 2 days, 3 days, or more), an antagonist of PD1 / PD-L1 binding (e.g., an antagonist of human PD1 or human PD-L1) and / or an antagonist of human CTLA4. For example, in a method of treating cancer, cancer or a disease that can be treated by promoting an immune response may be treated, the method including first administering to a subject in need thereof an antagonist of PD1 / PD-L1 binding (e.g., an antagonist of human PD1 or human PD-L1) and / or an antagonist of human CTLA4, and then later (e.g., 6 hours, 12 hours, 24 hours, 2 days, 3 days, or more), administering an inhibitor of DGKα and / or DGKζ. For example, in a method of treating cancer, the inhibitor of DGKα and / or DGKζ may be administered first, and then later (e.g., 6 hours, 12 hours, 24 hours, 2 days, 3 days, or more), administering simultaneously an antagonist of PD1 / PD-L1 binding (e.g., an antagonist of human PD1 or human PD-L1) and an antagonist of human CTLA4. For example, in a method of treating cancer, an inhibitor of DGKα and / or DGKζ may be administered first, and then later (e.g., 6 hours, 12 hours, 24 hours, 2 days, 3 days or more) an antagonist of PD1 / PD-L1 binding (e.g., an antagonist of human PD1 or human PD-L1) and an antagonist of human CTLA4 may be administered simultaneously.For example, in a method of treating cancer, an antagonist of PD1 / PD-L1 binding (e.g., an antagonist of human PD1 or human PD-L1) and an antagonist of human CTLA4 may be administered simultaneously first, followed by administration of an inhibitor of DGKα and / or DGKζ at a later time (e.g., 6 hours, 12 hours, 24 hours, 2 days, 3 days or more).

[0056] The methods described herein may be used to treat cancer, including, for example, advanced cancer, metastatic cancer, solid tumor, advanced solid tumor, hematological tumor, cancer that is refractory to a checkpoint inhibitor (or checkpoint antagonist), or that has progressed after treatment with a checkpoint inhibitor.

[0057] Examples of cancers to be treated include, but are not limited to, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate cancer), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer (also malignant tumors), gastric cancer, germ cell tumors, childhood sarcomas, sinonasal natural killer tumors, melanoma (e.g., metastatic melanoma, e.g., cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary central nervous system lymphoma, tumor angiogenesis, axial tumors, brain tumors, brainstem gliomas, ptosis Environmentally-driven cancers such as somatic adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, asbestos-related, virus-associated or virus-derived cancers (e.g., human papillomavirus (HPV-associated or HPV-derived tumors)), and hematological malignancies (e.g., all leukemias, lymphomas and myelomas, e.g., acute, chronic, and metastatic) arising from either of the two major blood cell lineages (i.e., myeloid cells (which give rise to granulocytes, erythrocytes, platelets, macrophages and mast cells) or lymphoid cells (which give rise to B, T, NK cells and plasma)). myeloid, lymphoid, and / or myeloid leukemias, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; mature cells), promyelocytic leukemia (M3 or M3 subtype [M3V]), myelomonocytic leukemia (M4 or M4 subtype with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroma;Lymphoma (e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL)), B-cell hematologic malignancies (e.g., B-cell lymphoma), T-cell lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma (MBCL), mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic large cell lymphoma (e.g., Ki-1 positive), adult T-cell leukemia / lymphoma, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma lymphoma, angiocentric lymphoma, enteropathic T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-cell lymphoblastic lymphoma, T-lymphoblastic lymphoma; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, histiocytic lymphoma, primary central nervous system malignant lymphoma, primary effusion lymphoma, B-cell lymphoma, B-lymphoblastic lymphoma Lymphoid hematopoietic malignancies include acute lymphoblastic leukemia (LBL), diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome) and lymphoplasmacytic lymphoma (LPL) / Waldenström syndrome. Myeloma (e.g., IgG myeloma, light-chain myeloma, non-secretory myeloma, smoldering myeloma (also called silent myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; myeloid hematopoietic tumors, mesenchymal tumors (e.g., fibrosarcoma and rhabdomyosarcoma); seminoma, malignant teratoma, central and peripheral nervous system tumors (e.g., astrocytoma, schwannoma); Mesenchymal tumors (such as fibrosarcoma, rhabdomyosarcoma, and osteosarcoma); and other tumors (such as melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and malignant teratoma), lymphoid and hematopoietic tumors (T-cell and B-cell tumors, including but not limited to T-cell disorders (e.g., T-prolymphocytic leukemia (T-PLL) (including small cell and cerebriform cell types); T-cell large granular lymphocyte leukemia (LGL); a / d T-NHL hepatosplenic T-cell lymphoma; peripheral / mature T-cell lymphomas (pleomorphic and immunoblastic T-cell lymphoma); angiocentric (nasal) T-cell lymphoma, etc.)); head and neck cancer, kidney cancer, rectal cancer, and thyroid cancer;and acute myeloid lymphoma, and any combination of the foregoing cancers. The methods described herein may also be used to treat metastatic cancer, unresectable cancer, refractory cancer (e.g., cancer that has not responded to previous immunotherapy, such as CTLA-4 or PD-1 antibody blockade), and / or recurrent cancer.

[0058] In some embodiments, the combination therapies described herein are administered to cancer patients who have shown an inadequate response or improvement to previous treatments (e.g., treatment with immuno-oncology or immunotherapeutic agents). In some embodiments, the cancer is refractory or resistant to previous treatments, either congenitally refractory or resistant (e.g., refractory to PD-1 pathway antagonists) or has acquired a resistant or refractory state. For example, the combination therapies described herein can be administered to subjects who did not respond or did not adequately respond to initial treatments, or whose disease has progressed, such as with treatment with an anti-PD-1 pathway antagonist alone or in combination with another treatment. In other embodiments, the combination therapies described herein are administered to patients who have not previously been treated with an immuno-oncology agent (e.g., a PD-1 pathway antagonist).

[0059] The combination treatment may further include one or more other treatments, such as radiation therapy, surgery or chemotherapy.

[0060] The methods described herein can also be used to treat patients (e.g., patients with infectious diseases) who have been exposed to a particular toxin or pathogen. Accordingly, the present disclosure also contemplates a method of treating an infectious disease in a subject, comprising administering a combination therapy described herein. Similar to the tumor application described above, the combination therapy may be used alone or as an adjuvant in combination with a vaccine to stimulate an immune response to pathogens, toxins, and autoantigens. Examples of pathogens for which the present treatment methods may be particularly useful include those for which there are currently no effective vaccines or for which conventional vaccines are not fully effective. These include, but are not limited to, HIV, hepatitis (types A, B, and C), influenza, herpes, giardia, malaria, leishmaniasis, Staphylococcus aureus, and Pseudomonas aeruginosa. Combination therapy may also be useful against established infections (e.g., HIV, which undergoes antigenic changes during the course of infection).

[0061] Some examples of pathogenic viruses that cause infections that can be treated by the methods described herein include HIV, hepatitis (types A, B, or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, cowpox virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0062] Some examples of pathogenic bacteria that cause infections that can be treated by the methods described herein include Chlamydia, Rickettsia, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, Bacillus, Vibrio cholerae, Clostridium tetani, Clostridium botulinum, Bacillus anthracis, Yersinia pestis, Leptospiraceae, and Lyme disease bacteria.

[0063] Some examples of pathogenic fungi that cause infections that can be treated with the methods described herein include Candida (e.g., Candida albicans, Krusei, Glabrata, Tropicalis), Cryptococcus neoformans, Aspergillus (e.g., Fumigatus, Niger), Mucor (e.g., Mucor, E. niger, Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, South American fungus, Coccidioides immitis, and Histoplasma capsulatum.

[0064] Some examples of pathogenic parasites that cause infections that can be treated with the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba spp., Giardia spp., Cryptosporidium spp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Ancylostoma brasiliensis.

[0065] All of the above methods may be combined with other forms of immunotherapy, such as cytokine (e.g., interferon, GM-CSF, G-CSF, IL-2) therapy or bispecific antibody therapy as described herein, in anticipation of the emergence of potent tumor antigens (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123).

[0066] In some embodiments, the methods involve combining an inhibitor of DGKα and / or DGKζ with an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4, and CD4 + Drugs that inhibit T cells and / or CD8 + In some embodiments, the antibody is administered to a subject in need thereof (e.g., a cancer patient) in combination with an agent that enhances T cells. + T-cell-blocking drugs and CD8 + The agent that enhances T cells can be an agent that acts locally at the tumor site.

[0067] Examples of inhibitors of DGKα and / or DGKζ enzyme activity In some embodiments, the inhibitor of DGKα and / or DGKζ is an inhibitor of DGKα. In some embodiments, the inhibitor of DGKα and / or DGKζ is an inhibitor of DGKζ. In some embodiments, the inhibitor of DGKα and / or DGKζ inhibits both enzymes. The level of enzyme inhibition can be measured as described herein below. In some embodiments, the inhibitor of DGKα and / or DGKζ does not significantly inhibit other DGK enzymes.

[0068] In some embodiments, inhibitors of DGKα and / or DGKζ promote immune responses, for example, by activating T cells. For example, inhibitors of DGKα and / or DGKζ can activate primary T cell signaling by increasing pERK / pPKC signaling, as determined, for example, by assays described herein below. In some embodiments, inhibitors of DGKα and / or DGKζ have one or more of the following properties: (i) lowering the threshold for antigen stimulation; (ii) activating CTL effector function; and (iii) enhancing tumor cell killing. When inhibitors of DGKα and / or DGKζ enhance tumor cell killing, this activity is associated with an increase in CD8+ T cell proliferation, as demonstrated, for example, in a CT26 animal model. +T cells may be involved. When inhibitors of DGKα and / or DGKζ enhance tumor cell killing, this activity may be involved by NK cells, as shown, for example, in the CT26 animal model. When inhibitors of DGKα and / or DGKζ enhance tumor cell killing, this activity may be involved by CD8 + T cells and NK cells may be affected. When DGKα and / or DGKζ inhibitors enhance tumor cell killing activity, this activity may be enhanced by a decrease in CD4 cells, for example, in a CT26 animal model. In some embodiments, DGKα and / or DGKζ inhibitors promote the appearance of AH1+ tetramer antigen in a CT26 animal model. DGKα and / or DGKζ inhibitors preferably have one or more, or all, of the above characteristics. The expression of these characteristics can be determined, for example, by performing assays as described in the section entitled "Biological Assays" herein.

[0069] A method of treating a disease (e.g., cancer) may comprise administering to a subject in need thereof an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4 and an inhibitor of DGKα and / or DGKζ, wherein the inhibitor of DGKα and / or DGKζ is a compound represented by formula (I): [ka] [In the formula, R1 is H, F, Cl, Br, -CN, or 0 to 4 R 1a C replaced with 1-3 Alkyl, 0 to 4 R 1a C replaced with 3-4 Cycloalkyl, 0 to 4 R 1a C replaced with 1-3 Alkoxy, -NR a R a , -S(O) n R e or -P(O)R e R e and; Each R 1aare independently F, Cl, -CN, -OH, -OCH3, or -NR a R a and; Each R a are independently H or C 1-3 is alkyl; Each R e independently, C 3-4 Cycloalkyl or 0 to 4 R 1a C replaced with 1-3 is alkyl; R2 is H, 0 to 4 R 2a C replaced with 1-3 Alkyl or 0 to 4 R 2a C replaced with 3-4 is cycloalkyl; Each R 2a are independently F, Cl, -CN, -OH, -O(C 1-2 alkyl), C 3-4 Cycloalkyl, C 3-4 Alkenyl or C 3-4 is alkynyl; R3 is H, F, Cl, Br, -CN, C 1-3 Alkyl, C 1-2 Fluoroalkyl, C 3-4 Cycloalkyl, C 3-4 fluorocycloalkyl or -NO2; R4 is -CH2R 4a , -CH2CH2R 4a , -CH2CHR 4a R 4d , -CHR 4a R 4b or -CR 4a R 4b R 4c and; R 4a and R 4b independently (I C 1-6 Alkyl, F, Cl, -CN, -OH, -OCH3, -SCH3, C 1-3 Fluoroalkoxy, -NR a R a , -S(O)2R e or -NR a S(O)2Re substituted with 0 to 4 substituents independently selected from: (ii) C 3-6 cycloalkyl, heterocyclyl, phenyl, or heteroaryl, each of which is F, Cl, Br, -CN, -OH, or C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-4 Hydroxyalkyl, -(CH2) 1-2 O(C 1-3 alkyl), C 1-4 Alkoxy, -O(C 1-4 hydroxyalkyl), -O(CH) 1-3 O(C 1-3 alkyl), C 1-3 Fluoroalkoxy, -O(CH) 1-3 NR c R c , -OCH2CH=CH2, -OCH2C≡CH, -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -NR c R c , -NR a S(O)2(C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl), -P(O)(C 1-3 alkyl)2, -S(O)2(C 1-3 alkyl), -O(CH2) 1-2 (C 3-6 cycloalkyl), -O(CH2) 1-2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butoxycarbonyl)azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thiophenyl, methylpiperidinyl, and R d or (iii) C substituted with one cyclic group 1-4 alkyl, and the cyclic group is C 3-6cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the cyclic group is selected from F, Cl, Br, —OH, —CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c , -NR a S(O)2(C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl) and C 3-6 substituted with 0 to 3 substituents independently selected from cycloalkyl; R 4a and R 4b together with the carbon atoms to which they are bonded, 3-6 cycloalkyl or 3- to 6-membered heterocyclyl, each of which has 0 to 3 R f is replaced by; Each R f are independently F, Cl, Br, -OH, -CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c or a cyclic group, the cyclic group being C 3-6 cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being selected from F, Cl, Br, —OH, —CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy and -NR c R c substituted with 0 to 3 substituents independently selected from: R 4c is C 1-6 Alkyl or C3-6 cycloalkyl, F, Cl, -OH, C 1-2 Alkoxy, C 1-2 substituted with 0 to 4 substituents independently selected from fluoroalkoxy and —CN; R 4d is -OCH3; Each R c are independently H or C 1-2 is alkyl; R d is phenyl and is substituted with 0-1 substituents selected from F, Cl, —CN, —CH3, and —OCH3; Each R5 is independently selected from -CN, 0 to 4 R g C replaced with 1-6 Alkyl, 0 to 4 R g C replaced with 2-4 Alkenyl, 0 to 4 R g C replaced with 2-4 Alkynyl, 0 to 4 R g C replaced with 3-4 Cycloalkyl, 0 to 4 R g phenyl substituted with 0 to 3 R g oxadiazolyl substituted with 0 to 4 R g Pyridinyl substituted with -(CH2) 1-2 (0 to 4 R g heterocyclyl substituted with -(CH2) 1-2 NR c C(O)(C 1-4 alkyl), -(CH2) 1-2 NR c C(O)O(C 1-4 alkyl), -(CH2) 1-2 NR c S(O)2(C 1-4 alkyl), -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -C(O)O(C 3-4 cycloalkyl), -C(O)NR a R a or -C(O)NR a (C 3-4 cycloalkyl); Each R g are independently F, Cl, -CN, -OH, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -O(CH2) 1-2 O(C 1-2 alkyl) or -NR c R c and; m is 0, 1, 2 or 3; and n is 0, 1, or 2. or a pharmaceutically acceptable salt thereof.

[0070] A method of treating a disease (e.g., cancer) may comprise administering to a subject in need thereof an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4 and an inhibitor of DGKα and / or DGKζ, wherein the inhibitor of DGKα and / or DGKζ is an antagonist of the formula: R1 is H, F, Cl, Br, -CN, 0 to 4 R 1a C replaced with 1-3 Alkyl, 0 to 3 R 1a cyclopropyl substituted with 0-3 R 1a C replaced with 1-3 Alkoxy, -NR a R a , -S(O) n CH3 or -P(O)(CH3)2; Each R 1a is independently F, Cl, or —CN; Each R a are independently H or C 1-3 is alkyl; R2 is H or 0 to 2 R 2a C replaced with 1-2 is alkyl; Each R 2a are independently F, Cl, -CN, -OH, -O(C 1-2 alkyl), cyclopropyl, C 3-4 Alkenyl or C 3-4 is alkynyl; R3 is H, F, Cl, Br, -CN, C 1-2alkyl, -CF3, cyclopropyl, or -NO2; R 4a and R 4b But independently, (I C 1-4 Alkyl, F, Cl, -CN, -OH, -OCH3, -SCH3, C 1-3 Fluoroalkoxy and -NR a R a substituted with 0 to 4 substituents independently selected from: (ii) C 3-6 cycloalkyl, heterocyclyl, phenyl, or heteroaryl, each of which is F, Cl, Br, -CN, -OH, or C 1-6 Alkyl, C 1-3 Fluoroalkyl, -CH2OH, -(CH2) 1-2 O(C 1-2 alkyl), C 1-4 Alkoxy, -O(C 1-4 hydroxyalkyl), -O(CH) 1-2 O(C 1-2 alkyl), C 1-3 Fluoroalkoxy, -O(CH) 1-2 NR c R c , -OCH2CH=CH2, -OCH2C≡CH, -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -NR c R c , -NR a S(O)2(C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl), -P(O)(C 1-2 alkyl)2, -S(O)2(C 1-3 alkyl), -O(CH2) 1-2 (C 3-4 cycloalkyl), -O(CH2) 1-2(morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butoxycarbonyl)azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thiophenyl, methylpiperidinyl, and R d or (iii) C substituted with one cyclic group 1-3 alkyl, and the cyclic group is C 3-6 cycloalkyl, heterocyclyl, phenyl, and heteroaryl, wherein the cyclic group is selected from F, Cl, Br, —OH, —CN, C 1-3 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c , -NR a S(O)2(C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl) and C 3-4 substituted with 0 to 3 substituents independently selected from cycloalkyl; R 4a and R 4b together with the carbon atoms to which they are bonded, 3-6 cycloalkyl or 3- to 6-membered heterocyclyl, each of which has 0 to 3 R f is replaced by; Each R f are independently F, Cl, Br, -OH, -CN, C 1-4 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c or a cyclic group, the cyclic group being C 3-6cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being selected from F, Cl, Br, —OH, —CN, C 1-4 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy and -NR c R c substituted with 0 to 3 substituents independently selected from: R 4c But C 1-4 Alkyl or C 3-6 cycloalkyl, F, Cl, -OH, C 1-2 Alkoxy, C 1-2 substituted with 0 to 4 substituents independently selected from fluoroalkoxy and —CN; and Each R5 is independently -CN, 0 to 4 R g C replaced with 1-5 Alkyl, 0 to 4 R g C replaced with 2-3 Alkenyl, 0 to 4 R g C replaced with 2-3 Alkynyl, 0 to 4 R g C replaced with 3-4 Cycloalkyl, 0 to 3 R g phenyl substituted with 0 to 3 R g oxadiazolyl substituted with 0-3 R g Pyridinyl substituted with -(CH2) 1-2 (0 to 4 R g heterocyclyl substituted with -(CH2) 1-2 NR c C(O)(C 1-4 alkyl), -(CH2) 1-2 NR c C(O)O(C 1-4 alkyl), -(CH2) 1-2 NR c S(O)2(C 1-4 alkyl), -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -C(O)O(C 3-4cycloalkyl), -C(O)NR a R a or -C(O)NR a (C 3-4 cycloalkyl), A compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0071] A method of treating a disease (e.g., cancer) may comprise administering to a subject in need thereof an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4 and an inhibitor of DGKα and / or DGKζ, wherein the inhibitor of DGKα and / or DGKζ is a compound represented by formula (I): [ka] [where: R1 is -CN; R2 is -CH3; R3 is H, F or -CN; R4 is [ka] is] or a pharmaceutically acceptable salt thereof.

[0072] A method for treating a disease (e.g., cancer) may comprise administering to a subject in need thereof an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4 and an inhibitor of DGKα and / or DGKζ, wherein the inhibitor of DGKα and / or DGKζ is a compound of formula (I) having one of the following structures or formulas (or an isomer thereof), or a pharmaceutically acceptable salt thereof: 1-(bis(4-fluorophenyl)methyl)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yl)piperazine-2-carboxylate methyl ester [ka] ; 4-((2R,5S)-4-(bis(4-fluorophenyl)methyl)-2,5-dimethylpiperazin-1-yl)-6-bromo-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-3-carbonitrile [ka] ; (R)-8-(4-(bis(4-fluorophenyl)methyl)-3-methylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2,7-dicarbonitrile [ka] ; 8-[(2S,5R)-4-[(4-chlorophenyl)(5-methylpyridin-2-yl)methyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] ; 4-[(2S,5R)-4-[(4-chlorophenyl)(4-fluorophenyl)methyl]-2,5-dimethylpiperazin-1-yl]-6-methoxy-1-methyl-1,2-dihydro-1,5-naphthyridin-2-one [ka] ; 8-[(2S,5R)-4-{[2-(difluoromethyl)-4-fluorophenyl]methyl}-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] ; 8-[(2S,5R)-4-[(4-fluorophenyl)(4-methylphenyl)methyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] ; 8-[(2S,5R)-4-[1-(2,6-difluorophenyl)ethyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] ; 8-((3R)-4-((4-chlorophenyl)(5-fluoropyridin-2-yl)methyl)-3-methylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2,7-dicarbonitrile [ka] ; 8-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] ; and 8-[(2S,5R)-4-[bis(4-methylphenyl)methyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka]

[0073] A method of treating a disease (e.g., cancer) may comprise administering to a subject in need thereof an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4 and an inhibitor of DGKα and / or DGKζ, wherein the inhibitor of DGKα and / or DGKζ is a compound represented by formula (II): [ka] [In the formula, R1 is H, F, Cl, Br, -CN, -OH, or 0 to 4 R 1a C replaced with 1-3 Alkyl, 0 to 4 R 1a C replaced with 3-4 Cycloalkyl, 0 to 4 R 1a C replaced with 1-3 Alkoxy, -NR a R a , -S(O) n R e or -P(O)R e R e and; Each R 1a are independently F, Cl, -CN, -OH, -OCH3, or -NR a R a and; Each R a are independently H or C 1-3 is alkyl; Each R e independently, C 3-4 Cycloalkyl or 0 to 4 R 1a C replaced with 1-3 is alkyl; R2 is H, 0 to 4 R 2a C replaced with 1-3 Alkyl or 0 to 4 R 2a C replaced with 3-4 is cycloalkyl; Each R 2a are independently F, Cl, -CN, -OH, -O(C 1-2 alkyl), C 3-4 Cycloalkyl, C 3-4 Alkenyl or C 3-4 is alkynyl; R4 is -CH2R 4a , -CH2CH2R 4a , -CH2CHR 4a R 4d , -CHR 4a R 4b or -CR 4a R 4b R 4c and; R 4a and R 4b independently (i) -CN or C 1-6 Alkyl, F, Cl, -CN, -OH, -OCH3, -SCH3, C 1-3 Fluoroalkoxy, -NR a R a , -S(O)2R e or -NR a S(O)2R e substituted with 0 to 4 substituents independently selected from: (ii) C 3-6 cycloalkyl, 4- to 10-membered heterocyclyl, phenyl, or 5- to 10-membered heteroaryl, each of which is F, Cl, Br, —CN, —OH, or C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-2 Bromoalkyl, C 1-2 Cyanoalkyl, C 1-4 Hydroxyalkyl, -(CH2) 1-2 O(C 1-3 alkyl), C 1-4 Alkoxy, C 1-3 Fluoroalkoxy, C 1-3 Cyanoalkoxy, -O(C 1-4 hydroxyalkyl), -O(CR x R x ) 1-3 O(C 1-3 alkyl), C 1-3 Fluoroalkoxy, -O(CH2) 1-3 NR c R c , -OCH2CH=CH2, -OCH2C≡CH, -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -NRc R c , -CH2NR a R a , -NR a S(O)2(C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -(CR x R x ) 0-2 NR a C(O)O(C 1-4 alkyl), -P(O)(C 1-3 alkyl)2, -S(O)2(C 1-3 alkyl), -(CR x R x ) 1-2 (C 3-4 cycloalkyl), -(CR x R x ) 1-2 (morpholinyl), -(CR x R x ) 1-2 (difluoromorpholinyl), -(CR x R x ) 1-2 (dimethylmorpholinyl), -(CR x R x ) 1-2 (oxazabicyclo[2.2.1]heptanyl), (CR x R x ) 1-2 (oxazaspiro[3.3]heptanyl), -(CR x R x ) 1-2 (methylpiperazinonyl), -(CR x R x ) 1-2 (acetylpiperazinyl), -(CR x R x ) 1-2 (piperidinyl), -(CR x R x ) 1-2 (difluoropiperidinyl), -(CR x R x ) 1-2 (Methoxypiperidinyl), -(CR x R x ) 1-2(hydroxypiperidinyl), -O(CR x R x ) 0-2 (C 3-6 cycloalkyl), -O(CR x R x ) 0-2 (methylcyclopropyl), -O(CR x R x ) 0-2 ((ethoxycarbonyl)cyclopropyl), -O(CR x R x ) 0-2 (oxetanyl), -O(CR x R x ) 0-2 (methylazetidinyl), -O(CR x R x ) 0-2 (tetrahydropyranyl), -O(CR x R x ) 1-2 (morpholinyl), -O(CR x R x ) 0-2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butoxycarbonyl)azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thiophenyl, methylpiperidinyl, dioxolanyl, pyrrolidinonyl and R d or (iii) C substituted with one cyclic group 1-4 alkyl, and the cyclic group is C 3-6 cycloalkyl, 4- to 10-membered heterocyclyl, monocyclic or bicyclic aryl or 5- to 10-membered heteroaryl, wherein the cyclic group is selected from F, Cl, Br, —OH, —CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c , -NR a S(O)2(C 1-3 alkyl), -NR aC(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl) and C 3-6 substituted with 0 to 3 substituents independently selected from cycloalkyl; R 4a and R 4b together with the carbon atoms to which they are bonded, 3-6 cycloalkyl or 3- to 6-membered heterocyclyl, each of which has 0 to 3 R f is replaced by; Each R f are independently F, Cl, Br, -OH, -CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c or a cyclic group, the cyclic group being C 3-6 cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being selected from F, Cl, Br, —OH, —CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy and -NR c R c substituted with 0 to 3 substituents independently selected from: R 4c is C 1-6 Alkyl or C 3-6 cycloalkyl, F, Cl, -OH, C 1-2 Alkoxy, C 1-2 substituted with 0 to 4 substituents independently selected from fluoroalkoxy and —CN; R 4d is -OCH3; Each R c are independently H or C 1-2 is alkyl; R dis phenyl and is substituted with 0-1 substituents selected from F, Cl, —CN, —CH3, and —OCH3; Each R5 is independently selected from -CN, 0 to 4 R g C replaced with 1-6 Alkyl, 0 to 4 R g C replaced with 2-4 Alkenyl, 0 to 4 R g C replaced with 2-4 Alkynyl, 0 to 4 R g C replaced with 3-4 Cycloalkyl, 0 to 4 R g phenyl substituted with 0 to 3 R g oxadiazolyl substituted with 0 to 4 R g Pyridinyl substituted with -(CH2) 1-2 (0 to 4 R g 4-10 membered heterocyclyl substituted with -(CH2) 1-2 NR c C(O)(C 1-4 alkyl), -(CH2) 1-2 NR c C(O)O(C 1-4 alkyl), -(CH2) 1-2 NR c S(O)2(C 1-4 alkyl), -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -C(O)O(C 3-4 cycloalkyl), -C(O)NR a R a or -C(O)NR a (C 3-4 cycloalkyl); Each R g are independently F, Cl, -CN, -OH, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -O(CH2) 1-2 O(C 1-2 alkyl) or -NR c R c and; m is 0, 1, 2 or 3; and n is 0, 1, or 2. or a salt thereof.

[0074] A method of treating a disease (e.g., cancer) may comprise administering to a subject in need thereof an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4 and an inhibitor of DGKα and / or DGKζ, wherein the inhibitor of DGKα and / or DGKζ is an antagonist of the formula: R1 is H, F, Cl, Br, -CN, -OH, 0 to 4 R 1a C replaced with 1-3 Alkyl, 0 to 3 R 1a cyclopropyl substituted with 0-3 R 1a C replaced with 1-3 Alkoxy, -NR a R a , -S(O) n CH3 or -P(O)(CH3)2; R2 is H or 0 to 2 R 2a C replaced with 1-2 is alkyl; Each R 2a are independently F, Cl, -CN, -OH, -O(C 1-2 alkyl), cyclopropyl, C 3-4 Alkenyl or C 3-4 is alkynyl; R 4a and R 4b But independently (i) -CN or C 1-4 Alkyl, F, Cl, -CN, -OH, -OCH3, -SCH3, C 1-3 Fluoroalkoxy and -NR a R a substituted with 0 to 4 substituents independently selected from: (ii) C 3-6 cycloalkyl, 4- to 10-membered heterocyclyl, phenyl, or 5- to 10-membered heteroaryl, each of which is F, Cl, Br, —CN, —OH, or C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-2 Bromoalkyl, C 1-2 Cyanoalkyl, C1-2 Hydroxyalkyl, -CHNR a R a , -(CH2) 1-2 O(C 1-2 alkyl), -(CH2) 1-2 NR x C(O)O(C 1-2 alkyl), C 1-4 Alkoxy, -O(C 1-4 hydroxyalkyl), -O(CR x R x ) 1-2 O(C 1-2 alkyl), C 1-3 Fluoroalkoxy, C 1-3 Cyanoalkoxy, -O(CH2) 1-2 NR c R c , -OCH2CH=CH2, -OCH2C≡CH, -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -NR c R c , -NR a S(O)2(C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl), -P(O)(C 1-2 alkyl)2, -S(O)2(C 1-3 alkyl), -(CH2) 1-2 (C 3-4 cycloalkyl), -CR x R x (morpholinyl), -CR x R x (difluoromorpholinyl), -CR x R x (dimethylmorpholinyl), -CR x R x (oxazabicyclo[2.2.1]heptanyl), -CR x R x (oxazaspiro[3.3]heptanyl), -CR x R x (methylpiperazinonyl), -CR x Rx (acetylpiperazinyl), -CR x R x (piperidinyl), -CR x R x (difluoropiperidinyl), -CR x R x (methoxypiperidinyl), -CR x R x (hydroxypiperidinyl), -O(CH2) 0-2 (C 3-4 cycloalkyl), -O(CH2) 0-2 (methylcyclopropyl), -O(CH2) 0-2 ((ethoxycarbonyl)cyclopropyl), -O(CH2) 0-2 (oxetanyl), -O(CH2) 0-2 (methylazetidinyl), -O(CH2) 1-2 (morpholinyl), -O(CH2) 0-2 (tetrahydropyranyl), -O(CH2) 0-2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butoxycarbonyl)azetidinyl, dioxolanyl, pyrrolidinonyl, triazolyl, tetrahydropyranyl, morpholinyl, thiophenyl, methylpiperidinyl, and R d or (iii) C substituted with one cyclic group 1-3 alkyl, and the cyclic group is C 3-6 cycloalkyl, 4- to 10-membered heterocyclyl, monocyclic or bicyclic aryl or 5- to 10-membered heteroaryl, wherein the cyclic group is selected from F, Cl, Br, —OH, —CN, C 1-3 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c , -NR a S(O)2(C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NRa C(O)O(C 1-4 alkyl) and C 3-4 substituted with 0 to 3 substituents independently selected from cycloalkyl; R 4a and R 4b together with the carbon atoms to which they are bonded, 3-6 cycloalkyl or 3- to 6-membered heterocyclyl, each of which has 0 to 3 R f is replaced by; Each R f are independently F, Cl, Br, -OH, -CN, C 1-4 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c or a cyclic group, C 3-6 cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being selected from F, Cl, Br, —OH, —CN, C 1-4 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy and -NR c R c substituted with 0 to 3 substituents independently selected from: R 4c But C 1-4 Alkyl or C 3-6 cycloalkyl, F, Cl, -OH, C 1-2 Alkoxy, C 1-2 substituted with 0 to 4 substituents independently selected from fluoroalkoxy and —CN; Each R5 is independently -CN, 0 to 4 R g C replaced with 1-5 Alkyl, 0 to 4 R g C replaced with 2-3 Alkenyl, 0 to 4 R g C replaced with 2-3 Alkynyl, 0 to 4 R gC replaced with 3-4 Cycloalkyl, 0 to 3 R g phenyl substituted with 0 to 3 R g oxadiazolyl substituted with 0-3 R g Pyridinyl substituted with -(CH2) 1-2 (0 to 4 R g 4-10 membered heterocyclyl substituted with -(CH2) 1-2 NR c C(O)(C 1-4 alkyl), -(CH2) 1-2 NR c C(O)O(C 1-4 alkyl), -(CH2) 1-2 NR c S(O)2(C 1-4 alkyl), -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -C(O)O(C 3-4 cycloalkyl), -C(O)NR a R a or -C(O)NR a (C 3-4 cycloalkyl); Each R x is independently H or -CH3; and m is 1, 2 or 3; A compound of formula (II) or a pharmaceutically acceptable salt thereof:

[0075] A method of treating a disease (e.g., cancer) can feature administering to a subject in need thereof an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4 and an inhibitor of DGKα and / or DGKζ, wherein m is 2; one R5 is R 5a and the other R5 is R 5c and formula (III): [ka] [In the formula, R 5ais -CH3 or -CH2CH3; and R 5c is -CH3, -CH2CH3 or -CH2CH2CH3] or a pharmaceutically acceptable salt thereof.

[0076] A method of treating a disease (e.g., cancer) may comprise administering to a subject in need thereof an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4 and an inhibitor of DGKα and / or DGKζ, wherein the inhibitor of DGKα and / or DGKζ has the formula (III): [ka] [In the formula, R1 is -CN; R2 is -CH3; R 5a is -CH3 or -CH2CH3; and R 5c is -CH3, -CH2CH3 or -CH2CH2CH3] or a pharmaceutically acceptable salt thereof.

[0077] A method for treating a disease (e.g., cancer) may comprise administering to a subject in need thereof an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4 and an inhibitor of DGKα and / or DGKζ, wherein the inhibitor of DGKα and / or DGKζ is a compound of formula (II) having one of the following structures, or a pharmaceutically acceptable salt thereof:

[0078] 4-((2S,5R)-2,5-diethyl-4-(1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 4-((2S,5R)-2,5-diethyl-4-((S)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 4-((2S,5R)-2,5-diethyl-4-((R)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0079] 4-((2S,5R)-5-ethyl-2-methyl-4-(1-(4-(trifluoromethyl)phenyl)ethyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 4-((2S,5R)-5-ethyl-2-methyl-4-((S)-1-(4-(trifluoromethyl)phenyl)ethyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 4-((2S,5R)-5-ethyl-2-methyl-4-((R)-1-(4-(trifluoromethyl)phenyl)ethyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0080] 4-((2S,5R)-5-ethyl-4-((4-fluorophenyl)(5-(trifluoromethyl)pyridin-2-yl)methyl)-2-methylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 4-((2S,5R)-5-ethyl-4-((S)-(4-fluorophenyl)(5-(trifluoromethyl)pyridin-2-yl)methyl)-2-methylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 4-((2S,5R)-5-ethyl-4-((R)-(4-fluorophenyl)(5-(trifluoromethyl)pyridin-2-yl)methyl)-2-methylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0081] 4-((2S,5R)-5-ethyl-2-methyl-4-(1-(4-(trifluoromethoxy)phenyl)ethyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 4-((2S,5R)-5-ethyl-2-methyl-4-((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 4-((2S,5R)-5-ethyl-2-methyl-4-((R)-1-(4-(trifluoromethoxy)phenyl)ethyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0082] 4-((2S,5R)-5-ethyl-2-methyl-4-(1-(4-(trifluoromethoxy)phenyl)propyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 4-((2S,5R)-5-ethyl-2-methyl-4-((S)-1-(4-(trifluoromethoxy)phenyl)propyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 4-((2S,5R)-5-ethyl-2-methyl-4-((R)-1-(4-(trifluoromethoxy)phenyl)propyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0083] 4-((2S,5R)-5-ethyl-2-methyl-4-(1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 4-((2S,5R)-5-ethyl-2-methyl-4-((S)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 4-((2S,5R)-5-ethyl-2-methyl-4-((R)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0084] 4-((2S,5R)-4-((4-chlorophenyl)(pyridin-2-yl)methyl)-5-ethyl-2-methylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 4-((2S,5R)-4-((R)-(4-chlorophenyl)(pyridin-2-yl)methyl)-5-ethyl-2-methylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 4-((2S,5R)-4-((S)-(4-chlorophenyl)(pyridin-2-yl)methyl)-5-ethyl-2-methylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0085] 4-((2S,5R)-4-((3-cyclopropyl-1,2,4-oxadiazol-5-yl)(4-fluorophenyl)methyl)-2,5-dimethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 4-((2S,5R)-4-((R)-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)(4-fluorophenyl)methyl)-2,5-diethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 4-((2S,5R)-4-((S)-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)(4-fluorophenyl)methyl)-2,5-diethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0086] 4-((2S,5R)-4-((4-fluorophenyl)(5-(trifluoromethyl)pyridin-2-yl)methyl)-2,5-dimethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 4-((2S,5R)-4-((S)-(4-fluorophenyl)(5-(trifluoromethyl)pyridin-2-yl)methyl)-2,5-dimethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 4-((2S,5R)-4-((R)-(4-fluorophenyl)(5-(trifluoromethyl)pyridin-2-yl)methyl)-2,5-dimethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0087] 4-((2S,5R)-4-(1-(4-(cyclopropylmethoxy)-2-fluorophenyl)propyl)-2,5-diethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 4-((2S,5R)-4-((S)-1-(4-(cyclopropylmethoxy)-2-fluorophenyl)propyl)-2,5-diethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 4-((2S,5R)-4-((R)-1-(4-(cyclopropylmethoxy)-2-fluorophenyl)propyl)-2,5-diethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0088] 4-((2S,5R)-2,5-diethyl-4-(1-(4-(trifluoromethyl)phenyl)butyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 4-((2S,5R)-2,5-diethyl-4-((S)-1-(4-(trifluoromethyl)phenyl)butyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 4-((2S,5R)-2,5-diethyl-4-((R)-1-(4-(trifluoromethyl)phenyl)butyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0089] 1-methyl-4-((2S,5R)-2-methyl-5-propyl-4-(1-(4-(trifluoromethyl)phenyl)ethyl)piperazin-1-yl)-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The compounds include 1-methyl-4-((2S,5R)-2-methyl-5-propyl-4-((S)-1-(4-(trifluoromethyl)phenyl)ethyl)piperazin-1-yl)-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile and 1-methyl-4-((2S,5R)-2-methyl-5-propyl-4-((R)-1-(4-(trifluoromethyl)phenyl)ethyl)piperazin-1-yl)-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile.

[0090] PD1 / PD-L1 binding antagonists Antagonists of PD1 / PD-L1 binding that may be combined with DGK inhibitors include:

[0091] The antagonist of PD1 / PD-L1 binding is an antagonist of human PD1 or an antagonist of human PD-L1 that stimulates an immune response by inhibiting negative checkpoints. The antagonist can be various types of molecules, such as proteins, nucleic acids, or small molecules. In some embodiments, the antagonist of PD1 / PD-L1 binding is an antibody that specifically binds to human PD1 or human PD-L1.

[0092] Anti-PD-1 antibodies known to those skilled in the art can be used in the methods described herein. Various human monoclonal antibodies that specifically bind to PD-1 with high affinity are disclosed in U.S. Patent No. 8,008,449. The anti-PD-1 human antibodies disclosed in U.S. Patent No. 8,008,449 have been shown to exhibit one or more of the following characteristics: (a) a 1x10 -7 (b) binds to human PD-1 with a KD of 1 M or less (as determined by surface plasmon resonance using a Biacore biosensor system); (c) does not substantially bind to human CD28, CTLA-4, or ICOS; (d) stimulates T-cell proliferation in a mixed lymphocyte reaction (MLR) assay; (e) increases interferon-γ production in an MLR assay; (f) binds to human PD-1 and cynomolgus PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) generates immunological memory against a specific antigen; (i) stimulates antibody responses; and (j) inhibits tumor cell growth in vivo. Anti-PD-1 antibodies that can be used in the methods of the disclosure include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in some embodiments, at least five, of the above characteristics.

[0093] Other anti-PD-1 monoclonal antibodies are described in, e.g., U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, U.S. Publication No. 2016 / 0272708, and PCT Publication Nos. WO 2012 / 145493, WO 2008 / 156712, WO 2015 / 112900, WO 2012 / 145493, WO 2015 / 112800, WO 2014 / 206107, WO 2015 / 35606, WO 2015 / 085847, WO 2014 / 179664, WO 2017 / 020291, WO 2017 / 020858, WO 2016 / 197367, WO 2017 / 024515, WO 2017 / 025051, WO 2017 / 123557, WO 2016 / 106159, WO 2014 / 194302, WO 2017 / 040790, WO 2017 / 133540, WO 2017 / 132827, WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 106061, WO 2017 / 19846, WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540, the contents of each of which are incorporated by reference in their entirety.

[0094] In some embodiments, the anti-PD-1 antibody is nivolumab (OPDIVO®, also known as 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO 2008 / 156712), PDR001 (Novartis; see WO 2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012 / 145493), cemiplimab (Regeneron; also known as REGN-2810; see WO 2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; also known as toripalimab; Si-Yang Liu et al. al., J. Hematol. Oncol. 10:136 (2017)), sintilimab, BGB-A317 (Beigene; also known as tislelizumab; see WO 2015 / 35606 and US 2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; WO 2015 / 085847; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics; WO 2017 / 19846), BCD-100 (Biocad; Kaplon et al., mAbs 10(2):183-203 (2018)) and IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540).

[0095] In some embodiments, the anti-PD-1 antibody is nivolumab, a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively blocks interaction with PD-1 ligands (PD-L1 and PD-L2), thereby inhibiting the suppression of anti-tumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0096] In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody against the human cell membrane receptor PD-1 (programmed death 1 or programmed cell death 1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0097] Anti-PD-1 antibodies that can be used in the methods of the present disclosure also include isolated antibodies that specifically bind to human PD-1 and cross-compete with any of the anti-PD-1 antibodies of the present disclosure that bind to human PD-1 (e.g., nivolumab; see U.S. Patent Nos. 8,008,449 and 8,779,105; WO 2013 / 173223). In some embodiments, the anti-PD-1 antibody binds to the same epitope as any of the anti-PD-1 antibodies described herein (e.g., nivolumab). Antibodies that cross-compete for binding to an antigen indicate that the monoclonal antibodies bind to the same epitope region of the antigen and sterically inhibit the binding of other cross-competing antibodies to that particular epitope region. Because these cross-competing antibodies bind to the same epitope region of PD-1, they are expected to have functional properties very similar to those of the reference antibody (e.g., nivolumab). Cross-competing antibodies can be readily identified by standard PD-1 binding assays, such as Biacore analysis, ELISA assays or flow cytometry, based on their ability to cross-compete with nivolumab (see, e.g., WO 2013 / 173223).

[0098] In some embodiments, the antibody that binds to human PD-1 by cross-competing with the human PD-1 antibody (nivolumab) or by binding to the same epitope region is a monoclonal antibody. For administration to humans, these cross-competing antibodies are chimeric, engineered, humanized, or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be produced and isolated by methods well known to those skilled in the art.

[0099] Anti-PD-1 antibodies that can be used in the methods of the disclosure also include antigen-binding sites of such antibodies. It has been well demonstrated that the antigen-binding ability of antibodies can be exerted by fragments of full-length antibodies.

[0100] Anti-PD-1 antibodies suitable for use in the disclosed compositions and methods are those that bind to PD-1 with high specificity and affinity, inhibit binding to PD-L1 and / or PD-L2, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the disclosed compositions or methods, the anti-PD-1 "antibody" includes an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits functional properties similar to those of whole antibodies in inhibiting ligand binding and stimulating the immune system. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1.

[0101] In one embodiment, the antagonist of PD1 / PD-L1 binding is an antagonist of PD-L1. Anti-PD-L1 antibodies known to those of skill in the art can be used in the compositions and methods disclosed herein. Examples of anti-PD-L1 antibodies useful in the compositions and methods disclosed herein include those disclosed in U.S. Patent No. 9,580,507. The anti-PD-L1 human monoclonal antibody disclosed in U.S. Patent No. 9,580,507 has been shown to exhibit one or more of the following characteristics: (a) a 1x10 -7 (b) bind to human PD-1 with a KD of 1 M or less (as determined by surface plasmon resonance using a Biacore biosensor system); (b) promote T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (c) increase interferon-γ production in an MLR assay; (d) increase IL-2 secretion in an MLR assay; (e) stimulate antibody responses; and (f) reverse the effects of regulatory T cells on effector T cells and / or dendritic cells. Anti-PD-L1 antibodies that can be used in the present disclosure include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, and in some embodiments, at least five, of the above characteristics.

[0102] In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of BMS-936559 (12A4, also known as MDX-1105; see, e.g., U.S. Patent No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; TECENTRIQ®, also known as MPDL3280A, RG7446; see U.S. Patent No. 8,217,149 and Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca; IMFINZI TM , also known as MEDI-4736; see WO 2011 / 066389), avelumab (Pfizer; also known as BAVENCIO®, MSB-0010718C; see WO 2013 / 079174), STI-1014 (Sorrento; see WO 2013 / 181634), CX-072 (Cytomx; see WO 2016 / 149201), KN035 (3D Med / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (Eli Lilly Co.; see, e.g., WO 2017 / 034916), BGB-A333 (BeiGene; Desai et al., JCO 36 (15suppl):TPS3113 (2018)) and CK-301 (Checkpoint Therapeutics; Gorelik et al., AACR:Abstract 4606 (Apr 2016)).

[0103] In one embodiment, the PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0104] In one embodiment, the PD-L1 antibody is durvalumab (IMFINZ). TM Durvalumab is a human IgG1κ monoclonal anti-PD-L1 antibody.

[0105] In some embodiments, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

[0106] Anti-PD-L1 antibodies that can be used in the methods of the disclosure also include isolated antibodies that specifically bind to human PD-L1 and cross-compete with any of the anti-PD-L1 antibodies of the disclosure that bind to human PD-L1 (e.g., atezolizumab, durvalumab, and / or avelumab). In some embodiments, the anti-PD-L1 antibody binds to the same epitope as any of the anti-PD-L1 antibodies described herein (e.g., atezolizumab, durvalumab, and / or avelumab). Antibodies that cross-compete for binding to an antigen indicate that they bind to the same epitope region of the antigen and sterically inhibit the binding of other cross-competing antibodies to that particular epitope region. Because these cross-competing antibodies bind to the same epitope region of PD-L1, they are expected to have functional properties very similar to the reference antibody (e.g., atezolizumab and / or avelumab). Cross-competing antibodies can be readily identified by standard PD-L1 binding assays, such as Biacore analysis, ELISA assays or flow cytometry, based on their ability to cross-compete with atezolizumab and / or avelumab (see, e.g., WO 2013 / 173223).

[0107] In some embodiments, antibodies that cross-compete with, or bind to the same epitope region as, human PD-L1 antibodies (atezolizumab, durvalumab, and / or avelumab) are monoclonal antibodies. For administration to humans, these cross-competing antibodies are chimeric, engineered, or humanized or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be produced and isolated by methods well known to those skilled in the art.

[0108] Anti-PD-L1 antibodies that can be used in the methods of the disclosure also include antigen-binding portions of such antibodies. It has been well demonstrated that the antigen-binding ability of antibodies can be exerted by fragments of full-length antibodies.

[0109] Suitable anti-PD-L1 antibodies for use in the methods of the disclosure are those that bind to PD-L1 with high specificity and affinity, inhibit PD-1 binding, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the methods of the disclosure, the anti-PD-L1 "antibody" includes an antigen-binding portion or fragment that binds to PD-L1 and exhibits functional properties similar to those of whole antibodies in inhibiting receptor binding and stimulating the immune system. In some embodiments, the anti-PD-L1 antibody, or antigen-binding portion thereof, cross-competes with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1.

[0110] Anti-PD-L1 antibodies useful in the present disclosure can be any PD-L1 antibody that specifically binds to PD-L1 (e.g., an antibody that cross-competes with durvalumab, avelumab, or atezolizumab for binding to human PD-1 (e.g., an antibody that binds to the same epitope as durvalumab, avelumab, or atezolizumab). In some embodiments, the anti-PD-L1 antibody is durvalumab. In other embodiments, the anti-PD-L1 antibody is avelumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab.

[0111] CTLA4 antagonists Antagonists of CTLA4 that may be combined with DGK inhibitors include:

[0112] The CTLA-4 antagonist is an antagonist of human CTLA-4, which stimulates immune response by inhibiting negative checkpoint.Antagonist can be various kinds of molecules, such as protein, nucleic acid or small molecule.In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds to human CTLA-4.

[0113] Anti-CTLA-4 antibodies known to those skilled in the art can be used in the methods of the present disclosure. The anti-CTLA-4 antibodies of the present disclosure bind to human CTLA-4 to prevent CTLA-4 from interacting with the human B7 receptor. Because the interaction between CTLA-4 and B7 transmits a signal that inactivates T cells bearing the CTLA-4 receptor, inhibiting this interaction effectively triggers, enhances, and prolongs T cell activation, thereby inducing, enhancing, or prolonging an immune response.

[0114] Human monoclonal antibodies that specifically bind to CTLA-4 with high affinity are disclosed in U.S. Patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies are described, for example, in U.S. Patent Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121 and International Publication Nos. WO 2012 / 122444, WO 2007 / 113648, WO 2016 / 196237, and WO 2000 / 037504, the contents of each of which are incorporated by reference in their entirety. The anti-CTLA-4 human monoclonal antibodies disclosed in U.S. Patent No. 6,984,720 have been shown to exhibit one or more of the following characteristics: (a) an equilibrium association constant (Ka) of at least about 10 7 M -1 or about 10 9 M -1 or about 10 10 M -1 ~10 11 M -1 (b) specifically binds to human CTLA-4 with a binding affinity of at least about 10 (as determined by Biacore analysis); 3 , about 10 4 or about 10 5 m -1 s -1 (c) a kinetic dissociation constant (kd) of at least about 10 3 , about 10 4 or about 10 5 m -1 s -1and (d) inhibit the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies useful in the present disclosure include monoclonal antibodies that specifically bind to human CTLA-4 and exhibit at least one, at least two, or at least three of the above characteristics.

[0115] In some embodiments, the CTLA-4 antibody is selected from the group consisting of ipilimumab (also known as YERVOY®, MDX-010, 10D1; see U.S. Pat. No. 6,984,720), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; see WO 2016 / 196237), and tremelimumab (AstraZeneca; also known as ticilimumab, CP-675,206; see WO 2000 / 037504 and Ribas, Update Cancer Ther. 2(3): 133-39 (2007)). In particular embodiments, the anti-CTLA-4 antibody is ipilimumab.

[0116] In certain embodiments, the CTLA-4 antibody used in the methods of the present disclosure is ipilimumab, a fully human IgG1 monoclonal antibody that inhibits CTLA-4 binding to the B7 ligand, thereby activating T cells and improving overall survival (OS) in patients with advanced melanoma.

[0117] In certain embodiments, the CTLA-4 antibody is tremelimumab.

[0118] In a particular embodiment, the CTLA-4 antibody is MK-1308.

[0119] In certain embodiments, the CTLA-4 antibody is AGEN-1884.

[0120] Anti-CTLA-4 antibodies that can be used in the methods of the present disclosure also include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with any of the anti-CTLA-4 antibodies of the present disclosure that bind to human CTLA-4 (e.g., ipilimumab and / or tremelimumab). In some embodiments, the anti-CTLA-4 antibody binds to the same epitope as any of the anti-CTLA-4 antibodies described herein (e.g., ipilimumab and / or tremelimumab). Antibodies that cross-compete for binding to an antigen indicate that they bind to the same epitope region of the antigen and sterically inhibit the binding of other cross-competing antibodies to that particular epitope region. Because these cross-competing antibodies bind to the same epitope region as CTLA-4, they are expected to have functional properties very similar to those of the reference antibody (e.g., ipilimumab and / or tremelimumab). Cross-competing antibodies can be readily identified by standard CTLA-4 binding assays, such as Biacore analysis, ELISA assays or flow cytometry, based on their ability to cross-compete with ipilimumab and / or tremelimumab (see, for example, WO 2013 / 173223).

[0121] In some embodiments, antibodies that cross-compete with human CTLA-4 antibodies (ipilimumab and / or tremelimumab) to bind to human CTLA-4, or antibodies that bind to the same epitope region, are monoclonal antibodies. For administration to humans, these cross-competing antibodies are chimeric, engineered, or humanized or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be produced and isolated by methods well known to those skilled in the art.

[0122] Anti-CTLA-4 antibodies that can be used in the methods of the present disclosure also include antigen-binding sites of such antibodies. It has been well demonstrated that the antigen-binding ability of antibodies can be exerted by fragments of full-length antibodies.

[0123] Anti-CTLA-4 antibodies suitable for use in the methods of the present disclosure are antibodies that bind to CTLA-4 with high specificity and affinity, inhibit the activity of CTLA-4, and prevent CTLA-4 from interacting with the human B7 receptor. In any of the compositions or methods of the present disclosure, the anti-CTLA-4 "antibody" includes an antigen-binding portion or fragment that binds to CTLA-4 and exhibits functional properties similar to those of a whole antibody in inhibiting the interaction of CTLA-4 with the human B7 receptor and promoting the immune system. In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof cross-competes with ipilimumab and / or tremelimumab for binding to human CTLA-4.

[0124] CTLA4 antagonists also include variants of CTLA4 antibodies. Examples of variants of CTLA4 antibodies include non-fucosylated anti-CTLA4 antibodies (e.g., non-fucosylated ipilimumab), and activating CTLA4 antibodies with a coating that is selectively shed within tumors (e.g., non-fucosylated activated ipilimumab or non-fucosylated activated CTLA-4 antibodies). Examples of non-fucosylated and / or activating anti-CTLA4 antibodies (e.g., ipilimumab) are disclosed in WO2014 / 089113 and WO2018 / 085555.

[0125] Administration of inhibitors of DGKα and / or DGKζ and antagonists of PD1 / PD-L1 binding or CTLA4 The desired compound according to Formula (I) or (II) (a compound selected from Compounds 1-34) and / or a pharmaceutically acceptable salt thereof may be administered by any means appropriate to the condition to be treated, which may dictate the need for site-specific treatment or the amount of compound to be delivered.

[0126] Also included herein is a class of pharmaceutical compositions comprising a compound of Formula (I) or (II) (selected from Compounds 1-34) and / or a pharmaceutically acceptable salt thereof; and one or more nontoxic, pharmaceutically acceptable carriers and / or diluents and / or adjuvants (collectively referred to herein as "carriers"), and, optionally, other active ingredients. A compound of Formula (I) or (II) (selected from Compounds 1-34) may be administered by any appropriate route, preferably in the form of a pharmaceutical composition adapted for such route, and in a dosage effective for the intended treatment. The compounds and compositions described herein may be administered, for example, orally, transmucosally, or parenterally, including intravascularly, intravenously, intraperitoneally, subcutaneously, intramuscularly, and intrasternally, in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. For example, pharmaceutical carriers may include mannitol or a mixture of lactose and microcrystalline cellulose. The mixture may include additional ingredients such as lubricants (e.g., magnesium stearate) and disintegrants (e.g., crospovidone). The carrier mixture may be filled into gelatin capsules or compressed into tablets. The pharmaceutical composition may be administered, for example, as an oral dosage form or by infusion.

[0127] For oral administration, the pharmaceutical compositions described herein may be in the form of, for example, tablets, capsules, liquid capsules, suspensions, or liquids. Pharmaceutical compositions are preferably formulated in dosage unit forms containing a specific amount of active ingredient. For example, pharmaceutical compositions may be provided as tablets or capsules containing an amount of active ingredient ranging from about 0.1 to 1000 mg, preferably from about 0.25 to 250 mg, and more preferably from about 0.5 to 100 mg. The appropriate daily dose for administration to humans or other mammals may vary greatly depending on the patient's condition and other factors, but can be determined using conventional methods.

[0128] Any pharmaceutical composition discussed herein can be orally delivered, for example, via any acceptable and suitable oral formulation. Examples of oral formulations include, but are not limited to, tablets, troches, lozenges, aqueous and oily suspensions, dispersible powders or granules, emulsions, hard and soft capsules, liquid capsules, syrups, and elixirs. Pharmaceutical compositions for oral administration can be prepared according to any method known in the art for preparing pharmaceutical compositions for oral administration. To provide a pharmaceutically easy-to-take formulation, the pharmaceutical composition can include at least one substance selected from sweeteners, flavoring agents, coloring agents, demulcents, antioxidants, and preservatives.

[0129] Tablets can be prepared, for example, by mixing at least one compound of Formula (I) or (II) (selected from Compounds 1-34) and / or at least one pharmaceutically acceptable salt thereof with at least one non-toxic, pharmaceutically acceptable excipient suitable for tablet manufacture. Examples of excipients include, but are not limited to, inert diluents (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate, and sodium phosphate), granulating and disintegrating agents (e.g., microcrystalline cellulose, croscarmellose sodium, cornstarch, and alginic acid), binders (e.g., starch, gelatin, polyvinylpyrrolidone, and gum arabic), and lubricants (e.g., magnesium stearate, stearic acid, and talc). Furthermore, tablets can be uncoated or coated by known techniques to mask unpleasant drug tastes or to delay disintegration and absorption of the active ingredient in the gastrointestinal tract, thereby prolonging the effect of the active ingredient. Examples of water soluble taste masking materials include, but are not limited to, hydroxypropyl methylcellulose and hydroxypropyl cellulose. Examples of time delay materials include, but are not limited to, ethyl cellulose and cellulose acetate butyrate.

[0130] Hard gelatin capsules can be prepared, for example, by mixing at least one compound of Formula (I) or (II) (selected from Compounds 1-34) and / or at least one pharmaceutically acceptable salt thereof with at least one inert solid diluent (e.g., calcium carbonate, calcium phosphate, and kaolin).

[0131] Soft gelatin capsules can be prepared, for example, by mixing at least one compound of Formula (I) or (II) (selected from Compounds 1-34) and / or at least one pharmaceutically acceptable salt thereof with at least one water-soluble carrier (e.g., polyethylene glycol) and at least one oil medium (e.g., peanut oil, liquid paraffin, and olive oil).

[0132] Aqueous suspensions can be prepared, for example, by mixing at least one compound of Formula (I) or (II) (selected from Compounds 1-34) and / or at least one pharmaceutically acceptable salt thereof with at least one additive suitable for preparing aqueous suspensions. Examples of additives suitable for preparing aqueous suspensions include, but are not limited to, suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, alginic acid, polyvinylpyrrolidone, tragacanth gum, and gum arabic), dispersing or wetting agents (e.g., naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide and long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide and fatty acids and and partial esters derived from hexitols (e.g., polyoxyethylene sorbitol monooleate), and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). The aqueous suspension may also contain at least one preservative (e.g., ethyl p-hydroxybenzoate and n-propyl p-hydroxybenzoate), at least one coloring agent, at least one flavoring agent, and / or at least one sweetening agent (e.g., but not limited to, sucrose, saccharin, and aspartame).

[0133] Oily suspensions can be prepared, for example, by suspending at least one compound of formula (I) or (II) (selected from compounds 1-34) and / or at least one pharmaceutically acceptable salt thereof in either a vegetable oil (e.g., peanut oil, olive oil, sesame oil, and coconut oil) or a mineral oil (e.g., liquid paraffin). Oily suspensions can also contain at least one thickening agent (e.g., beeswax, hard paraffin, and cetyl alcohol). To provide a palatable oily suspension, at least one sweetener and / or at least one flavoring agent as described above can be added to the oily suspension. Oily suspensions can further contain at least one preservative, including, but not limited to, an antioxidant (e.g., butylhydroxyanisole and α-tocopherol).

[0134] Dispersible powders and granules can be prepared, for example, by mixing at least one compound of formula (I) or (II) (selected from compounds 1-34) and / or at least one pharmaceutically acceptable salt thereof with at least one dispersing agent and / or wetting agent, at least one suspending agent, and / or at least one preservative. Suitable dispersing agents, wetting agents, and suspending agents have already been described above. Examples of preservatives include, but are not limited to, antioxidants (e.g., ascorbic acid). Furthermore, dispersible powders and granules may also contain at least one excipient (e.g., but not limited to, sweeteners, flavoring agents, and coloring agents).

[0135] Emulsions of at least one compound of Formula (I) or (II) (selected from Compounds 1-34) and / or at least one pharmaceutically acceptable salt thereof can be prepared, for example, as oil-in-water emulsions. The oil phase of emulsions containing a compound of Formula (I) or (II) (selected from Compounds 1-34) can be composed of known ingredients in a known manner. The oil phase can be provided by, for example, but not limited to, vegetable oils (e.g., olive oil and peanut oil), mineral oils (e.g., liquid paraffin), and mixtures thereof. The oil phase can include only an emulsifier, or a mixture of at least one emulsifier and a fat or oil, or both a fat and an oil. Suitable emulsifiers include, but are not limited to, naturally occurring phosphatides (e.g., soybean lecithin), esters or partial esters derived from fatty acids and hexitol anhydrides (e.g., sorbitan monooleate), and condensation products of partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier, which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier, with or without a stabilizer, makes up the so-called emulsifying wax, which, together with the oil and fat, forms the oily dispersed phase of the cream formulation, making up the so-called emulsifying ointment base. The emulsion may also contain sweeteners, flavoring agents, preservatives, and / or antioxidants. Suitable emulsifiers and emulsion stabilizers for use in the formulations used in the present therapeutic methods include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate, alone or in combination with wax; or other materials known in the art.

[0136] In addition, the compound of formula (I) or (II) (selected from Compounds 1-34) and / or at least one pharmaceutically acceptable salt thereof can be delivered, for example, intravenously, subcutaneously, and / or intramuscularly via any pharmaceutically acceptable and suitable injection form. Examples of injection forms include, but are not limited to, sterile aqueous solutions containing acceptable vehicles and solvents (e.g., water, Ringer's solution, and isotonic sodium chloride solution), sterile oil-in-water microemulsions, and aqueous or oily suspensions.

[0137] Preparations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules by using one or more of the carriers or diluents described for use in oral preparations, or by using other suitable dispersing or wetting agents and suspending agents. The compound may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth gum, and / or various buffers. Other adjuvants and administration methods are well known and widely known in the pharmaceutical field. The active ingredient may also be administered by injection in a composition with a suitable carrier (e.g., saline, dextrose, or water), or cyclodextrins (e.g., Captisol), solubilizing cosolvents (e.g., propylene glycol), or solubilizing micelles (e.g., Tween 80).

[0138] Alternatively, a sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Among the acceptable vehicles and solvents that may be used, water, Ringer's solution, and isotonic sodium chloride solution are used. Furthermore, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any sterile, fixed oil may be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids such as oleic acid are used in injectable preparations.

[0139] Sterile injectable oil-in-water microemulsions can be prepared, for example, by: 1) dissolving at least one compound of Formula (I) or (II) (selected from Compounds 1-34) in an oil phase (e.g., a mixture of soybean oil and lecithin), 2) combining the Formula (I) or (II) (selected from Compounds 1-34) containing oil phase with a mixture of water and glycerol, and 3) treating the combination to form a microemulsion.

[0140] Sterile aqueous suspensions or sterile oily suspensions can be prepared according to methods known to those skilled in the art. For example, sterile aqueous solutions or sterile aqueous suspensions can be prepared using non-toxic, parenterally acceptable diluents or solvents (e.g., 1,3-butanediol), and sterile oily suspensions can be prepared using sterile, non-toxic, acceptable solvents or suspension media (e.g., sterile fixed oils (e.g., synthetic monoglycerides or diglycerides), and fatty acids (e.g., oleic acid).

[0141] Pharmaceutically acceptable carriers, adjuvants, and vehicles that may be used in pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) (e.g., d-α-tocopherol polyethylene glycol 1000 succinate), surfactants used in pharmaceutical dosage forms (e.g., Tween, polyethoxylated castor oil (e.g., CREMOPHOR surfactants (BASF), or other similar polymeric delivery matrices), serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts, or electrolytes (e.g., protamine sulfate). Examples of suitable carriers include cellulose-based materials, such as cellulose acetate, cellulose acetate dihydrate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. Cyclodextrins (e.g., α-, β-, and γ-cyclodextrin, or chemically modified derivatives (e.g., hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl cyclodextrin, or other solubilizing derivatives) may also be advantageously used to enhance delivery of compounds of the formulae described herein.

[0142] The pharmaceutically active compounds described herein can be processed according to conventional pharmaceutical methods to prepare medicaments for administration to patients (e.g., humans and other mammals). The pharmaceutical compositions may be subjected to conventional pharmaceutical operations (e.g., sterilization) and / or may contain conventional adjuvants (e.g., preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc.). Tablets and pills may additionally be prepared with enteric coatings. Such compositions may also contain adjuvants (e.g., wetting agents, sweeteners, flavoring agents, and fragrances).

[0143] The amount of compound and dosing regimen administered to treat a condition using the compounds and / or compositions described herein are influenced by various factors (e.g., age, weight, sex, the patient's condition, the type of disease, the severity of the disease, the route and frequency of administration, and the particular compound used). Therefore, the dosing regimen may vary widely but can be routinely determined using standard methods. A daily dose of about 0.001 to 100 mg / kg body weight, preferably about 0.0025 to about 50 mg / kg body weight, and most preferably about 0.005 to 10 mg / kg body weight, may be appropriate. The daily dose may be administered one to four times daily. Other dosing regimens include weekly and biday cycles.

[0144] For therapeutic purposes, the active compound described herein is usually combined with one or more adjuvants suitable for intended administration route.When administered orally, the compound can be mixed with lactose, sucrose, starch powder, cellulose ester of alkanoic acid, cellulose alkyl ester, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphate and sulfate, gelatin, gum arabic, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, and then be tableted or encapsulated for convenient administration.Such capsules or tablets can also contain controlled-release formulations, and can also be provided by dispersing the active compound in hydroxypropylmethylcellulose.

[0145] Pharmaceutical compositions described herein include at least one compound of Formula (I) or (II) (selected from Compounds 1-34) and / or at least one pharmaceutically acceptable salt thereof, and optionally an additive selected from any pharmaceutically acceptable carrier, adjuvant, and vehicle. Other compositions described herein include a compound of Formula (I) or (II) (selected from Compounds 1-34) described herein, or a prodrug thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0146] In some embodiments, the anti-PD-L1 antibodies used in the treatment methods described herein are administered at a dose ranging from about 0.1 mg / kg of body weight to about 20.0 mg / kg, 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, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg, about once every 2, 3, 4, 5, 6, 7, or 8 weeks.

[0147] In some embodiments, the anti-PD-L1 antibody is administered at a dose of about 15 mg / kg body weight about once every three weeks. In other embodiments, the anti-PD-L1 antibody is administered at a dose of about 10 mg / kg body weight about once every two weeks.

[0148] In other embodiments, the anti-PD-L1 antibodies useful in the present disclosure are administered at a maximally effective dose of about 200 mg to about 1600 mg, about 200 mg to about 1500 mg, about 200 mg to about 1400 mg, about 200 mg to about 1300 mg, about 200 mg to about 1200 mg, about 200 mg to about 1100 mg, about 200 mg to about 1000 mg, about 200 mg to about 900 mg, about 200 mg to about 800 mg, about 200 mg to about 700 mg, about 200 mg to about 600 mg, about 700 mg to about 1300 mg, about 800 mg to about 1200 mg, about 700 mg to about 900 mg, or about 1100 mg to about 1300 mg. In some embodiments, the anti-PD-L1 antibody is administered at a maximally effective dose of at least about 240 mg, at least about 300 mg, at least about 320 mg, at least about 400 mg, at least about 480 mg, at least about 500 mg, at least about 560 mg, at least about 600 mg, at least about 640 mg, at least about 700 mg, at least 720 mg, at least about 800 mg, at least about 840 mg, at least about 880 mg, at least about 900 mg, at least 960 mg, at least about 1000 mg, at least about 1040 mg, at least about 1100 mg, at least about 1120 mg, at least about 1200 mg, at least about 1280 mg, at least about 1300 mg, at least about 1360 mg, or at least about 1400 mg, with a dosing interval of about 1, 2, 3, or 4 weeks. In some embodiments, the anti-PD-L1 antibody is administered at a maximally effective dose of about 1200 mg once every 3 weeks. In another embodiment, the anti-PD-L1 antibody is administered at a maximum effective dose of about 800 mg about once every two weeks. In another embodiment, the anti-PD-L1 antibody is administered at a maximum effective dose of about 840 mg about once every two weeks.

[0149] In some embodiments, atezolizumab is administered at a maximum effective dose of about 1200 mg about once every three weeks. In some embodiments, atezolizumab is administered at a maximum effective dose of about 800 mg about once every two weeks. In some embodiments, atezolizumab is administered at a maximum effective dose of about 840 mg about once every two weeks.

[0150] In some embodiments, avelumab is administered at a maximum effective dose of about 800 mg about once every two weeks.

[0151] In some embodiments, durvalumab is administered at a dose of about 10 mg / kg about once every two weeks. In some embodiments, durvalumab is administered at a maximum effective dose of about 800 mg / kg about once every two weeks. In some embodiments, durvalumab is administered at a maximum effective dose of about 1200 mg / kg about once every three weeks.

[0152] In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof used in the therapeutic methods described herein is administered at a dose ranging from 0.1 mg / kg to 10.0 mg / kg of body weight once every 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 1 mg / kg or 3 mg / kg of body weight once every 3, 4, 5, or 6 weeks. In one embodiment, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 3 mg / kg of body weight once every 2 weeks. In another embodiment, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of 1 mg / kg of body weight once every 6 weeks.

[0153] In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a maximally effective dose of about 10 to about 1,000 mg, about 10 to about 900 mg, about 10 to about 800 mg, about 10 to about 700 mg, about 10 to about 600 mg, about 10 to about 500 mg, about 100 mg to about 1,000 mg, about 100 mg to about 900 mg, about 100 mg to about 800 mg, about 100 mg to about 700 mg, about 100 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg to about 480 mg, or about 240 mg to about 480 mg. In certain embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is at least about 60 mg, at least about 80 mg, at least about 100 mg, at least about 120 mg, at least about 140 mg, at least about 160 mg, at least about 180 mg, at least about 200 mg, at least about 220 mg, at least about 240 mg, at least about 260 mg, at least about 280 mg, at least about 300 mg, at least about 320 mg, at least about 340 mg, at least about 360 mg, at least about 380 mg, at least about 400 mg, at least about 420 mg, at least about 440 mg, at least about 460 mg, at least about 480 mg, at least about 500 mg, at least about 520 mg In another embodiment, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a maximally effective dose of at least about 540 mg, at least about 550 mg, at least about 560 mg, at least about 580 mg, at least about 600 mg, at least about 620 mg, at least about 640 mg, at least about 660 mg, at least about 680 mg, at least about 700 mg, or at least about 720 mg. In another embodiment, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a maximally effective dose about once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0154] In some embodiments, ipilimumab is administered at a dose of about 3 mg / kg about once every 3 weeks. In some embodiments, ipilimumab is administered at a dose of about 10 mg / kg about once every 3 weeks. In some embodiments, ipilimumab is administered at a dose of about 10 mg / kg about once every 12 weeks. In some embodiments, ipilimumab is administered in four doses.

[0155] (Method of producing the compound) The compounds described herein can be synthesized by many methods available to those skilled in the art of organic chemistry. A general synthetic scheme for preparing the compounds included herein is described below. This scheme is illustrative and is not intended to limit the possible techniques that one skilled in the art can use to prepare the compounds described herein. Various methods for preparing the compounds included herein will be apparent to those skilled in the art. Examples of compounds prepared by the methods described in the general scheme are provided in the Examples section below. Preparation of homochiral examples can be carried out by techniques known to those skilled in the art. For example, homochiral compounds can be prepared by separating racemic products or diastereomers by chiral-phase preparative HPLC. Alternatively, compounds can be prepared by known methods that provide enantiomerically enriched or diastereomerically enriched products.

[0156] The reactions and techniques described in this section are carried out in solvents appropriate to the reagents and materials used and are suitable for the transformations being effected. It is also understood that in describing the synthetic methods set forth below, all proposed reaction conditions (including solvent selection, reaction atmosphere, reaction temperature, experimental time, and work-up method) have been selected to be standard conditions for the reactions, and should be readily recognized by those skilled in the art. Those skilled in the art of organic synthesis will understand that functional groups present on various portions of the molecule must be compatible with the proposed reagents and reactions. Such limitations on substituents compatible with the reaction conditions will be readily apparent to those skilled in the art, and alternatives may be required if the present substituents are not suitable. The reactions may require the determination to alter the order of synthetic steps or to select a different course of action for certain reactions to obtain the desired compound. It is also recognized that another important consideration in planning any synthetic route in this field is the judicious selection of protecting groups used to protect reactive functional groups present in the desired compounds described herein. For the experienced experimenter, an authoritative reference describing many protecting group alternatives is Greene's Protective Groups in Organic Synthesis by Wuts and Greene (Fourth Edition, Wiley & Sons, 2007).

[0157] (Example) The following examples illustrate specific and preferred embodiments of the present disclosure and are not intended to limit the scope of the present disclosure. Chemical and scientific abbreviations and symbols have their common and accustomed meanings unless otherwise specified. Additional abbreviations used in the examples and elsewhere in this specification are defined herein. Common intermediates are generally useful in the preparation of one or more examples and are sequentially named (e.g., Intermediate 1, Intermediate 2, etc.) and abbreviated (e.g., Int. 1 or I1, Int. 2 or I2, etc.). In some cases, alternative methods of preparation of intermediates or examples are described. Chemists skilled in the synthetic arts will frequently devise desirable alternative preparation methods based on one or more considerations (e.g., shorter reaction times, less expensive starting materials, ease of manipulation and purification, higher yields, ease of catalysis, avoidance of toxic reagents, availability of specialized equipment, and reduced number of steps). The intent of describing alternative preparation methods is to facilitate further preparation of the examples of the present disclosure. In some cases, some functional groups in the outlined examples and claims may be substituted with bioisosteric substitutions known to those skilled in the art (e.g., replacing a carboxylic acid group with a tetrazole or phosphate moiety). 1 H NMR data used water suppression in data processing. The spectra shown are not corrected for the effect of water suppression. Protons adjacent to the water suppression frequency at 3.35 ppm are shown with reduced signal intensity.

[0158] TIFF2026041710000031.tif238164

[0159] Example 1: DGKi activates nivolumab and ipilimumab in an alloreactive MLR assay This example demonstrates that DGK inhibition enhances the activity of PD-1 and CTLA-4 inhibitors, as demonstrated by increased secretion of interferon-γ (IFN-γ) in an MLR assay.

[0160] The assay was performed as follows. Peripheral blood mononuclear cells were isolated from EDTA-treated whole blood by cell separation (Ficoll). T cells were further isolated from the cells using the Stemcell EasySep Human T Cell Isolation Kit (Stemcell 19051). Pre-purchased frozen monocytes were thawed and differentiated into dendritic cells (DCs) by treatment with GMCSF and IL-4 for 6 days in a 37°C CO2 incubator. T cells were seeded at 100,000 cells / well in 10% FBS RPMI medium in a 96-well round-bottom plate. Allogeneic DCs were added to appropriate wells at a T cell:immature DC ratio of 10:1. The DGK inhibitor, DGKi Compound 15, was diluted in DMSO and then in 10% FBS RPMI medium and added to appropriate wells containing T cells:immature DCs at a final DMSO concentration of 0.1% in 250 μL (final volume). The mixed lymphocyte reaction was allowed to proceed in an incubator for 5 days. On day 5, 130 μL of medium was removed and 10 μL was used for an IFN-γ ELISA assay (BD cat 555142).

[0161] The results presented in Figure 1A and B show that T cells treated with PD-1 or CTLA-4 inhibitors secrete increased amounts of IFN-γ due to inhibition of DGK.

[0162] Example 2: Inhibition of DGK activates the combination of PD-1 and CTLA4 antagonists in a B16 animal tumor model This example demonstrates that administering a DGKi simultaneously with a PD-1 antagonist and a CTLA4 antagonist leads to tumor regression compared to the combination of a PD-1 antagonist and a CTLA4 antagonist. This assay was performed in the B16 tumor model (a human melanoma tumor model). Mice were administered anti-PD-1 antibody (mIgG1-D265A monoclonal antibody directed against murine PD-1), anti-CTLA4 antibody (mIgG2b monoclonal antibody directed against murine CTLA4), vehicle alone, and / or DGKi, and tumor growth was measured. The results, shown in Figure 2A-G, demonstrated that while no significant tumor regression was observed with either the individual agents or the combination of the two, the combination of anti-PD-1 antibody and anti-CTLA4 antibody with DGKi resulted in tumor regression (Figure 2G).

[0163] Example 3: Inhibition of DGK enhances PD-1 and / or CTLA-4 inhibitory activity in the CT26 animal tumor model This example demonstrates that administration of a DGK inhibitor enhances tumor regression induced by anti-PD-1 and / or anti-CTLA4 antibodies in the CT26 model. The assay was performed as follows. CT26 cells (a murine colon cancer cell line (ATCC)) were cultured in RPMI 1640 medium (Gibco / ThermoFisher Scientific) containing 10% fetal bovine serum (Invitrogen / ThermoFisher Scientific). Six- to eight-week-old female BALB / c mice were purchased from Envigo. For tumor implantation (day 0), a suspension of CT26 cells (1 x 10 7 The mice were subcutaneously injected (0.1 mL) of 1000 cells / mL into the right flank of the mice. 3) (usually about 10 days after implantation), mice were randomly selected and assigned to various control and treatment groups, and treatment was initiated. DGKi compound 16 was formulated in 90% PEG400, 5% ethanol, and 5% TPGS and administered orally at a volume of 10 mL / kg body weight. Anti-CTLA4 (anti-mCTLA4, mIgG2b), anti-PD1 (mIgG1-D265A monoclonal antibody against murine PD-1), and isotype controls were diluted in DPBS to a dose of 10 mg / kg. Antibody treatments were administered by intraperitoneal injection (IP) every 4 days for a total of three doses (Q4Dx3). Tumor volume was measured at 0 mm , indicating complete tumor regression (0 mm ). 3 ) or 1000mm 3 Blood was measured twice weekly with a digital caliper until the blood reached 100 μL and was euthanized. For AH1 tetramer staining, 100 μL of blood was collected from each mouse and added to a lithium heparin tube. Blood was stained with AH1 tetramer (MBL), anti-Cd3, anti-Cd4, and anti-Cd8 (Biolegend). Samples were lysed using Lyse / Fix buffer (BD), collected from a CantoX hemocytometer (BD), and analyzed using FlowJo (BD).

[0164] The results presented in Figure 3A–H show that DGKi enhances the tumor volume reduction effects of (i) PD1 inhibitors; (ii) CTLA-4 inhibitors; and (iii) PD1 and CTLA-4 inhibitors in the CT26 mouse model. The results presented in Figure 3I show that DGKi increases the percentage of CD8 cells positive for the AH1+ tetramer tumor antigen. Therefore, this combination treatment resulted in an increased complete remission rate in the CT26 model, correlating with the increase in AH1+ T cells. The combination of both CTLA4 and PD1 antagonists with a DGK inhibitor resulted in the highest number of complete remissions, with 10 in 10 patients achieving complete remission.

[0165] Example 4: Inhibition of DGK lowers the antigen threshold required for TCR activation This example demonstrates that inhibition of DGK (1) enhances T cell responses even with low affinity tumor antigens and (2) reduces the concentration of tumor antigen required for T cell activation. The assay was performed as follows: MC38 cells (murine colon adenocarcinoma cells) were cultured in RPMI 1640 medium (Gibco / ThermoFisher Scientific) containing 10% fetal bovine serum (Invitrogen / ThermoFisher Scientific). OVA and mutant peptide variants were purchased from AnaSpec and resuspended according to the manufacturer's instructions. MC38 cells were pulsed with peptide (1 μg / mL or the indicated concentration) for 3 hours, and then free peptide was washed away. OT1 mice (mice obtained by crossbreeding with C57BL / 6 mice and genetically engineered to have TCRs specific for ovalbumin (OVA (SIINFEKL) or the following OVA peptide derivatives: A2 (SAINFEKL), Q4 (SIIQFEKL), T4 (SIITFEKL), and Q4H7 (SIIQFEHL)), but not for recombinant peptides (FILKSINE)) were purchased from the Jackson Laboratory. The binding affinities of these peptides to the TCR are shown in the table below. CD8 T cells were purified from total splenocytes (StemCell) of OT1 mice, activated with CD3 / CD28 beads (Invitrogen), and then frozen. Frozen activated OT-1 CD8 T cells were thawed during peptide pulsing and plated with DGKi Compound 15, control compound, or DMSO for 1 hour. Protein-pulsed MC38 cells were added to the plate and cocultured overnight at 37°C. The supernatant was collected and IL-2 was measured using AlphaLISA (PerkinElmer). [Table 1] The results presented in Figures 4A-F demonstrate that DGKi compound 15 reduces the affinity and antigen concentration required for antigen recognition and activation by T cells.

[0166] Example 5: Inhibition of DGK activates human CTL effector function and enhances tumor cell killing This example demonstrates that inhibition of DGK enhances CTL effector function and tumor cell killing. The assay was performed as follows. HCT116-GFP (human colon cancer) cells were purchased from Cellomics. HCT116-GFP were pulsed with the indicated concentrations of A2 and B35 peptides (Astarte) for 1 hour, followed by washing. Cells were seeded and fixed overnight. CMV-specific human CD8 T cells (Astarte) were thawed and treated with DGKi compound 15 for 1 hour, then added to HCT116-GFP cells. After 24 hours of co-culture, supernatants were collected and IFNγ was measured using AlphaLISA (PerkinElmer). GFP images were captured under a fluorescence microscope. The results presented in Figures 5A and B demonstrate that DGKi compound 15 activates human CTL effector function and enhances tumor cell killing.

[0167] Example 6: Inhibition of DGK can restore the reduced T cell effector function caused by reduced B2M levels Many human tumors develop mutations that result in partial or complete loss of MHC class I, which is crucial for T cells to recognize and attack tumor cells. This example demonstrates that inhibition of DGK enables T cells to recognize tumor cells with low amounts of MHC. Without DGK inhibition, these target cells are not recognized by T cells. The assay was performed as follows: HCT116-GFP cells were purchased from Cellomics and cultured in RPMI 1640 medium (Gibco / ThermoFisher Scientific) containing 10% fetal bovine serum (Invitrogen / ThermoFisher Scientific). B2M guide RNA (Synthego) was introduced into HCT116-GFP cells by Nucleofection (Lonza). After recovery, cells were seeded into individual wells to generate single-cell clones. Clonal cells were stained with B2M (Biolegend) and evaluated by flow cytometry. Clonal cells were then pulsed with 1 mg / mL A2 or B35 peptide (Astarte) for 1 hour, followed by washing. Cells were seeded and fixed overnight. CMV-specific human CD8 T cells (Astarte) were thawed, treated with DGKi compound 15 for 1 hour, and then added to HCT116 cells. After 24 hours of co-culture, the supernatant was collected and IFN-γ was measured using AlphaLISA (PerkinElmer). The results presented in Figures 6A and B show that DGKi compound 15 increases the amount of IFN-γ from T cells that recognize tumor cells with reduced MHC class I antigens.

[0168] Example 7: Tumor treatment activity of DGK inhibitors and PD1 antagonists inhibits CD8 + T cells play a role This example demonstrates the tumor therapeutic activity of CD8 + This indicates that T cells play a role. The assay was performed as follows. CT26 cells (ATCC) were cultured in RPMI 1640 medium (Gibco / ThermoFisher Scientific) containing 10% fetal bovine serum (Invitrogen / ThermoFisher Scientific). Six- to eight-week-old female BALB / c mice were purchased from Envigo. For tumor implantation (day 0), a CT26 cell suspension (1 x 10 7Mice were subcutaneously injected (0.1 mL) with 100 μg of CD8-depleting antibody (2.43, Bio X Cell) diluted in PBS at 100 μg per mouse. Administration began on day 1 and continued every 3–4 days until the end of the experiment. When tumors reached a predetermined volume (∼100 mm 3 ) (generally about 10 days after implantation), mice were randomly selected and divided into various control and treatment groups, and treatment was initiated. DGKi compound 16 was formulated in 90% PEG400, 5% ethanol, and 5% TPGS and administered orally at 10 mL / kg body weight. Five doses (Q3Dx5) were administered every three days at a total dose of 5 mg / kg. Anti-PD1 antibody (mIgG1-D265A monoclonal antibody against mouse PD-1) and isotype control were diluted in DPBS to a dose of 10 mg / kg. Antibody treatments were administered by intraperitoneal injection (IP) every four days at a total dose of three doses (Q4Dx3). Tumor volume was measured at a level of 0 mm2, indicating complete tumor regression (0 mm2). 3 ) or 1000mm 3 The animals were measured twice weekly with a digital caliper until they reached a normal pH level and were euthanized. The results presented in Figure 7 show that CT26 mice treated with anti-PD-1 antagonist and DGKi Compound 16 showed increased CD8 + The reduction in cells indicates a reduction in tumor volume.

[0169] Example 8: Tumor volume reduction by DGK inhibition and PD1 antagonist is enhanced by CD4 cell depletion This example demonstrates that the combination of a DGK inhibitor and a PD-1 antagonist shrinks tumors, and that depletion of CD4 cells further shrinks tumors. The assay was performed as follows. CT26 cells (ATCC) were cultured in RPMI 1640 medium (Gibco / ThermoFisher Scientific) containing 10% fetal bovine serum (Invitrogen / ThermoFisher Scientific). Six- to eight-week-old female BALB / c mice were purchased from Envigo. For tumor implantation (day 0), a CT26 cell suspension (1 x 10 7Mice were subcutaneously injected (0.1 mL) with 100 μg of CD4-depleting antibody (GK1.5, Bio X Cell) diluted in PBS at 100 μg per mouse. Administration began on day 1 and continued every 3–4 days until the end of the experiment. When tumors reached a predetermined volume (∼100 mm 3 At the time of engraftment (generally approximately 10 days after implantation), mice were randomly selected and assigned to various control and treatment groups, and treatment was initiated. DGKi compound 16 was formulated in 90% PEG400, 5% ethanol, and 5% TPGS and administered orally at 10 mL / kg body weight. Five doses (Q3Dx5) were administered every three days at a total dose of 5 mg / kg. Anti-PD1 (mIgG1-D265A monoclonal antibody against mouse PD-1) and isotype control (MOPC-21, Bio X Cell) were diluted in DPBS to a dose of 10 mg / kg. Antibody treatments were administered by intraperitoneal injection (IP) every four days at a total dose of three doses (Q4Dx3). Tumor volume was assessed as the tumor completely regressed (0 mm). 3 ) or 1000mm 3 The animals were measured twice weekly with a digital caliper until they reached a normal pH level and were euthanized. The results presented in Figure 8 show that MC38 mice treated with anti-PD-1 antagonist and DGKi compound 16 showed a significant reduction in CD4 T cells, possibly due to a reduction in Treg cells. + Cell depletion further reduced tumor volume.

[0170] Example 9: NK cells are required for the efficacy of DGKi and anti-PD1 anti-tumor This example demonstrates that NK cells influence the tumor-reducing activity of DGKi and PD1 antagonists in the CT26 animal model. The assay was essentially performed as described in Examples 6 and 7, except that instead of antibodies binding to CD4 or CD8, anti-asialoGM1 (Life Technologies) was administered at 50 μg per mouse starting on day 4 after tumor implantation and continued every 7 days until the end of the experiment. The results presented in Figure 9 demonstrate that NK cells contribute to the antitumor activity of DGKi compound 16 in combination with a PD1 inhibitor in the CT26 mouse model.

[0171] Example 10: Combination of a DGKi of Formula II with either anti-PD-1 or anti-CTLA4 shows robust efficacy This example demonstrates that the combination of an example DGKi of formula II selected from the group consisting of compounds 17-34 with an anti-PD-1 antibody or an anti-CTLA4 antibody has potent anti-tumor activity in an MC38 animal model. The assay was performed as follows. The murine colon adenocarcinoma tumor cell line MC38 was cultured in a T75 flask in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) containing 10% fetal bovine serum (FBS, Invitrogen). Cells were cultured until subconfluent and passaged twice weekly by simply rinsing with DPBS (Dulbecco's phosphate-buffered saline, Gibco) and letting the cells sit for a few minutes before dislodging them from the flask. MC38 cells were passaged at densities ranging from 1:16 to 1:20, depending on the timing and cell density. For in vivo transplantation, cells were rinsed with DPBS and then collected in ice-cold HBSS (Hank's balanced salt solution, Gibco) in a conical tube (50 mL) on ice. The tube was centrifuged at 1300 rpm for 10 minutes, the supernatant carefully removed, and the pellet washed with HBSS and centrifuged again. The pellet was suspended in HBSS, approximately the transplant volume. The cell concentration was measured using Moxi-Z (Orflo) and the final concentration was adjusted with HBSS. Cell viability was measured by trypan blue exclusion using Countess II (Life Technologies). Six- to eight-week-old female C57Bl / 6 mice were purchased from Charles River Laboratories (Kingston, NY) and allowed to acclimate for 3 to 7 days. At the time of tumor implantation (day 0), MC38 cells (8.5 x 10 cells) were instilled into a tuberculin syringe (1 mL) fitted with a 25-gauge needle. 6Mice were subcutaneously injected (0.1 mL) with 1000 cells / mL of 10000 cells and implanted into both the left and right flanks. On day 6 (post-implantation), tumors had grown to a predetermined volume (~78 mm). 3 The animals were divided into various treatment and control groups (n=10 per group) based on the mean tumor volume at that time point. Treatment began on day 7 (post-implantation), at which point the tumors had grown to ~100 mm 3 The DGKi of Formula II selected from the group consisting of compounds 17-34 was formulated in 90% PEG400, 5% ethanol, and 5% TPGS and administered orally at a volume of 10 mL / kg body weight. A total of 28 doses (QDx28) were administered daily at 0.3 mg / kg. Anti-PD-1 (mIgG1-D265A monoclonal antibody against mouse PD-1), anti-CTLA4 (mIgG2b monoclonal antibody against mouse CTLA4), and the corresponding isotype controls (InVivoPlus mouse IgG1, clone MOPC-21 and InVivoMab mouse IgG2b, clone MPC-11 (respective anti-PD-1 and anti-CTLA4 isotype controls were purchased from Bio X Cell, West Lebanon, NH)) were diluted in DPBS to a dose of 10 mg / kg. The antibody treatments were administered by intraperitoneal injection (IP) every four days for a total of three doses (Q4Dx3). The tumor volume was determined as follows: complete regression of the tumor (0 mm 3 ) or 1000mm 3 The animals were measured twice weekly with a digital caliper until they reached a normal pH level and were euthanized. The results shown in Figure 10 indicate that while DGKi alone did not show significant activity (Figures 10B-D), the combination of DGKi with anti-PD-1 antibody or anti-CTLA4 antibody showed potent antitumor activity (Figures 10E and F, respectively).

[0172] Example 11: In both MC38 and CT26 animal models, the combination of the compound of Formula II with an anti-PD-1 antibody exhibits potent anti-tumor activity and sustains immunological memory. This example demonstrates that in both MC38 and CT26 animal models, the combination of an example DGKi of formula II selected from the group consisting of compounds 17-34 with anti-PD-1 has potent anti-tumor activity, can induce complete regression, and maintains immunological memory. The studies were performed as follows: MC38 animal model studies were performed as described in Example 10. CT26 animal model studies were performed as described in Example 3. DGKi and anti-PD-1 were prepared (as in Example 10) and administered as described in Example 10. Animals cured by this treatment regimen showed no change in tumor volume after 10 tumor volume doubling times (TVDT, 10 x 4.2 days = 42 days). These animals were implanted subcutaneously in the right flank with cells at 10 times the initial cell concentration, and secondary T cell responses were assessed by measuring twice a week for at least 42 days. The results shown in Figure 11A-H demonstrate that the combination of the DGKi of Formula II with an anti-PD-1 antibody leads to potent antitumor activity in animal models. Furthermore, reimplantation with tumor cells in the MC38 and CT26 models resulted in 100% rejection of the transplanted cells (Figures 11D and H).

[0173] Example 12: In the B16F10 animal model, the combination of the compound of Formula II with anti-PD-1 and anti-CTLA4 shows more potent activity compared to the combination of the compound of Formula II with anti-PD-1 or anti-CTLA4. This example shows the B16F10 (melanoma / MHCI lo ) In animal models, triple combinations of an example DGKi of formula II selected from the group consisting of compounds 17 to 34, an anti-PD-1 antibody, and an anti-CTLA4 antibody produce anti-tumor activity that is more potent than the combination of two of them. Animal model studies were performed as follows. The mouse melanoma cell line B16F10 was cultured in T75 flasks in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Invitrogen). Cells were cultured until subconfluent and passaged twice weekly by simply rinsing the flask with DPBS (Dulbecco's phosphate-buffered saline, Gibco) followed by trypsin (0.25% trypsin, Gibco) and allowing the cells to stand for a few minutes before being released from the flask. Depending on the timing and cell density, B16F10 cells were passaged at a density ranging from 1:18 to 1:20. For in vivo transplantation, cells were trypsinized as described above and then collected in ice-cold HBSS (Hank's balanced salt solution, Gibco) in a conical tube (50 mL) on ice. The tubes were centrifuged at 1300 rpm for 10 minutes, the supernatant carefully removed, and the pellet washed with HBSS and centrifuged again. The pellet was resuspended in an approximate transplant volume of HBSS. Cell concentration was measured using a Moxi-Z (Orflo) and the final concentration was adjusted with HBSS. Cell viability was measured by trypan blue exclusion using a Countess II (Life Technologies). Six- to eight-week-old female C57Bl / 6 mice were purchased from Charles River Laboratories (Raleigh, NC) and allowed to acclimate for 3 to 7 days. At the time of tumor implantation (day 0), B16F10 cells (1 x 10 cells) were instilled into the tumor cells using a tuberculin syringe (1 mL) fitted with a 25-gauge needle. 7 Mice were subcutaneously injected (0.1 mL) with 1000 cells / mL and implanted in the right flank. Tumors were grown to a predetermined volume (~50 mm). 3Treatment was initiated on day 8 (post-implantation), when tumors had grown to 10 mm. Animals were divided into various treatment and control groups (n=10 per group) based on the mean tumor volume at that time point. DGKi of formula II selected from the group consisting of compounds 17-34 were formulated in 90% PEG400, 5% ethanol, and 5% TPGS and administered orally at a volume of 10 mL / kg body weight. A total of 28 doses (QDx28) were administered daily at 0.3 mg / kg. Anti-PD-1, anti-CTLA4, and the corresponding isotype control were diluted in DPBS to a dose of 10 mg / kg (as in Example 10). Antibody therapeutics were administered by intraperitoneal injection (IP) at a total of three doses (Q4Dx3) every four days. Tumor volume was determined as the point at which the tumor completely regressed (0 mm). 3 ) or 1000mm 3 The animals were measured twice weekly with a digital caliper until they reached a normal pH level and were euthanized. The results presented in Figures 12A-F demonstrate improved responses with triple combination treatment compared to the binary combination in the B16F10 animal model.

[0174] Example 13: Synthesis of DGK inhibitors

[0175] DGKi compound 1 4-((2R,5S)-4-(bis(4-fluorophenyl)methyl)-2,5-dimethylpiperazin-1-yl)-6-bromo-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-3-carbonitrile [ka]

[0176] DGKi compound 2 1-(bis(4-fluorophenyl)methyl)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yl)piperazine-2-carboxylate methyl ester [ka]

[0177] DGKi compound 3 (R)-4-(4-(bis(4-fluorophenyl)methyl)-3-methylpiperazin-1-yl)-6-bromo-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-3-carbonitrile [ka]

[0178] DGKi compound 4 (R)-8-(4-(bis(4-fluorophenyl)methyl)-3-methylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2,7-dicarbonitrile [ka]

[0179] DGKi compound 5 8-[(2S,5R)-4-[(4-fluorophenyl)(phenyl)methyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka]

[0180] DGKi compounds 6 and 7 8-[(2S,5R)-4-[(4-fluorophenyl)(phenyl)methyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka]

[0181] DGKi compound 8 4-[(2S,5R)-4-[(4-chlorophenyl)(4-fluorophenyl)methyl]-2,5-dimethylpiperazin-1-yl]-6-methoxy-1-methyl-1,2-dihydro-1,5-naphthyridin-2-one [ka]

[0182] DGKi compound 9 8-[(2S,5R)-4-{[2-(difluoromethyl)-4-fluorophenyl]methyl}-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka]

[0183] DGKi compound 10 8-[(2S,5R)-4-[(4-fluorophenyl)(4-methylphenyl)methyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka]

[0184] DGKi compound 11 8-[(2S,5R)-4-[1-(2,6-difluorophenyl)ethyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka]

[0185] DGKi compounds 12~14 8-((2S,5R)-4-(1-(2,4-difluorophenyl)propyl)-2,5-dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka]

[0186] Intermediate 1 6-cyano-3-(N-methylacetamido)picolinic acid ethyl ester [ka] To a stirred, pale yellow solution of ethyl 3-(N-methylacetamido)-1-(11-oxidanyl)-114-pyridine-2-carboxylate (50 g, 210 mmol) in DCM (500 mL) was added trimethylsilyl cyanide (39.4 mL, 294 mmol) at room temperature. The mixture was stirred for 10 minutes and cooled to -10 °C. Benzoyl chloride (34.1 mL, 294 mmol) was then added via a 50 mL addition funnel over 15 minutes, followed by the slow addition of TEA (41.0 mL, 294 mmol) via a 50 mL addition funnel over 20 minutes. An exothermic reaction was observed during the addition of TEA. After stirring at the same temperature for 2.5 hours, the mixture (TEA salt) became opaque. The reaction was quenched with 10% NaHCO3 solution (500 mL) and extracted with DCM (3 x 300 mL). The combined organic solutions were washed with brine (2x250 mL), then dried over Na2SO4 and concentrated to give a pale yellow crude product. The crude material was purified on an ISCO® normal phase RediSep silica column (eluent: EA / petroleum ether). The product was isolated (65-70% EA / petroleum ether) and the fractions were concentrated to give ethyl 6-cyano-3-(N-methylacetamido)picolinate as a pale brown liquid (43 g, 83% yield). LCMS: m / z = 248.0 (M+H); rt 1.255 min; LC-MS method: Column - KINETEX-XB-C18 (75 x 3 mm; 2.6 μm); Mobile phase A: 10 mM ammonium formate in water:acetonitrile (98:2); Mobile phase B: 10 mM ammonium formate in water:acetonitrile (2:98); Gradient: 20 to 100% B over 4 min (flow rate 1.0 mL / min), followed by 100% B for 0.6 min (flow rate 1.5 mL / min). Then, gradient elution from 100 to 20% B over 0.1 min (flow rate 1.5 mL / min).

[0187] Intermediate 2 8-Hydroxy-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile To a stirred solution of ethyl 6-cyano-3-(N-methylacetamido)picolinate (0.9 g, 3.64 mmol) in tetrahydrofuran (10 mL) was added KHMDS (4.80 mL, 4.37 mmol) at -78 °C over 10 min. The reaction mixture was stirred for 15 min, slowly warmed to room temperature over 30 min, and then stirred for an additional 90 min. The reaction mixture was cooled to 0 °C, and the reaction was quenched with saturated sodium bicarbonate solution (70 mL). The mixture was diluted with ethyl acetate (2 x 100 mL). The aqueous layer was collected and acidified with 1.5 N HCl to adjust the pH to ∼3.0. The mixture was stirred for 15 min, and a solid formed. This was filtered through a Buchner funnel to give 8-hydroxy-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (550 mg, 75% yield) as a brown solid. LCMS: m / z = 202.0 (M+H); rt 0.361 min; LC-MS method: Column-KINETEX-XB-C18 (75 x 3 mm; 2.6 μm); Mobile phase A: 10 mM aqueous ammonium formate:acetonitrile (98:2); Mobile phase B: 10 mM aqueous ammonium formate:acetonitrile (2:98); Gradient: 20–100% B over 4 min, flow rate 1.0 mL / min, followed by 100% B for 0.6 min (flow rate 1.5 mL / min). Then, the solution was eluted with a gradient of 100 to 20% B over 0.1 min (flow rate: 1.5 mL / min).

[0188] Intermediate 3 8-chloro-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] To a stirred solution of 8-hydroxy-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (0.55 g, 2.73 mmol) in acetonitrile (10 mL) was added POCl (1.53 mL, 16.4 mmol). The mixture was heated to 85 °C over 5 minutes and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure to give the crude product. The mixture was cooled to 0 °C, and the reaction was quenched with saturated sodium bicarbonate solution (50 mL). The reaction was diluted with DCM (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 8-chloro-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (0.25 g, 29.1% yield) as a brown solid. LCMS: m / z = 220.2 (M+H); rt 1.528 min; LC-MS method: Column - KINETEX-XB-C18 (75 x 3 mm; 2.6 μm); Mobile phase A: 10 mM ammonium formate in water:acetonitrile (98:2); Mobile phase B: 10 mM ammonium formate in water:acetonitrile (2:98); Gradient: 20 to 100% B over 4 min (flow rate 1.0 mL / min), followed by 100% B for 0.6 min (flow rate 1.5 mL / min). Then, gradient elution from 100 to 20% B over 0.1 min (flow rate 1.5 mL / min).

[0189] Intermediate 4 Stereochemistry: A (Cyanomethyl)trimethylphosphonium iodide [ka] (Cyanomethyl)trimethylphosphonium iodide was prepared according to the general method described in Zaragoza, F., et al., J. Org. Chem. 2001, 66, 2518-2521. In a round-bottom flask (1 L), trimethylphosphine / toluene (100 mL, 100 mmol) was diluted with THF (50.0 mL) and toluene (50.0 mL) and cooled in an ice bath. While vigorously stirring, iodoacetonitrile (7 mL, 16.7 g, 68.3 mmol) was added dropwise, resulting in a tan precipitate. The cooling bath was removed, and the reaction mixture was stirred at room temperature overnight. The flask was placed in a sonicator to break up any clumped solids. The reaction mixture was stirred for an additional 4 hours, and the solid was collected by filtration and dried under vacuum to give (cyanomethyl)trimethylphosphonium iodide (16.6 g, 68.3 mmol, 68.3% yield). 1 H NMR (400MHz, DMSO-d6) δ 4.03 (d, J=16.4Hz, 2H), 2.05 (d, J=15.4Hz, 9H)

[0190] Intermediate 5 Stereochemistry: Homochiral 8-((2S,5R)-2,5-Dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile·TFA [ka] To a solution of 6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yltrifluoromethanesulfonic acid (65 g, 195 mmol) and tert-butyl (2R,5S)-2,5-dimethylpiperazine-1-carboxylate (43.9 g, 205 mmol) in acetonitrile (1.3 L) was added DIPEA (0.102 L, 585 mmol). The solution was stirred at 80 °C for 6 h, the solvent was removed, and the crude residue was purified by silica gel chromatography (product Rf: 0.4 in 100% ethyl acetate). The product, tert-butyl (2R,5S)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yl)-2,5-dimethylpiperazine-1-carboxylate (75 g, 189 mmol, 97% yield), was obtained. LCMS: m / z = 398.2 (M + H); rt 2.7 min; Method: Column - Kinetex XB-C18 (75 x 3 mm; 2.6 μm), flow rate 1 mL / min; Gradient: 20% B to 100% B in 4 min; Detection wavelength: 254 nm (Solvent A: 98% water: 2% acetonitrile (10 mM ammonium formate); Solvent B: 2% water: 98% acetonitrile (10 mM ammonium formate)). To a solution of tert-butyl (2R,5S)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yl)-2,5-dimethylpiperazine-1-carboxylate (30 g, 75 mmol) in ethyl acetate (1000 mL) was added HCl (4 M dioxane solution, 189 mL, 755 mmol) at 0 °C and stirred for 6 h while allowing the temperature to return to room temperature. LC / MS analysis showed ~90% product mass at 0.60 RT along with ~4% mass of amide by-product (consistent with nitrile hydrolysis) at 0.44 RT. The reaction mixture was diluted with methyl tert-butyl ether (MTBE, 2000 mL), stirred for 15 min, and the HCl salt of the product was filtered and washed with MTBE (100 mL). The HCl salt was dissolved in water (300 mL) and the pH was adjusted to ~8 with 10% aqueous sodium bicarbonate. The organic layer was extracted with DCM (5x250 mL) and the combined organic layers were washed with water (2x300 mL), dried over sodium sulfate and concentrated to give 8-((2S,5R)-2,5-dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (20 g, 65.2 mmol, 86% yield). LCMS: m / z=298.2 (M+H); rt 0.5 min; Method: Column-Kinetex XB-C18 (75x3 mm; 2.6 μm), flow rate 1 mL / min; gradient time 4 min; 20% to 100% B; Detection wavelength: 254 nm (Solvent A: 98% water: 2% acetonitrile (10 mM ammonium formate); Solvent B: 2% water: 98% acetonitrile (10 mM ammonium formate); 1H NMR(400MHz, CDCl3) δ 7.79 (d, J= 8.8Hz, 1H), 7.70 (d, J= 12, 3.2Hz, 1H), 6.29 (s, 1H), 3.80 (dd, J= 8.8Hz, 1H) 3.70 (m, 1H), 3.65 (s, 13C NMR (75MHz, Chloroform-d) δ 161.9, 155.0, 138.5, 137.0, 128.2, 125.0, 122.2, 117.2, 111.3, 56.5, 51.9, 50.0, 49.5, 29.0, 18.8, 15.4

[0191] Intermediate 6 8-chloro-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] A 2-dram vial containing 8-hydroxy-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (192 mg, 0.780 mmol) was added to a stir bar and acetonitrile (3.1 mL). DIEA (0.272 mL, 1.560 mmol) was then added to the suspension, and the mixture was stirred for 1-2 minutes until a homogeneous yellow solution was formed. To this reaction mixture, phosphoryl chloride (0.131 mL, 1.404 mmol) was added. Under a nitrogen atmosphere, a bubbler tube containing oil was attached, and the vial was capped. The mixture was stirred at room temperature for 1.5 hours, and then benzyltriethylammonium chloride (200 mg, 0.878 mmol) was added. Under a nitrogen atmosphere, the vial was capped and heated in an oil bath (65 °C) for 1 hour. The reaction mixture was cooled, and the volatiles were removed under reduced pressure using a rotary evaporator. The reaction residue was dissolved in ethyl acetate and poured into a beaker containing ice (~10 mL), then transferred to a separatory funnel. The aqueous phase was extracted with ethyl acetate. The combined organic extracts were washed sequentially with 1.5 M KHPO, saturated aqueous sodium bicarbonate, and brine. The organic extract was dried over magnesium sulfate, filtered, and the solvent removed under reduced pressure to yield a brownish crystalline solid (204 mg). LCMS: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, particle size: 1.7 μm; Mobile phase A: 100% water (containing 0.05% trifluoroacetic acid); Mobile phase B: 100% acetonitrile (containing 0.05% trifluoroacetic acid); Temperature: 40 °C; Gradient: 2 to 98% B for 1.5 min, followed by 98% B for 0.5 min; Flow rate: 0.8 mL / min; Detection: UV (220 nm); Retention time = 1.01 min; Observed adduct: [M+H]; Observed molecular weight: 265.0 (weakly ionized); 1 H NMR (chloroform-d) δ 8.03 (d, J=8.8Hz, 1H), 7.89-7.97 (m, 1H), 3.82 (s, 3H)

[0192] Intermediate 7 8-((2S,5R)-2,5-Dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile·TFA [ka] To a solution of 6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yltrifluoromethanesulfonic acid (65 g, 195 mmol) and tert-butyl (2R,5S)-2,5-dimethylpiperazine-1-carboxylate (43.9 g, 205 mmol) in acetonitrile (1.3 L) was added DIPEA (0.102 L, 585 mmol). The solution was stirred at 80 °C for 6 h, the solvent was removed, and the crude residue was chromatographed on silica gel (product Rf: 0.4; 100% ethyl acetate). The product, tert-butyl (2R,5S)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yl)-2,5-dimethylpiperazine-1-carboxylate (75 g, 189 mmol, 97% yield), was obtained. LCMS: m / z=398.2 (M+H); rt 2.7 min; Method: Column-Kinetex XB-C18 (75 x 3 mm; 2.6 μm), flow rate 1 mL / min; gradient time 4 min; 20% to 100% B; Detection wavelength: 254 nm (Solvent A: 98% water:2% acetonitrile (10 mM ammonium formate); Solvent B: 2% water:98% acetonitrile (10 mM ammonium formate) To a solution of tert-butyl (2R,5S)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yl)-2,5-dimethylpiperazine-1-carboxylate (30 g, 75 mmol) in ethyl acetate (1000 mL) was added HCl (4 M in dioxane, 189 mL, 755 mmol) at 0 °C and stirred for 6 h while the mixture was allowed to warm to room temperature. LC / MS analysis showed ∼90% mass of product at 0.60 RT along with ∼4% mass of amide by-product (consistent with nitrile hydrolysis) at 0.44 RT. The reaction mixture was diluted with methyl t-butyl ether (MTBE, 2000 mL), stirred for 15 min, and the HCl salt of the product was filtered and washed with MTBE (100 mL). The HCl salt was dissolved in water (300 mL) and the pH was adjusted to ∼8 with 10% aqueous sodium bicarbonate. The organic layer was extracted with DCM (5x250 mL) and the combined organic layers were washed with water (2x300 mL), dried over sodium sulfate and concentrated to give 8-((2S,5R)-2,5-dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (20 g, 65.2 mmol, 86% yield). LCMS: m / z=298.2 (M+H); rt 0.5 min; Method: Column-Kinetex XB-C18 (75x3 mm; 2.6 μm), flow rate 1 mL / min; gradient time 4 min; 20% to 100% B; Detection wavelength: 254 nm (Solvent A: 98% water:2% acetonitrile (10 mM ammonium formate); Solvent B: 2% water:98% acetonitrile (10 mM ammonium formate); 1 H NMR(400MHz, CDCl3) δ 7.79 (d, J= 8.8Hz, 1H), 7.70 (d, J= 12,3.2Hz, 1H), 6.29 (s, 1H), 3.80 (dd, J= 8.8Hz, 1H) 3.70 (m, 1H), 3.65 (s, 3H), 3.29 (m, 2H), 2.80 (m, 2H), 1.19 (d, J= 6Hz, 3H), 1.15 (d, J= 6Hz, 3H); 13C NMR (75 MHz, chloroform-d) δ 161.9, 155.0, 138.5, 137.0, 128.2, 125.0, 122.2, 117.2, 111.3, 56.5, 51.9, 50.0, 49.5, 29.0, 18.8, 15.4; stereochemistry: homochiral

[0193] Synthesis of DGKi compound 1 4-((2R,5S)-4-(bis(4-fluorophenyl)methyl)-2,5-dimethylpiperazin-1-yl)-6-bromo-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-3-carbonitrile [ka] To a stirred solution of 6-bromo-3-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yltrifluoromethanesulfonic acid (80 mg, 0.194 mmol) in acetonitrile (5 mL) was added DIPEA (0.102 mL, 0.582 mmol) and (2S,5R)-1-(bis(4-fluorophenyl)methyl)-2,5-dimethylpiperazine HCl salt (75 mg, 0.214 mmol). The mixture was stirred at 85 °C overnight. The solvent was removed under reduced pressure, and the resulting residue was dissolved in ethyl acetate (15 mL). The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (24 g flash column, elution solvent: 50-80% EtOAc / petroleum ether). The fractions were concentrated in vacuo to give 4-((2R,5S)-4-(bis(4-fluorophenyl)methyl)-2,5-dimethylpiperazin-1-yl)-6-bromo-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-3-carbonitrile (95 mg, 85% yield). LCMS: m / z=578.2 (M+H); rt 3.916 min

[0194] Synthesis of DGKi compound 2 1-(bis(4-fluorophenyl)methyl)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yl)piperazine-2-carboxylate methyl ester [ka] To a stirred solution of 8-chloro-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (22.90 mg, 0.104 mmol) in DMA (1 mL) and t-butanol (4 mL), methyl 1-(bis(4-fluorophenyl)methyl)piperazine-2-carboxylate (TFA salt) (40 mg, 0.087 mmol) and cesium carbonate (85 mg, 0.261 mmol) were added under a nitrogen atmosphere, followed by the addition of chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (3.37 mg, 4.34 μmol). The reaction vessel was immersed in a 70 °C oil bath and heated to 90 °C over 2 minutes. The mixture was stirred for 16 hours. The reaction mixture was filtered through Celite and concentrated under high vacuum to give a brown viscous material. The crude material was purified by preparative HPLC (conditions: column: Sunfire C18, 19 x 150 mm, particle size: 5 μm; mobile phase A: 10 mM ammonium acetate (adjusted to pH 4.5 with acetic acid); mobile phase B: acetonitrile; gradient: 30 to 100% B over 15 min, followed by 100% B for 5 min; flow rate: 17 mL / min). The product-containing fractions were combined and dried in a centrifugal evaporator to give methyl 1-(bis(4-fluorophenyl)methyl)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yl)piperazine-2-carboxylate (3.5 mg, 6.23 μmol, 7.17% yield). LCMS: m / z=530.2 (M+H); rt 2.20 min; LCMS method: Column-X Bridge BEH XP C18 (50x2.1 mm 2.5 μm; flow rate 1.1 mL / min; gradient time 3 min; temperature: 50 °C; solvent: 0% B to 100% B; detection wavelength: 220 nm (Solvent A: 95% water: 5% acetonitrile (containing 10 mM ammonium acetate); Solvent B: 5% water: 95% acetonitrile (containing 10 mM ammonium acetate)); 1H NMR (400MHz, DMSO-d6) δ ppm 8.16 (d, J=8.8Hz, 1H), 8.08 (d, J=9.0Hz, 1H), 7.57 (dd, J=8.8, 5.6Hz, 2H), 7.42-7.28 (m, 2H), 7.22-7.08 (m, 4H), 6.14 (s, 1H), 5.17 (s, 1H), 4.78 (d, J=12.2Hz, 1H), 3.64 (d, J=12.0Hz, 1H), 3.59 (s, 3H), 3.54 (s, 3H), 3.45-3.35 (m, 2H), 3.15(dd, J=12.5, 3.9Hz, 1H), 3.04 (td, J=11.7, 2.9Hz, 1H), 2.71-2.63 (m, 1H)

[0195] Synthesis of DGKi compound 3 (R)-4-(4-(bis(4-fluorophenyl)methyl)-3-methylpiperazin-1-yl)-6-bromo-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-3-carbonitrile [ka] To a stirred solution of 6-bromo-3-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridin-4-yltrifluoromethanesulfonic acid (100 mg, 0.243 mmol) in acetonitrile (8 mL) was added DIPEA (0.127 mL, 0.728 mmol) and (R)-1-(bis(4-fluorophenyl)methyl)-2-methylpiperazine HCl salt (82 mg, 0.243 mmol). The reaction was heated to 85° C. over 5 minutes, and the mixture was stirred for 1 hour. The reaction mixture was concentrated under high vacuum to give a brown gum. The resulting crude compound was purified by ISCO™ (12 g silica gel column; 60–67% ethyl acetate / petroleum ether) to give (R)-4-(4-(bis(4-fluorophenyl)methyl)-3-methylpiperazin-1-yl)-6-bromo-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-3-carbonitrile (90 mg, 42.7% yield) as a brown viscous material. LCMS: m / z=566.0 (M+2H); rt 2.23 min; LCMS method: Column: AQUITY UPLC BEH C18 (3.0x50mM) 1.7 μm; Mobile phase A: Buffer:acetonitrile (95:5); Mobile phase B: Buffer:acetonitrile (5:95), buffer: 10mM ammonium acetate; Gradient: 20-100% B over 2.0 min, followed by 100% B for 0.2 min; Flow rate 0.7 mL / min.

[0196] Synthesis of DGKi compound 4 (R)-8-(4-(bis(4-fluorophenyl)methyl)-3-methylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2,7-dicarbonitrile [ka] To a stirred solution of (R)-4-(4-(bis(4-fluorophenyl)methyl)-3-methylpiperazin-1-yl)-6-bromo-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-3-carbonitrile (90 mg, 0.159 mmol) in NMP (5 mL) under a nitrogen atmosphere, zinc (2.085 mg, 0.032 mmol) and zinc cyanide (37.4 mg, 0.319 mmol) were added. After purging with nitrogen for 3 minutes, dppf (5.30 mg, 9.57 μmol) and Pd(dba) (14.6 mg, 0.016 mmol) were added. The mixture was heated to 80° C. over 5 minutes and stirred for 4 hours. The reaction mixture was filtered through Celite and concentrated under high vacuum to give a brown gum. The crude material was purified by preparative HPLC (HPLC method: Column: SUNFIRE C18 (150 mm x 19 mm ID, 5 μm); Mobile phase A: 10 mM ammonium acetate in water; Mobile phase B: acetonitrile; Gradient: 40–60% B over 3.0 min (flow rate 17 mL / min), followed by 60–100% B over 17 min (flow rate 17 mL / min)). Product-containing fractions were combined and concentrated under high vacuum. The sample was then diluted (EtOH / HO, 1:3) and lyophilized overnight to give (R)-8-(4-(bis(4-fluorophenyl)methyl)-3-methylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2,7-dicarbonitrile (50 mg, 61.4% yield) as a pale yellow solid. LCMS: m / z=511.2 (M+H); rt 3.520 min; LCMS method: Column-KINETEX-XB-C18 (75x3 mm; 2.6 μ); Mobile phase A: 10 mM ammonium formate in water:acetonitrile (98:2); Mobile phase B: 10 mM ammonium formate in water:acetonitrile (2:98); Gradient: 20–100% B over 4 min (flow rate 1.0 mL / min), followed by 100% B for 0.6 min (flow rate 1.5 mL / min), followed by a gradient of 100–20% B over 0.1 min (flow rate 1.5 mL / min); 1H NMR (400MHz, DMSO-d6) δ ppm 8.26 (d, J=8.8Hz, 1H), 8.15 (d, J=9.0Hz, 1H), 7.56 (dd, J=11.9, 8.7Hz, 2H), 7.57 (dd, J=11.7, 8.8Hz, 2H), 7.16 (t, J=8.9Hz, 4H), 4.90 (s, 1H), 4.10 (d,J=13.0Hz, 1H), 4.01 (d, J=12.5Hz, 1H), 3.86 (dd, J=12.2, 2.9Hz, 1H), 3.66-3.55 (m, 1H), 3.53 (s, 3H), 3.08-2.97 (m, 1H), 2.97-2.90 (m, 1H), 2.90 (s, 1H), 1.03 (d, J=6.6Hz, 3H)

[0197] Synthesis of DGKi compound 5 8-[(2S,5R)-4-[(4-fluorophenyl)(phenyl)methyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] A mixture of 8-((2S,5R)-2,5-dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile·TFA (41.1 mg, 100 μmol), (4-fluorophenyl)(phenyl)methanol (28.3 mg, 140 μmol), and (cyanomethyl)trimethylphosphonium iodide (48.6 mg, 200 μmol) in acetonitrile (200 μL) was added to a 2-drum reaction vessel and sealed. Hunig's base (75 μL, 429 μmol) was added, and the mixture was heated at 110 °C for 2 h. The reaction mixture was injected directly onto a 12 g silica gel column and eluted with 20–100% ethyl acetate / hexane to give Example 182 as a diastereomeric mixture. Analytical LC / MS conditions: injection volume = 3 μL, initial %B: 0, final %B: 100, gradient time 2 min, flow rate 1 mL / min, wavelength 220 nm, solvent: acetonitrile / water / TFA, solvent A: 10% acetonitrile / 90% water / 0.05% TFA; solvent B: 10% water / 90% acetonitrile / 0.05% TFA, column: Acquity BEH C18 21x50 mm 1.7 μm, column oven temperature = 40 °C; LC / MS results: retention time 1.4 min; observed mass: 482.5 (M + ) The crude material was further purified by preparative LC / MS (conditions: column: XBridge C18, 200 mm x 19 mm, particle size: 5 μm; mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); gradient: 47% B at 0 min, 47–87% B over 20 min, followed by 100% B for 4 min; flow rate: 20 mL / min; column temperature: 25°C). Fractions were collected as determined by MS signal. The product-containing fractions were combined and dried using a centrifugal evaporator to give the title compound (14.4 mg, 30% yield). Theoretical molecular weight 481.575; LC / MS conditions: QC-ACN-TFA-XB: Observed MS ion: 482.2, retention time 1.6 minutes; 1H NMR (500MHz, DMSO-d6) δ 8.18-8.10 (m, 1H), 8.06 (d, J=8.8Hz, 1H), 7.68-7.48 (m, 4H), 7.39-7.26 (m, 2H), 7.25-7.08 (m, 3H), 6.00 (s, 1H), 4.67 (br s, 1H), 4.59 (br d, J=6.7Hz, 1H), 3.76-3.62 (m, 1H), 3.55 (br d, J=12.8Hz, 1H), 3.15-3.04 (m, 1H), 2.90-2.81 (m, 1H), 2.36 (br dd, J=17.4, 11.9Hz, 1H), 1.35-1.28 (m, 3H), 1.24 (s, 1H), 1.07 (br t, J=5.6Hz, 3H)

[0198] Synthetic methods for DGKi compounds 6 and 7 8-[(2S,5R)-4-[(4-fluorophenyl)(phenyl)methyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] Example 5 was separated into its diastereomers using chiral solid-phase chromatography (column: Chiralpak OJ-H, 21x250mm; 5μ, mobile phase: 90% CO2 / 10% methanol, flow rate conditions: 45mL / min, detection wavelength: 225nm, injection details: 500μL (15mg dissolved in 1mL methanol / acetonitrile)). The first eluting diastereomer, Example 6 (66.4 mg), was isolated in 20.2% yield. Analytical LC / MS was used to determine the final purity. Injection 1 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile Phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0% B to 100% B over 3 minutes, followed by 100% B for 0.50 minutes; Flow Rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 1 Results: Purity: 100.0%; Observed Mass: 482.1; Retention Time: 2.49 minutes; Injection 2 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 Acetonitrile:water (containing 0.1% trifluoroacetic acid); Mobile phase B: 95:5 acetonitrile:water (containing 0.1% trifluoroacetic acid); Temperature: 50°C; Gradient: 0% B to 100% B over 3 minutes, followed by 100% B for 0.50 minutes; Flow rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 2 results: Purity: 100.0%; Observed mass: 482.11; Retention time: 1.75 minutes The second eluting diastereomer, Example 7 (71.7 mg), was isolated in 21.9% yield. Analytical LC / MS was used to determine final purity. Injection 1 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile Phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0% B to 100% B over 3 minutes, followed by 100% B for 0.50 minutes; Flow Rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 1 Results: Purity: 100.0%; Observed Mass: 482.11; Retention Time: 2.51 minutes; Injection 2 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 Acetonitrile:water (containing 0.1% trifluoroacetic acid); Mobile phase B: 95:5 acetonitrile:water (containing 0.1% trifluoroacetic acid); Temperature: 50°C; Gradient: 0% B to 100% B over 3 minutes, followed by 100% B for 0.50 minutes; Flow rate: 1 mL / min; Detection: MS and UV (220 nm). Injection 2 Results: Purity: 100.0%; Observed mass: 482.1; Retention time: 1.76 minutes.

[0199] Synthesis of DGKi compound 8 4-[(2S,5R)-4-[(4-chlorophenyl)(4-fluorophenyl)methyl]-2,5-dimethylpiperazin-1-yl]-6-methoxy-1-methyl-1,2-dihydro-1,5-naphthyridin-2-one [ka] 4-((2S,5R)-2,5-Dimethylpiperazin-1-yl)-6-methoxy-1-methyl-1,5-naphthyridin-2(1H)-one (50 mg, 0.165 mmol) and 1-(bromo(4-chlorophenyl)methyl)-4-fluorobenzene (49.5 mg, 0.165 mmol) were combined with diisopropylethylamine (0.173 mL, 0.992 mmol) in acetonitrile (3 mL), and the reaction mixture was heated at 55° C. overnight. LC / MS showed the reaction was complete. The crude material was purified by preparative LC / MS (conditions: column: XBridge C18, 200 mm x 19 mm, particle size: 5 μm; mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); gradient: 42% B for 0 min, followed by 42–82% B over 25 min, then 100% B for 5 min; flow rate: 20 mL / min; column temperature: 25°C). Fractions were collected based on MS signal, and the product-containing fractions were combined and dried in a centrifugal evaporator. Theoretical molecular weight: 521.03; LC / MS conditions: QC-ACN-AA-XB: observed MS ion 521.1, retention time 2.77 min.

[0200] Synthesis of DGKi compound 9 8-[(2S,5R)-4-{[2-(difluoromethyl)-4-fluorophenyl]methyl}-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] To a solution of 8-((2S,5R)-2,5-dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (30 mg, 0.081 mmol) in DMF (2 mL) was added 2-(difluoromethyl)-4-fluorobenzaldehyde (16.86 mg, 0.097 mmol) and stirred at room temperature for 1 hour. Sodium cyanoborohydride (15.22 mg, 0.242 mmol) was added, and the mixture was stirred at room temperature overnight. LC / MS analysis indicated the reaction was complete. The crude material was purified by preparative LC / MS (conditions: column: XBridge C18, 200 mm x 19 mm, particle size: 5 μm; mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); gradient: 31% B for 0 min, 31–71% B for 25 min, then 100% B for 5 min; flow rate: 20 mL / min; column temperature: 25 °C). Fractions were collected as determined by MS and UV signals. The product-containing fractions were combined and dried in a centrifugal evaporator. The product yield was 13.0 mg, and the estimated purity by LC / MS analysis was 100%. Analytical LC / MS was used to determine the final purity.Injection 1 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 acetonitrile:water (containing 0.1% trifluoroacetic acid); Mobile Phase B: 95:5 acetonitrile:water (containing 0.1% trifluoroacetic acid); Temperature: 50 °C; Gradient: 0% B to 100% B over 3 min, followed by 100% B for 0.50 min; Flow Rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 1 Results: Purity: 100.0%; Observed Mass: 456.08; Retention Time: 1.39 min; Injection 2 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 Acetonitrile:water (containing 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0% B to 100% B over 3 minutes, followed by 100% B for 0.50 minutes; Flow rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 2 results: Purity: 100.0%; Observed mass: 456.07; Retention time: 2.22 minutes; 0% B to 100% B over 3 minutes, followed by 100% B for 0.50 minutes; Flow rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 2 results: Purity: 100.0%; Observed mass: 456.07; Retention time: 2.22 minutes.

[0201] Synthesis of DGKi compound 10 8-[(2S,5R)-4-[(4-fluorophenyl)(4-methylphenyl)methyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] To a mixture of 8-((2S,5R)-2,5-dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile·TFA (68.6 mg, 60 wt%, 0.1 mmol), (cyanomethyl)trimethylphosphonium iodide (48.6 mg, 0.200 mmol), and (4-fluorophenyl)(p-tolyl)methanol (26.0 mg, 0.120 mmol) in acetonitrile (0.3 mL) was added Hunig's base (0.105 mL, 0.600 mmol). The reaction mixture was stirred at 110 °C for 2 h, followed by the addition of (cyanomethyl)trimethylphosphonium iodide (48.6 mg, 0.200 mmol), (4-fluorophenyl)(p-tolyl)methanol (26.0 mg, 0.120 mmol), and Hunig's base (0.058 mL, 0.300 mmol). The reaction mixture was stirred at 110 °C for an additional 2 h. The resulting crude reaction mixture was injected directly onto a Si-RediSep Rf (12 g) and subjected to flash chromatography (20–100% ethyl acetate / hexane). The product-containing fractions were combined and dried under vacuum. The resulting material was further purified by preparative LC / MS (conditions: column: XBridge C18, 200 mm x 19 mm, particle size: 5 μm; mobile phase A: 5:95 acetonitrile:water (containing 0.1% trifluoroacetic acid); mobile phase B: 95:5 acetonitrile:water (containing 0.1% trifluoroacetic acid); gradient: 20% B for 0 min, followed by 20–60% B over 25 min, then 100% B for 5 min; flow rate: 20 mL / min; column temperature: 25°C). Fractions were collected as determined by MS and UV signals. The product-containing fractions were combined and dried using a centrifugal evaporator. The yield of the diastereomeric product as the TFA salt was 47.1 mg. The diastereomeric product was resolved into two diastereomers using SFC chiral chromatography (conditions: column: Chiral AD, 30x250mm, particle size: 5μ; mobile phase: 80% CO2 / 20% IPA (containing 0.1% DEA); flow rate: 100 mL / min; column temperature: 25°C). The title compound was recovered in the second eluting peak (>91% de, theoretical molecular weight 495.602). Analytical LC / MS was used to determine the final purity. Injection 1 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile Phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0% B to 100% B over 3 minutes, followed by 100% B for 0.50 minutes; Flow Rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 1 Results: Purity: 97.6%; Observed Mass: 496.26; Retention Time: 2.52 minutes; Injection 2 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 Acetonitrile:water (containing 0.1% trifluoroacetic acid); Mobile phase B: 95:5 acetonitrile:water (containing 0.1% trifluoroacetic acid); Temperature: 50°C; Gradient: 0% B to 100% B over 3 minutes, followed by 100% B for 0.50 minutes; Flow rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 2 results: Purity: 98.2%; Observed mass: 496.28; Retention time: 1.73 minutes

[0202] Synthesis of DGKi compound 11 8-[(2S,5R)-4-[1-(2,6-difluorophenyl)ethyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] To a mixture of 2-(1-bromoethyl)-1,3-difluorobenzene (15.12 mg, 0.065 mmol) and 5-methyl-6-oxo-8-(piperazin-1-yl)-5,6-dihydro-1,5-naphthyridine-2,7-dicarbonitrile·TFA (34.0 mg, 60% wt, 0.05 mmol) in acetonitrile (0.3 mL) was added Hunig's base (0.052 mL, 0.300 mmol) and stirred at 55 °C for 2 hours. LCMS showed complete conversion to the product. The crude material was purified by preparative LC / MS (conditions: column: XBridge C18, 200 mm x 19 mm, particle size: 5 μm; mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); gradient: 37% B for 0 min, 37–77% B over 20 min, then 100% B for 5 min; flow rate: 20 mL / min; column temperature: 25 °C), and fractions were collected as determined by MS and UV signals. The product-containing fractions were combined and dried in a centrifugal evaporator to give the product (12.1 mg; theoretical molecular weight 437.495). Analytical LC / MS was used to determine the final purity.Injection 1 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile Phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0% B to 100% B over 3 minutes, followed by 100% B for 0.50 minutes; Flow Rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 1 Results: Purity: 100.0%; Observed Mass: 438.14; Retention Time: 2.36 minutes; Injection 2 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 Acetonitrile:water (containing 0.1% trifluoroacetic acid); Mobile phase B: 95:5 acetonitrile:water (containing 0.1% trifluoroacetic acid); Temperature: 50°C; Gradient: 0% B to 100% B over 3 minutes, followed by 100% B for 0.50 minutes; Flow rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 2 results: Purity: 100.0%; Observed mass: 438.14; Retention time: 1.2 minutes.

[0203] Synthetic method for DGKi compounds 12-14 8-((2S,5R)-4-(1-(2,4-difluorophenyl)propyl)-2,5-dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] To a mixture of 8-((2S,5R)-2,5-dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (29.7 mg, 0.1 mmol) and 1-(1-bromopropyl)-2,4-difluorobenzene (25.9 mg, 0.110 mmol) in acetonitrile (0.3 mL), Hunig's base (87 μL, 0.500 mmol) was added and the mixture was stirred on a hot plate at 55° C. for 16 hours. The crude material was purified by preparative LC / MS (conditions: column: XBridge C18, 200 mm x 19 mm, particle size: 5 μm; mobile phase A: 5:95 acetonitrile:water (containing 0.1% trifluoroacetic acid); mobile phase B: 95:5 acetonitrile:water (containing 0.1% trifluoroacetic acid); gradient: 3% B for 0 min, 3–43% B over 25 min, then 100% B for 5 min; flow rate: 20 mL / min; column temperature: 25 °C). Fractions were collected as determined by MS and UV signals. The product-containing fractions were combined and dried on a centrifugal evaporator (stereochemistry: diastereomeric mixture). The mixture of synthetic diastereomers of DGKi compound 12 was further separated into two homochiral diastereomers using SFC chiral chromatography (conditions: column: Chiral OD, 30x250mm; particle size: 5μ; mobile phase: 15% IPA / 85% CO2 (containing 0.1% DEA); flow rate: 100mL / min; detection wavelength: 220nm). DGKi compound 13 (isomer 1) was recovered as the first eluting peak (95% de; stereochemistry: homochiral). DGKi compound 14 (isomer 2) was recovered as the second eluting peak (95% de; stereochemistry: homochiral).

[0204] Synthesis of DGKi compound 15 8-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] DMF was sparged with nitrogen for 1 hour. Zinc (0.95 mg, 0.015 mmol), bromo(tri-tert-butylphosphine)palladium(I) dimer (9.96 mg, 0.013 mmol), and 4-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-6-bromo-1-methyl-3-nitro-1,5-naphthyridin-2(1H)-one (21.38 mg, 0.037 mmol) were added to a 1-dram vial. Sparged DMF (0.3 mL) was added, and the mixture was capped under a nitrogen atmosphere and placed in a 50°C oil bath for 15 minutes. Zinc cyanide (2.86 mg, 0.024 mmol) was added, and the mixture was capped under a nitrogen atmosphere and placed in a 50°C oil bath for 3 hours. LC / MS analysis indicated the reaction was complete. The crude material was purified by preparative LC / MS (conditions: column: XBridge C18, 19x200mm, particle size: 5µm; mobile phase A: 5:95 acetonitrile:water (containing 10mM ammonium acetate); mobile phase B: 95:5 acetonitrile:water (containing 10mM ammonium acetate); gradient: 50-90% B over 15min, followed by 100% B for 5min; flow rate: 20mL / min), and the product-containing fractions were combined and dried by centrifugal evaporation. The title compound (11.4mg) was isolated in 59.7% yield. Alternative synthesis: A solution of 8-chloro-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (750 mg, 2.83 mmol) in DMF (6 mL) was combined with 1-(bis(4-fluorophenyl)methyl)piperazine (899 mg, 3.12 mmol), followed by Hunig's base (0.990 mL, 5.67 mmol) and stirred at room temperature overnight. LC / MS analysis indicated the reaction was complete. The crude material was filtered and purified by preparative HPLC (aqueous acetonitrile with ammonium acetate as a buffer) to give a yellow solid (1.02 g). Two analytical LC / MS injections were used to determine the final purity. Injection 1 conditions: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, particle size: 1.7 μm; Mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 50 °C; Gradient: 0–100% B over 3 min, followed by 100% B for 0.75 min; Flow rate: 1.0 mL / min; Detection: UV (220 nm); Injection 2 conditions: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, particle size: 1.7 μm; Mobile phase A: 5:95 acetonitrile:water (containing 0.1% trifluoroacetic acid); Mobile phase B: 95:5 acetonitrile:water (containing 0.1% trifluoroacetic acid); Temperature: 50°C; Gradient: 0–100% B over 3 min, followed by 100% B for 0.75 min; Flow rate: 1.0 mL / min; Detection: UV (220 nm); Injection 1 results: Purity: 100%; Observed mass: 517.0; Retention time: 2.4 min; Injection 2 results: Purity: 98.4%; Observed mass: 517.0; Retention time: 1.7 min. 1H NMR (500MHz, chloroform-d) δ 7.88 (d, J=8.7Hz, 1H), 7.76 (d, J=8.9Hz, 1H), 7.40 (dd, J=8.5, 5.5Hz, 4H), 7.02 (t, J=8.7Hz, 4H), 4.34 (s, 1H), 3.68 (s, 3H), 3.62-3.55 (m, 4H), 2.64 (br s, 4H); 13 C NMR (126 MHz, chloroform-d) δ 163.0, 161.0, 155.4, 147.0, 138.0, 137.7, 137.7, 135.9, 132.4, 129.5, 129.2, 129.2, 126.0, 123.1, 116.5, 115.8, 115.6, 74.3, 51.6, 51.2, 29.7

[0205] Synthesis of DGKi compound 16 8-[(2S,5R)-4-[bis(4-methylphenyl)methyl]-2,5-dimethylpiperazin-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] To a mixture of (cyanomethyl)trimethylphosphonium iodide (46.2 mg, 0.19 mmol), di-p-tolylmethanol (23.46 mg, 0.108 mmol), and 8-((2S,5R)-2,5-dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile·TFA (72.4 mg, 54% wt, 0.095 mmol) in acetonitrile (0.3 mL), Hunig's base (0.10 mL, 0.57 mmol) was added and the mixture was stirred at 110°C for 2 hours. The crude material was purified by preparative LC / MS (conditions: column: XBridge C18, 200 mm x 19 mm, particle size: 5 μm; mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); gradient: 55% B at 0 min, followed by 55–95% B over 20 min, then 100% B at 4 min; flow rate: 20 mL / min; column temperature: 25 °C), and fractions were collected as determined by MS and UV signals. The product-containing fractions were combined and dried in a centrifugal evaporator to give the product (23.4 mg; theoretical molecular weight 491.639). Analytical LC / MS was used to determine the final purity.Injection 1 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile Phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 50 °C; Gradient: 0% B to 100% B over 3 minutes, followed by 100% B for 0.75 minutes; Flow Rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 1 Results: Purity: 100.0%; Observed Mass: 492.21; Retention Time: 2.77 minutes; Injection 2 Conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, Particle Size: 1.7 μm; Mobile Phase A: 5:95 Acetonitrile:water (containing 0.1% trifluoroacetic acid); Mobile phase B: 95:5 acetonitrile:water (containing 0.1% trifluoroacetic acid); Temperature: 50°C; Gradient: 0% B to 100% B over 3 minutes, followed by 100% B for 0.75 minutes; Flow rate: 1 mL / min; Detection: MS and UV (220 nm); Injection 2 results: Purity: 100.0%; Observed mass: 492.2; Retention time: 1.71 minutes. 1 H NMR (400MHz, DMSO-d6) δ ppm 8.15 (d, J=8.5Hz, 1 H), 8.04-8.09 (m, 1 H), 7.81 (s, 4 H), 7.57-7.63 (m, 2 H), 7.12-7.19 (m, 2 H), 6.00 (s, 1 H), 4.82 (s, 1 H), 4.52-4.63 (m, 1 H), 3.64-3.76 (m, 1 H), 3.51-3.58 (m, 4 H), 2.99-3.10 (m, 1 H), 2.86 (br d, J=8.5Hz, 1 H), 2.28-2.37 (m, 1 H), 1.31 (d, J=6.5Hz, 3 H), 1.07 (d, J=6.5Hz, 3 H); 13C NMR (100.66MHz, DMSO-d6) δ ppm 162.4, 160.9, 159.9, 153.5, 148.0, 138.7, 138.6, 135.0, 132.6, 129.3 (d, J=8.0 Hz), 128.8 (d, J=10.0 Hz), 124.0, 122.8, 118.6, 117.5, 115.6, 115.4, 109.8, 104.8, 69.0, 51.8, 49.4, 48.9, 47.2, 28.6, 13.4, 7.4 Reference: PCT / US2020 / 048070

[0206] DGKi compounds 17 and 18 4-((2S,5R)-2,5-diethyl-4-(1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] To a stirred solution of 4-((2S,5R)-2,5-diethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile·TFA (0.12 g, 0.27 mmol) in acetonitrile (10 mL) was added DIPEA (0.14 mL, 0.82 mmol), 1-(1-chloropropyl)-4-(trifluoromethyl)benzene (0.12 g, 0.55 mmol), and sodium iodide (0.04 g, 0.27 mmol) and heated at 85° C. for 16 h. The reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure to give the crude product. This was purified by preparative HPLC [HPLC method: Column: Sunfire C18, 150x19mm ID, 5µm; Mobile phase A: 10mM aqueous ammonium acetate; Mobile phase B: acetonitrile; Gradient: 0-100% B over 18min, followed by 100% B for 5min; Flow rate: 17mL / min]. The fractions were concentrated under reduced pressure and lyophilized from EtOH / HO (1:5) to give compounds 17 and 18. Compound 17: (10 mg, 7% yield); LCMS: m / z=513.3 (M+H); rt 2.52 min; (LCMS method: Column: XBridge BEH XP C18 (50x2.1) mm, 2.5 μm Mobile phase A: 95% water:5% acetonitrile (containing 10 mM ammonium formate); Mobile phase B: 5% water:95% acetonitrile (containing 10 mM ammonium formate); Flow rate: 1.1 mL / min; Temperature: 50°C; Time (min): 0-4; %B: 0-100; 1 H NMR (400MHz, DMSO-d6) δ 8.24 (d, J=6.6Hz, 1H), 7.98 (d, J=9.0Hz, 1H), 7.73 (d, J=8.1Hz, 2H), 7.56 (d, J=7.1Hz, 2H), 5.83-5.48 (m, 1H), 4.98-4.86 (m, 1H), 3.64 (br. s., 1H), 3.43 (s, 3H), 3.08 (d, J=9.8Hz, 1H), 2.93-2.82 (m, 2H), 2.42-2.26 (m, 1H), 2.13-2.08 (m, 1H), 1.98-1.82 (m, 3H), 1.66-1.54 (m, 1H), 1.44-1.31 (m, 1H), 0.98-0.91 (br. s., 3H), 0.69-0.53 (m, 6H) Compound 18: (3 mg, 2% yield); LCMS: m / z=513.3 (M+H); rt 2.54 min; (LCMS method: Column: XBridge BEH XP C18 (50x2.1) mm, 2.5 μm Mobile phase A: 95% water:5% acetonitrile (containing 10 mM ammonium formate); Mobile phase B: 5% water:95% acetonitrile (containing 10 mM ammonium formate); Flow rate: 1.1 mL / min; Temperature: 50°C; Time (min): 0-4; %B: 0-100; 1H NMR (400MHz, DMSO-d6) δ 8.28-8.19 (m, 1H), 8.01-7.95 (m, 1H), 7.72 (d, J=7.8Hz, 2H), 7.58 (d, J=8.6Hz, 2H), 6.06-5.28 (m, 1H), 5.08-4.76 (m, 1H), 3.64-3.50 (m, 2H), 3.43 (s, 3H), 3.16-3.08 (m, 1H), 2.25-2.14 (m, 2H), 2.00-1.83 (m, 3H), 1.57-1.53 ​​(m, 3H), 1.03-0.89 (m, 3H), 0.65-0.54 (m, 6H)

[0207] DGKi compounds 19 and 20 4-((2S,5R)-5-ethyl-2-methyl-4-(1-(4-(trifluoromethyl)phenyl)ethyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] To a stirred solution of 4-((2S,5R)-5-ethyl-2-methylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile·TFA (70 mg, 0.22 mmol) in acetonitrile (2 mL) was added DIPEA (0.12 mL, 0.67 mmol), 1-(1-chloroethyl)-4-(trifluoromethyl)benzene (93 mg, 0.45 mmol), and sodium iodide (33.6 mg, 0.22 mmol) at room temperature and heated at 85 °C for 16 h. The reaction mixture was cooled to room temperature, the solvent was removed under reduced pressure, and the resulting residue was dissolved in ethyl acetate (100 mL). The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to give the crude product. This was purified by preparative HPLC [HPLC method: column: Sunfire C18 (150 mm × 19.2 mm ID, 5 μm), mobile phase A = 10 mM ammonium acetate aqueous solution, mobile phase B = acetonitrile, flow rate: 19 mL / min], and the fractions were concentrated under reduced pressure, diluted with EtOH / HO (1:5), and lyophilized to give compounds 19 and 20. Compound 19: (9 mg, 8% yield); LCMS: m / z=485.1 (M+H); rt 2.34 min; (LCMS method: Column: XBridge BEH XP C18 (50x2.1 mm), 2.5 μm; Mobile phase A: 95% water:5% acetonitrile (10 mM ammonium acetate); Mobile phase B: 5% water:95% acetonitrile (10 mM ammonium acetate); Flow rate: 1.1 mL / min; Temperature: 50°C; Time (min): 0-3; %B: 0-100; 1H NMR(400MHz, DMSO-d6) δ ppm 8.32-8.17 (m, 1H), 8.05-7.94 (m, 1H), 7.76-7.66 (m, 2H), 7.66-7.55 (m, 2H), 6.11-5.42 (m, 1H), 5.10-4.79 (m, 1H), 3.78-3.59 (m, 2H), 3.44 (s, 3H), 3.17-3.05 (m, 1H), 2.64-2.55 (m, 1H), 2.26-2.09 (m, 1H), 1.65-1.34 (m, 3H), 1.31-1.16 (m, 5H), 1.01 (br t, J=7.1Hz, 3H) Compound 20: (9mg, 8% yield); LCMS: m / z=485.1(M+H); rt 2.29 minutes; (LCMS method: カラム: XBridge BEH XP C18 (50x2.1mm), 2.5μm; mobile phase A: 95% water: 5% アセトニトリル (10mM アセンモニウム); mobile phase B: 5% water: 95% アセトニトリル (10mM アンモニウム); flow rate: 1.1mL / min; temperature: 50℃; time (min): 0~3; %B: 0~100%); 1 H NMR (400MHz, DMSO-d6) δ ppm 8.24 (br d, J=8.6Hz, 1H), 7.99 (d, J=9.0Hz, 1H), 7.73 (d, J=8.3Hz, 2H), 7.61 (br d, J=8.3Hz, 2H), 5.87-5.63 (m, 1H), 5.10-4.79 (m, 1H), 3.90-3.80 (m, 1H), 3.44 (s, 3H), 3.46-3.15 (m, 1H), 2.89-2.73 (m, 2H), 2.41-2.34 (m, 1H), 1.63-1.34 (m, 5H), 1.29 (br d, J=6.1Hz, 3H), 0.79-0.64 (m, 3H)

[0208] DGKi compound 21および22 4-((2S,5R)-5-ethyl-4-((4-fluorophenyl)(5-(trifluoromethyl)pyridin-2-yl)methyl)-2-methylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] To a stirred solution of 4-((2S,5R)-5-ethyl-2-methylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile·TFA (0.5 g, 1.17 mmol) in acetonitrile (10 mL) was added DIPEA (1.02 mL, 5.86 mmol) followed by 2-(bromo(4-fluorophenyl)methyl)-5-(trifluoromethyl)pyridine (0.78 mg, 2.35 mmol) and heated at 80° C. for 3 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure to give the crude product. This was purified by preparative HPLC (HPLC method: column: INERTSIL ODS 21.2 x 250 mm, 5 μm; mobile phase A: 0.1% TFA aqueous solution; mobile phase B: acetonitrile; gradient: 30 to 80% B over 14 min, followed by 100% B for 5 min; flow rate: 17 mL / min). The fractions were concentrated under reduced pressure and lyophilized from EtOH / HO (1:5) to give Compounds 21 and 22. Compound 21: 140 mg, 21% yield; LCMS: m / z=566.2 (M+H); rt 3.26 min; (LCMS method: Column: Column-Kinetex XB-C18 (75×3 mm; 2.6 μm), Mobile Phase A: 98% water:2% acetonitrile (containing 10 mM ammonium formate); Mobile Phase B: 2% water:98% acetonitrile (containing 10 mM ammonium formate); Flow rate: 1.0 mL / min; Temperature: 50° C.; Time (min): 0-4; %B: 0-100%); 1H NMR (400MHz, DMSO-d6) δ ppm 8.83 (br s, 1 H), 8.19-8.31 (m, 2 H), 7.95-8.12 (m, 2 H), 7.53-7.63 (m, 2 H), 7.12-7.26 (m, 2 H), 5.41-6.26 (m, 1 H), 4.79-5.20 (m, 2 H), 3.60-3.74 (m, 1 H), 3.44 (s, 3 H), 2.73-2.87 (m, 1 H), 2.22-2.42 (m, 2 H), 1.40-1.68 (m, 5 H), 0.53-0.71 (m, 3H) Compound 22: 155 mg, 23% yield; LCMS: m / z=566.2 (M+H); rt 3.25 min; (LCMS method: Column: Column-Kinetex XB-C18 (75x3 mm; 2.6 μm), Mobile Phase A: 98% water: 2% acetonitrile (containing 10 mM ammonium formate); Mobile Phase B: 2% water: 98% acetonitrile (containing 10 mM ammonium formate); Flow rate: 1.0 mL / min; Temperature: 50°C; Time (min): 0-4; %B: 0-100%); 1 H NMR (400MHz, DMSO-d6) δ ppm 8.92 (s, 1 H), 8.17-8.27 (m, 2 H), 7.90-8.02 (m, 2 H), 7.60-7.67 (m, 2 H), 7.14-7.22 (m, 2 H), 5.52-6.07 (m, 1 H), 4.87-5.08 (m, 2 H), 3.39-3.71 (m, 4 H), 2.69-2.78 (m, 1 H), 2.37-2.45 (m, 1 H), 1.37-1.69 (m, 5 H), 0.58-0.77 (m, 3 H)

[0209] DGKi compounds 23 and 24 (4-((2S,5R)-4-((4-chlorophenyl)(pyridin-2-yl)methyl)-5-ethyl-2-methylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] To a stirred solution of 4-((2S,5R)-5-ethyl-2-methylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile (100 mg, 0.32 mmol) in acetonitrile (5 mL) was added DIPEA (0.3 mL, 1.60 mmol) followed by 2-(bromo(4-chlorophenyl)methyl)pyridine (181 mg, 0.64 mmol) and heated at 80° C. for 3 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure to give the crude product. This was purified by preparative HPLC (HPLC method: column: Cellulose-5 (250*20 ID) 5μ; mobile phase A: 0.1% DEA / IPA; mobile phase B: 0.1% DEA / ACN; gradient: 90% B, then 100% B for 5 min; flow rate: 18 mL / min), and the fractions were concentrated under reduced pressure and lyophilized from EtOH / HO (1:5) to give Compounds 23 and 24. Compound 23: 24 mg, 14% yield; LCMS: m / z=514.2 (M+H); rt 2.94 min; (LCMS method: Column: Column-Kinetex XB-C18 (75×3 mm; 2.6 μm), Mobile Phase A: 98% water:2% acetonitrile (containing 10 mM ammonium formate); Mobile Phase B: 2% water:98% acetonitrile (containing 10 mM ammonium formate); Flow rate: 1.0 mL / min; Temperature: 50° C.; Time (min): 0-4; %B: 0-100%); 1H NMR (400MHz, DMSO-d6): δ ppm 8.52 (d, J=4.5Hz, 1 H), 8.23 ​​(d, J=9.0Hz, 1 H), 7.96-8.02 (m, 1 H), 7.75-7.81 (m, 1 H), 7.59-7.68 (m, 3 H), 7.39 (d, J=8.5Hz, 2 H), 7.22-7.29 (m, 1 H), 5.54-5.95 (m, 1 H), 4.81-5.07 (m, 2 H), 3.39-3.68 (m, 5 H), 2.69-2.76 (m, 1 H), 2.35-2.44 (m, 1 H), 1.37-1.67 (m, 5 H), 0.58–0.67 (m, 3 H) Compound 24: 22mg, 13% yield; LCMS: m / z=514.2(M+H); rt 2.94 minutes; (LCMS method: Kinetex XB-C18 (75X3mm; 2.6μm), mobile phase A: 98% water: 2% アセトニトリル (containing 10mM ギ acid アンモニウム); Mobile phase B: 2% water: 98% アセトニトリル (containing 10mM ギ acid アンモニウム); flow rate: 1.0mL / min; temperature: 50℃; time (min): 0~4; %B: 0~100%); 1 H NMR (400MHz, DMSO-d6): δ ppm 8.41-8.45 (m, 1 H), 8.23 ​​(d, J=9.0Hz, 1 H), 7.96-8.02 (m, 1 H), 7.78-7.85 (m, 2 H), 7.53-7.61 (m, 2 H), 7.40 (d, J=8.5Hz, 2 H), 7.20-7.26 (m, 1 H), 5.52-5.97 (m, 1 H), 4.87-5.04 (m, 1 H), 4.78-4.86 (m, 1 H), 3.37-3.71 (m, 4 H), 2.72-2.78 (m, 1 H), 2.54-2.63 (m, 1 H), 2.35-2.46 (m, 1 H), 1.40-1.64 (m, 5 H), 0.58-0.70 (m, 3 H)

[0210] DGKi compound 25および26 4-((2S,5R)-4-((3-cyclopropyl-1,2,4-oxadiazol-5-yl)(4-fluorophenyl)methyl)-2,5-dimethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] To a stirred solution of 2-((2R,5S)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidin-4-yl)-2,5-diethylpiperazin-1-yl)-2-(4-fluorophenyl)acetic acid (0.045 g, 0.09 mmol), N-hydroxycyclopropanecarboximidamide (9.4 mg, 0.09 mmol) in DMF (2 mL) was added BOP (0.01 g, 0.23 mmol) and triethylamine (0.04 mL, 0.23 mmol) at room temperature. After 2 hours, the mixture was heated at 110° C. for 3 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to give the crude product. This was purified by preparative HPLC (chiral separation method: column: DAD-1-Cellulose-2 (250×4.6 mM), 5 μm; mobile phase: 0.1% DEA / acetonitrile, flow rate: 2.0 mL / min). Compound 25: (1.9 mg, 6% yield): LCMS: m / z, 543.3 (M+H); rt 2.21 min; LCMS Method: Column: XBridge BEH XP C18 (50x2.1) mm, 2.5 μm; Mobile Phase A: 95% water:5% acetonitrile (10 mM ammonium acetate); Mobile Phase B: 5% water:95% acetonitrile (10 mM ammonium acetate); Flow Rate: 1.1 mL / min; Temperature: 50°C; Time (min): 0-3; %B: 0-100%; 1H NMR(400MHz, DMSO-d6) δ ppm 8.29-8.16 (m, 1H), 8.06-7.92 (m, 1H), 7.75-7.58 (m, 2H), 7.26 (m, 2H), 6.01-5.32 (m, 1H), 5.28 (br s, 1H), 5.00-4.79 (m, 1H), 3.66-3.56 (m, 1H), 3.43 (s, 3H), 2.65-2.57 (m, 1H), 2.44-2.34 (m, 2H), 2.18-2.00 (m, 1H), 1.95-1.74 (m, 2H), 1.68-1.34 (m, 2H), 1.15-1.02 (m, 2H), 0.93-0.83 (m, 2H), 0.81-0.62 (m, 6H) Compound 26: (1.0mg, 3% yield): LCMS: m / z, 543.3(M+H); rt 2.20 minutes; LCMS method: KARARA: XBridge BEH XP C18 (50x2.1)mm, 2.5μm; Mobile phase A: 95% water: 5% アセトニトリル (10mM アセンモニウム); mobile phase B: 5% water: 95% アセトニトリル (10mM アンモニウム); flow rate: 1.1mL / min; temperature: 50℃; time (min): 0~3; %B: 0~100%); 1 H NMR (400MHz, DMSO-d6) δ ppm 8.23 ​​(d, J=8.8Hz, 1H), 8.06-7.91 (m, 1H), 7.62 (dd, J=6.2, 7.5Hz, 2H), 7.26 (t, J=8.8Hz, 2H), 5.92-5.31 (m, 1H), 5.29 (s, 1H), 4.96-4.78 (m, 1H), 3.60-3.50 (m, 1H), 3.43 (s, 3H), 3.25-3.10 (m, 1H), 2.97-2.75 (m, 2H), 2.27-1.65 (m, 3H), 1.49-1.24 (m, 2H), 1.11-0.97 (m, 2H), 0.94-0.75 (m, 5H), 0.74-0.50 (m, 3H)

[0211] DGKi compound 27および28 4-((2S,5R)-4-((4-fluorophenyl)(5-(trifluoromethyl)pyridin-2-yl)methyl)-2,5-dimethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] To a stirred solution of 4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile (1 g, 3.35 mmol) in acetonitrile (10 mL) was added DIPEA (5.9 mL, 33.5 mmol) followed by 2-(bromo(4-fluorophenyl)methyl)-5-(trifluoromethyl)pyridine (2.24 g, 6.70 mmol) and heated at 80° C. for 4 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure to give the crude product. This was purified by preparative HPLC (HPLC method: column: Sunfire C18, 150x19mm ID, 5µm; mobile phase A: 0.1% TFA in water; mobile phase B: acetonitrile:MeOH (1:1); gradient: 50-100% B over 20 min, followed by 100% B for 5 min; flow rate: 19mL / min). The fractions were concentrated under reduced pressure and lyophilized from EtOH / HO (1:5) to give Compounds 27 and 28. Compound 27: 110 mg, 6% yield; LCMS: m / z=552.2 (M+H); rt 3.09 min; (LCMS method: Column: Column-Kinetex XB-C18 (75x3 mm; 2.6 μm), Mobile Phase A: 98% water:2% acetonitrile (containing 10 mM ammonium formate); Mobile Phase B: 2% water:98% acetonitrile (containing 10 mM ammonium formate); Flow rate: 1.0 mL / min; Temperature: 50°C; Time (min): 0-4; %B: 20-100%); 1H NMR (400MHz, DMSO-d6) δ ppm 8.83 (s, 1H), 8.22 (d, J=9.0Hz, 2H), 8.11-7.95 (m, 2H), 7.71-7.58 (m, 2H), 7.25-7.13 (m, 2H), 5.76-5.44 (m, 1H), 5.13-4.67 (m, 2H), 3.86-3.49 (m, 1H), 3.44 (s, 3H), 3.19-3.08 (m, 1H), 2.84 (dd, J=3.8, 12.3Hz, 1H), 2.38-2.26 (m, 1H), 1.67-1.39 (m, 3H), 1.11-0.86 (m, 3H) Compound 28: 145 mg, 8% yield; LCMS: m / z=552.2 (M+H); rt 3.09 min; (LCMS method: Column: Column-Kinetex XB-C18 (75x3 mm; 2.6 μm), Mobile Phase A: 98% water:2% acetonitrile (containing 10 mM ammonium formate); Mobile Phase B: 2% water:98% acetonitrile (containing 10 mM ammonium formate); Flow rate: 1.0 mL / min; Temperature: 50°C; Time (min): 0-4; %B: 0-100%); 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.91 (s, 1H), 8.27-8.16 (m, 2H), 7.99 (d, J=9.0 Hz, 2H), 7.69-7.57 (m, 2H), 7.23-7.13 (m, 2H), 5.77-5.41 (m, 1H), 5.09-4.62 (m, 2H), 3.90-3.65 (m, 1H), 3.44 (s, 3H), 3.14-3.02 (m, 1H), 2.80-2.74 (m, 1H), 1.61-1.40 (m, 3H), 1.10-0.93 (m, 3H) [One H is obscured by the solvent peak.]

[0212] DGKi compounds 29 and 30 4-((2S,5R)-4-(1-(4-(cyclopropylmethoxy)-2-fluorophenyl)propyl)-2,5-diethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] To a stirred solution of 4-((2S,5R)-2,5-diethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile·HCl (200 mg, 0.55 mmol) in acetonitrile (5 mL) was added DIPEA (0.3 mL, 1.65 mmol), sodium iodide (83 mg, 0.55 mmol), and 1-(1-chloropropyl)-4-(cyclopropylmethoxy)-2-fluorobenzene (268 mg, 1.1 mmol), heated at 80 °C for 16 h, and cooled to room temperature. 1-(1-chloropropyl)-4-(cyclopropylmethoxy)-2-fluorobenzene (268 mg, 1.102 mmol) was added again, and heating was continued for an additional 16 h. The reaction mixture was cooled, the solvent removed under reduced pressure, and the resulting residue was dissolved in ethyl acetate (10 × 20 mL). The organic layer was washed with brine, dried over NaSO, and concentrated under reduced pressure to give the crude product, which was purified by preparative HPLC (HPLC method: column: EXRS (20 × 250 mm, 5 μm), mobile phase A: 10 mM ammonium acetate aqueous solution, mobile phase AB: acetonitrile, flow rate: 20 mL / min). Fraction 1 was concentrated under reduced pressure, and the product was diluted with EtOH / HO (1:5) and lyophilized to give compound 29 (35 mg, 11.6% yield). LCMS: m / z, 533.4 [M+H] + , rt 1.57 min; LCMS method: Column: KINETIX XB C18 (75x3 mm, 2.6 μm); Mobile phase A: 10 mM ammonium acetate in water (pH 3.3), Mobile phase B: acetonitrile; 1H NMR (DMSO-d6, 400MHz) δ (ppm) 8.23 ​​(d, J=9.0Hz, 1H), 7.97 (d, J=9.0Hz, 1H), 7.33 (m, 1H), 6.62-6.92 (m, 2H), 5.29-6.06 (m, 1H), 4.70-5.05 (m, 1H), 3.82 (m, 3H), 3.43 (s, 3H), 2.99-3.10 (m, 1H), 2.80-2.87 (m, 1H), 2.63-2.78 (m, 1H), 2.33 (s, 1H), 1.74-2.11 (m, 3H), 1.51-1.66 (m, 1H), 1.17-1.46 (m, 3H), 0.84-1.01 (m, 3H), 0.61-0.78 (m, 6H), 0.53-0.61 (m, 2H), 0.29-0.35 (m, 2H) The product was concentrated under reduced pressure and the product EtOH / H2O (1:5) was freeze-dried. Compound 30 (37 mg, 12.35% yield) was obtained. LCMS: m / z, 533.4 [M+H] + , rt 2.72 minutes; LCMS method: KINETIX XB C18 (75x3mm, 2.6μm); Mobile phase A: 10mM ammonium anhydride aqueous solution (pH 3.3), mobile phase B:アセトニトリル; 1 H NMR (DMSO-d6, 400MHz): δ (ppm) 8.13-8.35 (m, 1H), 7.98 (m, 1H), 7.38 (m, 1H), 6.61-6.89 (m, 2H), 5.18-6.15 (m, 1H), 4.66-5.13 (m, 1H), 3.63-3.90 (m, 3H), 3.43 (s, 3H), 3.25 (m, 1H), 3.00-3.15 (m, 1H), 2.63-2.70 (m, 1H), 2.26-2.38 (m, 1H), 1.81 (m, 3H), 1.35-1.61 (m, 2H), 1.15-1.26 (m, 2H), 0.88-1.00 (m, 3H), 0.61-0.71 (m, 6H), 0.51-0.59 (m, 2H), 0.32 (m, 2H)

[0213] DGKi compounds 31 and 32 4-((2S,5R)-2,5-diethyl-4-(1-(4-(trifluoromethyl)phenyl)butyl)piperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] To a stirred solution of 4-((2S,5R)-2,5-diethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile·HCl (0.4 g, 1.1 mmol) in acetonitrile (10 mL) was added DIPEA (0.6 mL, 3.31 mmol), followed by 1-(1-chlorobutyl)-4-trifluoromethyl)benzene (0.783 g, 3.31 mmol) and sodium iodide (0.165 g, 1.102 mmol) and heated at 85 °C for 16 h. The reaction mixture was filtered through Celite, washing with ethyl acetate, and the filtrate was concentrated under reduced pressure to give the crude compound. This was purified by preparative HPLC [HPLC method: Column: YMC ExRS (250 mm × 21.2 mm, 5 μm) Mobile phase A = 10 mM ammonium acetate aqueous solution (pH 4.5); Mobile phase B = acetonitrile; Gradient: elution with 80% B over 2 min, followed by 100% B over 16 min; Flow rate: 19 mL / min] to give Compounds 31 and 32. Compound 31 (10 mg, 1.7% yield), LCMS: m / z=527.4 (M+H); rt 2.626 min; [LCMS method: Column: XBridge BEH XP C18 (50×2.1 mm), 2.5 μm; Mobile phase A: 95% water:5% acetonitrile; 10 mM NHOAC; Mobile phase B: 5% water:95% acetonitrile (10 mM NHOAc); Flow rate: 1.1 mL / min; Temperature: 50° C.; Time (min)] 1H NMR (400MHz, DMSO-d6) δ 8.30-8.16 (m, 1H), 7.98 (d, J=9.0Hz, 1H), 7.72 (d, J=8.3Hz, 2H), 7.56 (br d, J=7.8Hz, 2H), 5.86-5.44 (m, 1H),5.01-4.77 (m, 1H), 3.730-3.718(m, 1H), 3.46 (s, 3H), 3.43-3.35(m, 1H) 3.13-3.01 (m, 1H), 2.93-2.75 (m, 2H), 2.38-2.26 (m, 1H), 2.17-1.74 (m, 3H), 1.63-1.22 (m, 3H), 1.01-0.86 (m, 4H), 0.84-0.75 (m, 3H), 0.73-0.54 (m, 3H) Compound 32: (7.2mg, 1.23% yield), LCMS: m / z=527.3(M+H); rt 2.654 minutes; [LCMS method: カラム: XBridge BEH XP C18 (50x2.1)mm, 2.5μm; mobile phase A: 95% water: 5% アセトニトリル (10mM NH4OAC); Mobile phase B: 5% water: 95% アセトニトリル; 10mM NH4OAc; Flow rate: 1.1mL / min; Temperature: 50℃; Time (minutes)]; 1 H NMR (400MHz, DMSO-d6) δ=8.29-8.15 (m, 1H), 7.96-8.02 (m, 1H), 7.70 (d, J=8.1Hz, 2H), 7.58 (br d, J=8.1Hz, 2H), 6.09-5.22 (m,1H), 5.13-4.66 (m, 1H), 3.68-3.52 (m, 2H), 3.43 (s, 3H), 3.28-3.04 (m, 2H), 2.60-2.53 (m, 1H), 2.25-2.12 (m, 1H), 2.04-1.68 (m, 3H), 1.60-1.29 (m,3H), 1.05-0.74 (m, 7H), 0.59 (t, J=7.5Hz, 3H)

[0214] DGKi compound 33および34 1-methyl-4-((2S,5R)-2-methyl-5-propyl-4-(1-(4-(trifluoromethyl)phenyl)ethyl)piperazin-1-yl)-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] To a solution of 6-chloro-1-methyl-4-((2S,5R)-2-methyl-5-propyl-4-(1-(4-(trifluoromethyl)phenyl)ethyl)piperazin-1-yl)pyrido[3,2-d]pyrimidin-2(1H)-one (0.1 g, 0.19 mmol) in DMF (2 mL) under argon atmosphere was added zinc cyanide (0.046 g, 0.39 mmol), zinc (0.7 mg, 9.8 μmol), and triethylamine (0.1 mL, 0.59 mmol) followed by dichloro[9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene]palladium(II) (0.015 g, 0.02 mmol) at room temperature. The mixture was heated at 90° C. overnight, diluted with EtOAc (50 mL), filtered through Celite®, and washed with ethyl acetate (2×50 mL). The filtrate was washed with water (50 mL), brine, dried over NaSO, and concentrated under reduced pressure to give the crude product, which was purified by preparative HPLC (HPLC method: column: YMC EXRS (250 × 19 mm, 5 μm); mobile phase A: 10 mM ammonium acetate aqueous solution (pH 4.5); mobile phase B: acetonitrile, flow rate: 20 mL / min) to give Compounds 33 and 34. Compound 33: (13 mg, 14% yield); LCMS: m / z=499.3 [M+H] + ; rt 2.376 min; (LCMS method: Column: XBridge BEH XP C18 (50x2.1 mm, 2.5 μm); Mobile phase A: 95% water:5% acetonitrile (10 mM NH4OAc); Mobile phase B: 5% water:95% acetonitrile (10 mM NH4OAc); Flow rate: 1.1 mL / min; Temperature: 50 °C); 1H NMR (400MHz, DMSO-d6) δ(ppm)=8.22 (br d, J=8.8Hz, 1H), 7.98 (d, J=8.8Hz, 1H), 7.70-7.72 (m, 2H), 7.59-7.61 (m, 2H), 5.84-5.59 (m, 1H), 5.10-4.67 (m, 1H), 3.91-3.75 (m, 1H), 3.38-3.43 (m, 4H), 2.86-2.70 (m, 2H), 2.47-2.36 (m, 1H), 1.63-1.51 (m, 1H), 1.47-1.18 (m, 8H), 0.9-0.99 (m, 1H), 0.75-0.59 (m, 3H) Compound 34: (13 mg, 13% yield); LCMS: m / z=499.3 [M+H] + ; rt 2.436 min; (LCMS method: Column: XBridge BEH XP C18 (50x2.1 mm, 2.5 μm); Mobile phase A: 95% water:5% acetonitrile (10 mM NH4OAc); Mobile phase B: 5% water:95% acetonitrile (10 mM NH4OAc); Flow rate: 1.1 mL / min; Temperature: 50 °C); 1 H NMR (400MHz, DMSO-d6) δ(ppm)=8.25 (br d, J=2.4Hz, 1H), 8.06-7.92 (m, 1H), 7.77-7.65 (m, 2H), 7.65-7.54 (m, 2H), 6.09-5.44 (m, 1H), 5.04-4.68 (m, 1H), 3.81-3.59 (m, 2H), 3.44 (s, 3H), 3.28-3.13 (m, 1H), 2.52-2.61 (m, 1H), 2.24-2.05 (m, 1H), 1.72-1.48 (m, 2H), 1.47-1.15 (m, 8H), 0.98-0.75 (m, 3H)

[0215] (Biological Assays) The pharmacological properties of the compounds described herein may be confirmed by a number of biological assays.

[0216] 1. In vitro DGK inhibition assay DGKα and DGKζ reactions were performed using either extruded liposomes (DGKα and DGKζ LIPGLO assay) or detergent / lipid micelle substrates (DGKα and DGKζ assay). The reactions were performed in assay buffer (MOPS (50 mM, pH 7.5), NaCl (100 mM), MgCl2 (10 mM), CaCl2 (1 μM), and DTT (1 mM)). Reactions using the detergent / lipid micelle substrate also contained octyl β-D-glucopyranoside (50 mM). The lipid substrate concentrations were 11 mM PS and 1 mM DAG in the detergent / lipid micelle reactions. The lipid substrate concentrations were 2 mM PS, 0.25 mM DAG, and 2.75 mM PC in the extruded liposome reactions. The reactions were performed in ATP (150 μM). The enzyme concentrations of DGKα and DGKζ were 5 nM. The compound inhibition experiments were performed as follows: 50 nL droplets of each test compound dissolved in DMSO (11 3-fold serial dilutions of each compound starting from a top concentration of 10 mM) were transferred to wells of a white 1536-well plate (Corning 3725). A 2x final reaction concentration of enzyme / substrate solution (5 mL) was prepared by mixing 2.5 mL of 4x diluted enzyme solution (DGKα or DGKζ (20 nM) in assay buffer (preparation described below)) with 2.5 mL of either 4x diluted liposome solution or 4x diluted detergent / lipid micelle solution (compositions described below) and incubated for 10 min at room temperature. Next, 1 μL of the 2x diluted enzyme / substrate solution was added to the wells containing the test compound, and the reaction was initiated by adding 1 μL of ATP (300 μM). The reaction was continued for 1 h, after which 2 μL of Glo reagent (Promega V9101) was added and incubated for 40 min. Next, kinase detection reagent (4 μL) was added and incubated for 30 minutes. Luminescence was measured using a microplate reader (EnVision). Percent inhibition was calculated from the ATP conversion obtained by taking 100% inhibition from the control reaction without enzyme and 0% inhibition from the vehicle-only reaction. Test compounds were evaluated at 11 concentrations, and the IC 50 It was decided that:

[0217] Preparation of 4x diluted detergent / lipid micelles Detergent / lipid micelles were prepared in a 2 L round-bottom flask by combining phosphatidylserine (15 g, Avanti 840035P) and diacylglycerol (1 g, 800811O) and dissolving them in chloroform (150 mL). The chloroform was removed under high vacuum using a rotary evaporator. The resulting colorless, viscous oil was resuspended in 400 mL of MOPS (50 mM, pH 7.5), NaCl (100 mM), NaF (20 mM), MgCl2 (10 mM), CaCl2 (1 μM), and DTT (1 mM), and octyl glucoside (200 mM) with vigorous mixing. The lipid / detergent solution was divided into 5 mL aliquots and stored at -80°C.

[0218] Preparation of 4-fold diluted liposomes The lipid composition of the 4x diluted liposome solution was 5 mol% DAG (Avanti 800811O), 40 mol% PS (Avanti 840035P), and 55 mol% PC (Avanti 850457), for a total lipid concentration of 15.2 mg / mL. The PC, DAG, and PS were dissolved in chloroform, mixed, and dried under reduced pressure to obtain a thin film. These lipids were hydrated at 20 mM in a mixture of MOPS (50 mM, pH 7.5), NaCl (100 mM), and MgCl2 (5 mM) and subjected to five freeze-thaw cycles. The lipid suspension was extruded 11 times through a 100 nm polycarbonate filter. Dynamic light scattering was used to confirm liposome size (radius 50–60 nm). The liposome preparation was stored at 4°C for 4 weeks.

[0219] Baculovirus expression of human DGKα and DGKζ Baculovirus samples of human DGKα-TVMV-His-pFBgate and human DGKζ-transcriptional variant-2-TVMV-His-pFBgate were prepared using the Bac-to-Bac Baculovirus Expression System (Invitrogen) according to the manufacturer's protocol. The DNA sequences used for expression of DGKα and DGKζ are SEQ ID NOs. 1 and 3, respectively. Baculovirus amplification was performed using infected F9 cells at a virus / cell ratio of 1:1500. After transfection, the cells were grown at 27°C for 65 hours. Scale-up of expression of each protein was performed in a Cellbag 50L WAVE Bioreactor System 20 / 50 (GE Healthcare Bioscience). 2 x 10 cells were seeded in ESF921 insect cell medium (Expression System). 6 Sf9 cells (12L, Expression Systems, Davis, CA) were infected with virus stock solution at a virus / cell ratio of 1:200 and grown at 27°C for 66-68 hours post-infection. Infected cell cultures were obtained by centrifugation in a SORVALL® RC12BP centrifuge (2000 rpm, 20 minutes at 4°C). Pelleted cells were stored at -70°C until purification.

[0220] Purification of human DGKα and DGKζ Full-length human DGKα and DGKζ, containing a TVMV-cleavable C-terminal Hex-His tag sequence (SEQ ID NOs. 2 and 4, respectively), were expressed and purified from Sf9 baculovirus-infected insect cell paste, as described above. Cells were lysed using nitrogen cavitation in a nitrogen disrupter (Parr Instruments), and the lysate was clarified by centrifugation. The clarified lysate was purified to ~90% homogeneity using three sequential column chromatography steps on an AKTA Purifier Plus system. The three-step column chromatography involved nickel affinity resin capture (HisTrap FF crude, GE Healthcare) followed by size exclusion chromatography (DGK-α: HiLoad 26 / 600 Superdex 200 prep grade, GE Healthcare; DGK-ζ: HiPrep 26 / 600 Sephacryl S 300 HR, GE Healthcare). The third step was ion exchange chromatography, which differed between the two isoforms. DGKα was purified using Q Sepharose anion exchange chromatography (GE Healthcare). DGKζ was purified using SP Sepharose cation exchange chromatography (GE Healthcare). These proteins were purified to a concentration of ≥2 mg / mL. The formulation buffer was the same for both proteins: Hepes (50 mM, pH 7.2), NaCl (500 mM), glycerol (10% v / v), TCEP (1 mM), and EDTA (0.5 mM).

[0221] 2. Raji CD4 T Cell IL2 Assay A 1536-well IL-2 assay was performed in a volume of 4 μL using preactivated CD4 T cells and Raji cells. Prior to the assay, CD4 T cells were preactivated by treatment with α-CD3, α-CD28, and PHA (1.5 μg / mL, 1 μg / mL, and 10 μg / mL, respectively). Raji cells were treated with staphylococcal enterotoxin B (SEB, 10,000 ng / mL). Serially diluted compounds were first transferred to a 1536-well assay plate (Corning, #3727), followed by the addition of preactivated CD4 T cells (2 μL, final density: 6000 cells / well) and SEB-treated Raji cells (2 μL, 2000 cells / well). After 24 hours of incubation at 37°C / 5% CO2 incubator, IL-2 detection reagent (4 μL) was added to the assay plate (Cisbio, #64IL2PEC). The assay plate was read on an Envision reader. To assess compound cytotoxicity, either Raji cells or CD4 T cells were incubated with serially diluted compounds. After 24 hours of incubation, CellTiter-Glo (4 μL, Promega, #G7572) was added and the plate was read on an Envision reader. The 50% effective concentration (IC 50 ) was calculated using a four-parameter logistic equation: y=A+((B A) / (1+((C / x)^D))), where A and B represent the minimum and maximum % activation or inhibition, respectively, and C is the IC 50 , D represents the slope of the curve, and x represents the compound concentration.

[0222] 3. CellTiter-Glo CD8 T Cell Proliferation Assay Frozen naive human CD8 T cells were thawed in RPMI + 10% FBS and incubated for 2 hours at 37°C, followed by cell counting. 384-well tissue culture plates were coated overnight at 4°C with anti-human CD3 (20 μL, 0.1 μg / mL in plain RPMI), which was then removed from the plate. CD8 T cells (20 kJ / 40 μL) were added to each well along with soluble anti-human CD28 (0.5 μg / mL). Test compounds were dispensed into the cell plate immediately after cell fixation. After 72 hours of incubation in a 37°C incubator, CellTiter-Glo reagent (10 μL, Promega catalog number G7570) was added to each well. The plate was shaken vigorously for 5 minutes and incubated at room temperature for an additional 15 minutes. CD8 T cell proliferation was measured using the Envision™. In the analysis, the CD8 T cell signal stimulated with anti-CD3 (0.1 μg / mL) and anti-CD28 (0.5 μg / mL) was background. A reference compound (8-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile, 3 μM) was used to set the range of 100% inhibition, and the EC 50 was used to normalize the data to absolute 50%.

[0223] 4. DGK-AP1 Reporter Assay Jurkat AP1 luciferase reporter was performed using the Cignal Lenti AP1 reporter (luc) kit (SABiosciences (CLS-011L)). Test compounds were transferred from the Echo LDV plate to each well of a 384-well plate (white, solid-phase, opaque PE CulturPlate 6007768) using an Echo 550 instrument. The sample size was 30 nL / well, with one source plate per one destination plate. Cells were transferred to clean conical tubes (50 mL) and a cell suspension (40 mL, 2x dilution of 20 mL) was prepared. Cells were concentrated by centrifugation (1200 rpm, 5 min, ambient temperature). The supernatant was removed, and all cells were suspended in RPMI (Gibco 11875) + 10% FBS and diluted to 1.35 x 10 6 This cell suspension was manually added to a 384-well TC plate containing the test compound at 30 μL / well using a multichannel pipette, and the concentration was adjusted to 4.0 x 10 cells / mL. 4 The cell plate was incubated at 37°C and 5% CO2 for 20 minutes. During the incubation, αCD3 (3 μL, 1.3 mg / mL) was mixed with medium (10 mL) to prepare an anti-CD3 antibody (αCD3) solution [final concentration = 0.4 μg / mL]. Next, αCD3 (1.5 μL, 1.3 mg / mL) was mixed with medium (0.5 mL) [final concentration = 4 μg / mL]. After 20 min, medium (10 μL) was added to all wells in the first column (rows A–M). αCD3 (10 μL, 4 μg / mL) / well was added to rows N–P in the first column as a reference. Then, using a multichannel pipette, αCD3 (10 μL, 0.4 μg / mL) / well was added. αCD3-stimulated + / - compound-treated cells were incubated for 6 h at 37°C and 5% CO2. During this incubation, Steady-Glo (Promega E2520) reagent was slowly thawed to ambient temperature. Steady-Glo reagent was then added (20 μL / well) using a Multidrop Combi dispenser. Air bubbles were removed by centrifugation (2000 rpm, ambient temperature, 10 seconds). The cells were incubated at room temperature for 5 minutes. Samples were analyzed using an Envision plate reader instrument to measure luminescence units (RLU) according to the luminescence protocol. Data were analyzed by normalizing to 100% inhibition using the reference compound (8-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile).

[0224] 5. Mouse cytotoxic T lymphocyte assay An antigen-specific cytotoxic T cell (CTL) assay was developed to functionally evaluate the ability of DGKα and DGKζ inhibitors to enhance effector T cell-mediated tumor cell killing. + T cells recognize the OT-1 antigen-specific CD8 T cells, which express the ovalbumin-derived peptide SIINFEKL. + The cytotoxic activity of T cells is initiated. Functional CTL cells were prepared as follows. OT-1 splenocytes from 8- to 12-week-old mice were isolated and expanded in the presence of SIINFEKL peptide (1 μg / mL) and mIL2 (10 U / mL). After 3 days, fresh medium containing mIL2 (10 U / mL) was added. On day 5 of expansion, CD8 +T cells were isolated and prepared for use. Activated CTL cells can be cryopreserved for 6 months. Separately, 1 million MC38 tumor cells were pulsed with SIINFEKL-OVA peptide (1 μg / mL) for 3 hours at 37°C. The cells were washed three times with fresh medium to remove excess peptide. Finally, in a 96-well U-bottom plate, CTL cells pretreated with DGK inhibitor for 1 hour were mixed with antigen-loaded MC38 tumor cells (1:10 ratio). The cells were then centrifuged at 700 rpm for 5 minutes and placed in an incubator overnight at 37°C. After 24 hours, supernatants were collected for analysis of IFN-γ cytokine levels using an AlphaLisa (Perkin Elmer).

[0225] 6. PHA Proliferation Assay Frozen stock solutions of phytohemagglutinin (PHA)-stimulated blast cells were incubated for 1 hour in RPMI medium (Gibco, ThermoFisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO) and added to each well of a 384-well plate (10,000 cells / well). Test compounds were transferred to each well of the 384-well plate, and the treated blast cells were incubated in tissue culture medium containing human IL2 (20 ng / mL) at 37°C and 5% CO2 for 72 hours. The proliferation rate was measured using MTS reagent [3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega, Madison, WI) according to the manufacturer's instructions. Percent inhibition was calculated by comparing the value between IL2 stimulation (0% inhibition) and the unstimulated control (100% inhibition). The determined inhibitory concentration (IC 50 ) was calculated based on 50% inhibition by two-fold dilutions between IL2-stimulated and unstimulated treatments.

[0226] 7. Human CD8 T cell IFN-γ assay Frozen naive human CD8 T cells were thawed in AIM-V medium and incubated at 37°C for 2 hours, followed by cell counting. 384-well tissue culture plates were coated overnight at 4°C with anti-human CD3 / PBS (0.05 μg / mL, 20 μL), which was then removed from the plate. 40,000 cells / CD8 T cells (40 μL) and soluble anti-human CD28 (0.1 μg / mL) were added to each well. After fixing the cells, test compounds were immediately transferred to the cell plate using an Echo liquid handler. After 20 hours of incubation at 37°C, supernatants (3 μL / well) were transferred to new 384-well white assay plates, and cytokines were measured. Interferon-γ (IFN-γ) was quantified using an AlphaLISA kit (Cat#AL217) as described by the manufacturer (Perkin Elmer). The values ​​obtained from each well were converted to IFN-γ concentration (pg / mL). EC 50 Values ​​were determined using anti-CD3 (0.05 μg / mL) and anti-CD28 (0.1 μg / mL) as baseline and costimulation of Example 40 (3 μM) with anti-CD3 and anti-CD28 set as 100% activity.

[0227] 8. Human CD8 T cell pERK assay Frozen naive human CD8 T cells were thawed in AIM-V medium, incubated at 37°C for 2 hours, and counted. + T cells were added to 384-well tissue culture plates at a concentration of 20,000 cells / well in AIM-V medium. One compound was added to each well, followed by bead-immobilized anti-human CD3 and anti-CD28 mAbs at a final concentration of 0.3 μg / mL. Cells were incubated at 37°C for 10 minutes. The reaction was stopped by adding lysis buffer from the AlphaLISA Surefire kit (Perkin Elmer, cat# ALSU-PERK-A). Lysates (5 μL / well) were transferred to a new 384-well white assay plate to measure pERK activity. EC of the compound 50was determined using anti-CD3 and anti-CD28 as baseline and costimulation of 8-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (3 μM) with anti-CD3 and anti-CD28 set as 100% activity.

[0228] 9. Human whole blood IFN-γ assay Human venous whole blood (22.5 μL / well) obtained from healthy donors was pretreated with test compounds for 1 h at 37°C in a humidified incubator with 95% air / 5% CO2. The blood was stimulated with anti-human CD3 (2.5 μL) and anti-CD28 mAb (final concentration 1 μg / mL) for 24 h at 37°C. IFN-γ in the supernatant was measured using an AlphaLISA kit (Cat# AL217). EC of the compound 50 was determined using anti-CD3 and anti-CD28 as baseline and costimulation of 8-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile (3 μM) with anti-CD3 and anti-CD28 set as 100% activity.

[0229] [Table 2] [Table 3]

[0230] Table A shows the in vitro IC50 of DGK inhibition measured by the DGKα and DGKζ liposome (LIPGLO) assay. 50 Activity values ​​are listed. The compounds described herein have inhibitory activity against either or both of the DGKα and DGKζ enzymes and can therefore be used to treat diseases associated with the inhibition of DGKα and DGKζ activity. [Sequence List Free Text]

[0231] Nucleotide sequence of hDGKα-(M1-S735)-Ct-TVMV-His: 0001 ATGGCCAAGG AGAGGGGCCT AATAAGCCCC AGTGATTTTG CCCAGCTGCA 0051 AAAATACATG GAATACTCCA CCAAAAAGGT CAGTGATGTC CTAAAGCTCT 0101 TCGAGGATGG CGAGATGGCT AAATATGTCC AAGGAGATGC CATTGGGTAC 0151 GAGGGATTCC AGCAATTCCT GAAAATCTAT CTCGAAGTGG ATAATGTTCC 0201 CAGACACCTA AGCCTGGCAC TGTTTCAATC CTTTGAGACT GGTCACTGCT 0251 TAAATGAGAC AAATGTGACA AAAGATGTGG TGTGTCTCAA TGATGTTTCC 0301 TGCTACTTTT CCCTTCTGGA GGGTGGTCGG CCAGAAGACA AGTTAGAATT 0351 CACCTTCAAG CTGTACGACA CGGACAGAAA TGGGATCCTG GACAGCTCAG 0401 AAGTGGACAA AATTATCCTA CAGATGATGC GAGTGGCTGA ATACCTGGAT 0451 TGGGATGTGT CTGAGCTGAG GCCGATTCTT CAGGAGATGA TGAAAGAGAT 0501 TGACTATGAT GGCAGTGGCT CTGTCTCTCA AGCTGAGTGG GTCCGGGCTG 0551 GGGCCACCAC CGTGCCACTG CTAGTGCTGC TGGGTCTGGA GATGACTCTG 0601 AAGGACGACG GACAGCACAT GTGGAGGCCC AAGAGGTTCC CCAGACCAGT 0651 CTACTGCAAT CTGTGCGAGT CAAGCATTGG TCTTGGCAAA CAGGGACTGA 0701 GCTGTAACCT CTGTAAGTAC ACTGTTCACG ACCAGTGTGC CATGAAAGCC 0751 CTGCCTTGTG AAGTCAGCAC CTATGCCAAG TCTCGGAAGG ACATTGGTGT 0801 CCAATCACAT GTGTGGGTGC GAGGAGGCTG TGAGTCCGGG CGCTGCGACC 0851 GCTGTCAGAA AAAGATCGG ATCTACCACA GTCTGACCGG GCTGCATTGT 0901 GTATGGTGCC ACCTAGAGAT CCACGATGAC TGCCTGCAAG CGGTGGGCCA 0951 TGAGTGTGAC TGTGGGCTGC TCCGGGATCA CATCCTGCCT CCATCTTCCA 1001 TCTATCCCAG TGTCCTGGCC TCTGGACCGG ATCGTAAAAA TAGCAAAACA 1051 AGCCAGAAGA CCATGGATGA TTTAAATTTG AGCACCTCTG AGGCTCTGCG 1101 GATTGACCCT GTTCCTAACA CCCACCCCACT TCTCGTCTTT GTCAATCCTA 1151 AGAGTGGCGG GAAGCAGGGG CAGAGGGTGC TCTGGAAGTT CCAGTATATA 1201 TTAAACCCTC GACAGGTGTT CAACCTCCTA AAGGATGGTC CTGAGATAGG 1251 GCTCCGATTA TTCAAGGATG TTCCTGATAG CCGGATTTTG GTGTGTGGTG 1301 GAGACGGCAC AGTAGGCTGG ATTCTAGAGA CCATTGACAA AGCTAACTTG 1351 CCAGTTTTGC CTCCTGTTGC TGTGTTGCCC CTGGGTACTG GAAATGATCT 1401 GGCTCGATGC CTAAGATGGG GAGGAGGTTA TGAAGGACAG AATCTGGCAA 1451 AGATCCTCAA GGATTTAGAG ATGAGTAAAG TGGTACATAT GGATCGATGG 1501 TCTGTGGAGG TGATACCTCA ACAAACTGAA GAAAAAAGTG ACCCAGTCCC 1551 CTTTCAAATC ATCAATAACT ACTTCTCTAT TGGCGTGGAT GCCTCTATTG 1601 CTCATCGATT CCACATCATG CGAGAGAAAT ATCCGGAGAA GTTCAACAGC 1651 AGAATGAAGA ACAAGCTATG GTACTTCGAA TTTGCCACAT CTGAATCCAT 1701 CTTCTCCAACA TGCAAAAAGC TGGAGGAGTC TTTGACAGTT GAGATCTGTG 1751 GGAAAACCGCT GGATCTGAGC AACCTGTCCC TAGAAGGCAT CGCAGTGCTA 1801 AACATCCCTA GCATGCATGG TGGCTCCAAC CTCTGGGGTG ATACCAGGAG 1851 ACCCCATGGG GATATCTATG GGATCAACCA GGCCTTAGGT GCTACAGCTA 1901 AAGTCATCAC CGACCCTGAT ATCCTGAAAA CCTGTGTACC AGACCTAAGT 1951 GACAAGAGAC TGGAAGTGGT TGGGCTGGAG GGTGCAATTG AGATGGGCCA 2001 AATCTATACC AAGCTCAAGA ATGCTGGACG TCGGCTGGCC AAGTGCTCTG 2051 AGATCACCTT CCACACCACA AAAACCCTTC CCATGCAAAT TGACGGAGAA 2101 CCCTGGATGC AGACGCCCTG TACAATCAAG ATCACCCACA AGAACCAGAT 2151 GCCCATGCTC ATGGGCCCAC CCCCCCGCTC CACCAATTTC TTTGGCTTCT 2201 TGAGCGGATC CTCGGAGACA GTGCGGTTTC AGGGACACCA CCACCATCAC 2251 CACTGA (SEQ ID NO: 1)

[0232] Amino acid sequence of hDGKα-(M1-S735)-Ct-TVMV-His: 0001 MAKERGLISP SDFAQLQKYM EYSTKKVSDV LKLFEDGEMA KYVQGDAIGY EGFQQFLKIY 0060 0061 LEVDNVPRHL SLALFQSFET GHCLNETNVT KDVVCLNDVS CYFSLLEGGR PEDKLEFTFK 0120 0121 LYDTDRNGIL DSSEVDKIIL QMMRVAEYLD WDVSELRPIL QEMKEIDYD GSGSVSQAEW 0180 0181 VRAGATTVPL LVLLGLEMTL KDDGQHMWRP KRFPRPVYCN LCESSIGLGK QGLSCNLCKY 0240 0241 TVHDQCAMKA LPCEVSTYAK SRKDIGVQSH VWVRGGCESG RCDRCQKKIR IYHSLTGLHC 0300 0301 VWCHLEIHDD CLQAVGHECD CGLLRDHILP PSSIYPSVLA SGPDRKNSKT SQKTMDDLNL 0360 0361 STSEALRIDP VPNTHPLLVF VNPKSGGKQG QRVLWKFQYI LNPRQVFNLL KDGPEIGLRL 0420 0421 FKDVPDSRIL VCGGDGTVGW ILETIDKANL PVLPPVAVLP LGTGNDLARC LRWGGGYEGQ 0480 0481 NLAKILKDLE MSKVVHMDRW SVEVIPQQTE EKSDPVPFQI INNYFSIGVD ASIAHRFHIM 0540 0541 REKYPEKFNS RMKNKLWYFE FATSESIFST CKKLEESLTV EICGKPLDLS NLSLEGIAVL 0600 0601 NIPSMHGGSN LWGDTRRPHG DIYGINQALG ATAKVITDPD ILKTCVPDLS DKRLEVVGLE 0660 0661 GAIEMGQIYT KLKNAGRRLA KCSEITFHTT KTLPMQIDGE PWMQTPCTIK ITHKNQMPML 0720 0721 MGPPPRSTNF FGFLSGSSET VRFQGHHHHH H 0751 (SEQ ID NO: 2)

[0233] Nucleotide sequence of hDGKζ-(M1-A928)-transcript variant-2 Ct-TVMV-His: 0001 ATGGAGCCGC GGGACGGTAG CCCCGAGGCC CGGAGCAGCG ACTCCGAGTC 0051 GGCTTCCGCC TCGTCCAGCG GCTCCGAGCG CGACGCCGGT CCCGAGCCGG 0101 ACAAGGCGCC GCGGCGACTC AACAAGCGGC GCTTCCCGGG GCTGCGGCTC 0151 TTCGGGCACA GGAAAGCCAT CACGAAGTCG GGCCTCCAGC ACCTGGCCC 0201 CCCTCCGCCC ACCCCTGGGG CCCCGTGCAG CGAGTCAGAG CGGCAGATCC 0251 GGAGTACAGT GGACTGGAGC GAGTCAGCGA CATATGGGGGA GCACATCTGG 0301 TTCGAGACCA ACGTGTCCGG GGACTTCTGC TACGTTGGGG AGCAGTACTG 0351 TGTAGCCAGG ATGCTGCAGA AGTCAGTGTC TCGAAGAAAG TGGCCAGCCT 0401 GCAAGATTGT GGTGCACACG CCCTGCATCG AGCAGCTGGA GAAGATAAAT 0451 TTCCGCTGTA AGCCGTCCTT CCGTGAATCA GGCTCCAGGA ATGTCCGCGA 0501 GCCAACCTTT GTACGGCACC ACTGGGTACA CAGACGACGC CAGGACGGCA 0551 AGTGTCGGCA CTGTGGGAAG GGATTCCAGC AGAAGTTCAC CTTCCACAGC 0601 AAGGAGATTG TGGCCATCAG CTGCTCGTGG TGCAAGCAGG CATACCACAG 0651 CAAGGTGTCC TGCTTCATGC TGCAGCAGAT CGAGGAGCCG TGCTCGCTGG 0701 GGGTCCACGC AGCCGTGGTC ATCCCGCCCA CCTGGATCCT CCGCGCCCGG 0751 AGGCCCCAGA ATACTCTGAA AGCAAGCAAG AGAAAAGA GGGCATCCTT 0801 CAAGAGGAAG TCCAGCAAGA AAGGGCCTGA GGAGGGCCGC TGGAGACCCT 0851 TCATCATCAG GCCCACCCCC TCCCCGCTCA TGAAGCCCCT GCTGGTGTTT 0901 GTGAACCCCA AGAGTGGGGG CAACCAGGGT GCAAAGATCA TCCAGTCTTT 0951 CCTCTGGTAT CTCAATCCCC GACAAGTCTT CGACCTGAGC CAGGGAGGGC 1001 CCAAGGAGGC GCTGGAGATG TACCGCAAAG TGCACAACCT GCGGATCCTG 1051 GCGTGCGGGG GCGACGGCAC GGTGGGCTGG ATCCTCTCCA CCCTGGACCA 1101 GCTACGCCTG AAGCCGCCAC CCCCTGTTGC CATCCTGCCC CTGGGTACTG 1151 GCAACGACTT GGCCCGAACC CTCAACTGGG GTGGGGGCTA CACAGATGAG 1201 CCTGTGTCCA AGATCCTCTC CCACGTGGAG GAGGGGAACG TGGTACAGCT 1251 GGACCGCTGG GACCTCCACG CTGAGCCCAA CCCCGAGGCA GGGCCTGAGG 1301 ACCGAGATGA AGGCGCCACC GACCGGTTGC CCCTGGATGT CTTCAACAAC 1351 TACTTCAGCC TGGGCTTTGA CGCCCACGTC ACCCTGGAGT TCCACGAGTC 1401 TCGAGAGGCC AACCCAGAGA AATTCAACAG CCGCTTTCGG AATAAGATGT 1451 TCTACGCCGG GACAGCTTTC TCTGACTTCC TGATGGGCAG CTCCAAGGAC 1501 CTGGCCAAGC ACATCCGAGT GGTGTGTGAT GGAATGGACT TGACTCCCAA 1551 GATCCAGGAC CTGAAACCCC AGTGTGTTGT TTTCCTGAAC ATCCCCAGGT 1601 ACTGTGCGGG CACCATGCCC TGGGGCCACC CTGGGGAGCA CCACGACTTT 1651 GAGCCCCAGC GGCATGACGA CGGCTACCTC GAGGTCATTG GCTTCACCAT 1701 GACGTCGTTG GCCGCGCTGC AGGTGGGCGG ACACGGCGAG CGGCTGACGC 1751 AGTGTCGCGA GGTGGTGCTC ACCACATCCA AGGCCATCCC GGTGCAGGTG 1801 GATGGCGAGC CCTGCAAGCT TGCAGCCTCA CGCATCCGCA TCGCCCTGCG 1851 CAACCAGGCC ACCATGGTGC AGAAGGCCAA GCGGCGGAGC GCCGCCCCCC 1901 TGCACAGCGA CCAGCAGCCG GTGCCAGAGC AGTTGCGCAT CCAGGTGAGT 1951 CGCGTCAGCA TGCACGACTA TGAGGCCCTG CACTACGACA AGGAGCAGCT 2001 CAAGGAGGCC TCTGTGCCGC TGGGCACTGT GGTGGTCCCA GGAGACAGTG 2051 ACCTAGAGCT CTGCCGTGCC CACATTGAGA GACTCCAGCA GGAGCCCGAT 2101 GGTGCTGGAG CCAAGTCCCC GACATGCCAG AAACTGTCCC CCAAGTGGTG 2151 CTTCCTGGAC GCCACCACTG CCAGCCGCTT CTACAGGATC GACCGAGCCC 2201 AGGAGCACCT CAACTATGTG ACTGAGATCG CACAGGATGA GATTTATATC 2251 CTGGACCCTG AGCTGCTGGG GGCATCGGCC CGGCCTGACC TCCCAACCCC 2301 CACTTCCCCT CTCCCCACCT CACCCTGCTC ACCCACGCCC CGGTCACTGC 2351 AAGGGGATGC TGCACCCCCT CAAGGTGAAG AGCTGATTGA GGCTGCCAAG 2401 AGGAACGACT TCGTTAAGCT CCAGGAGCTG CACCGAGCTG GGGGCGACCT 2451 CATGCACCGA GACGAGCAGA GTCGCACGCT CCTGCACCAC GCAGTCAGCA 2501 CTGGCAGCAA GGATGTGGTC CGCTACCTGC TGGACCACGC CCCCCCAGAG 2551 ATCCTTGATG CGGTGGAGGA AAACGGGGAG ACCTGTTTGC ACCAAGCAGC 2601 GGCCCTGGGC CAGCGCACCA TCTGCCACTA CATCGTGGAG GCCGGGGCCT 2651 CGCTCATGAA GACAGACCAG CAGGGCGACA CTCCCCGGCA GCGGGCTGAG 2701 AAGGCTCAGG ACACCGAGCT GGCCGCCTAC CTGGAGAACC GGCAGCACTA 2751 CCAGATGATC CAGCGGGAGG ACCAGGAGAC GGCTGTGGGA TCCTCGGAGA 2801 CAGTGCGGTT TCAGGGACAC CACCACCATC ACCACTGA (SEQ ID NO: 3)

[0234] Amino acid sequence of hDGKζ-(M1-A928)-transcript variant-2 Ct-TVMV-His: 0001 MEPRDGSPEA RSSDSESASA SSSGSERDAG PEPDKAPRRL NKRRFPGLRL FGHRKAITKS 0060 0061 GLQHLAPPPP TPGAPCSESE RQIRSTVDWS ESATYGEHIW FETNVSGDFC YVGEQYCVAR 0120 0121 MLQKSVSRRK CAACKIVVHT PCIEQLEKIN FRCKPSFRES GSRNVREPTF VRHHWVHRRR 0180 0181 QDGKCRHCGK GFQQKFTFHS KEIVAISCSW CKQAYHSKVS CFMLQQIEEP CSLGVHAAVV 0240 0241 IPPTWILRAR RPQNTLKASK KKKRASFKRK SSKKGPEEGR WRPFIIRPTP SPLMKPLLVF 0300 0301 VNPKSGGNQG AKIIQSFLWY LNPRQVFDLS QGGPKEALEM YRKVHNLRIL ACGGDGTVGW 0360 0361 ILSTLDQLRL KPPPPVAILP LGTGNDLART LNWGGGYTDE PVSKILSHVE EGNVVQLDRW 0420 0421 DLHAEPNPEA GPEDRDEGAT DRLPLDVFNN YFSLGFDAHV TLEFHESREA NPEKFNSRFR 0480 0481 NKMFYAGTAF SDFLMGSSKD LAKHIRVVCD GMDLTPKIQD LKPQCVVFLN IPRYCAGTMP 0540 0541 WGHPGEHHDF EPQRHDDGYL EVIGFTMTSL AALQVGGHGE RLTQCREVVL TTSKAIPVQV 0600 0601 DGEPCKLAAS RIRIALRNQA TMVQKAKRRS AAPLHSDQQP VPEQLRIQVS RVSMHDYEAL 0660 0661 HYDKEQLKEA SVPLGTVVVP GDSDLELCRA HIERLQQEPD GAGAKSPTCQ KLSPKWCFLD 0720 0721 ATTASRFYRI DRAQEHLNYV TEIAQDEIYI LDPELLGASA RPDLPTPTSP LPTSPCSPTP 0780 0781 RSLQGDAAPP QGEELIEAAK RNDFCKLQEL HRAGGDLMHR DEQSRTLLHH AVSTGSKDVV 0840 0841 RYLLDHAPPE ILDAVEENGE TCLHQAAALG QRTICHYIVE AGASLMKTDQ QGDTPRQRAE 0900 0901 KAQDTELAAY LENRQHYQMI QREDQETAVG SSETVRFQGH HHHHH 0945 (SEQ ID NO: 4)

Claims

1. A method for treating cancer, comprising administering to a subject therapeutically effective amounts of an inhibitor of DGKα and / or DGKζ and an antagonist of PD1 / PD-L1 binding.

2. A method for treating cancer, comprising administering to a subject therapeutically effective amounts of a DGKα and / or DGKζ inhibitor and a CTLA4 antagonist.

3. A method for treating cancer, comprising administering to a subject therapeutically effective amounts of an inhibitor of DGKα and / or DGKζ, an antagonist of PD1 / PD-L1 binding, and an antagonist of CTLA4.

4. The method according to any one of claims 1 to 3, wherein the inhibitor of human DGKα and / or DGKζ is an inhibitor of DGKα and does not significantly inhibit DGKζ.

5. The method according to any one of claims 1 to 3, wherein the inhibitor of DGKα and / or DGKζ is an inhibitor of DGKζ and does not significantly inhibit DGKα.

6. The method according to any one of claims 1 to 5, wherein the inhibitor of DGKα and / or DGKζ is an inhibitor of DGKα and DGKζ.

7. The method according to any one of claims 1 to 6, wherein the inhibitor of DGKα and / or DGKζ does not significantly inhibit other DGKs.

8. The method of any one of claims 1 and 3 to 7, wherein the antagonist of PD1 / PD-L1 binding is a PD1 antagonist, for example of human PD1.

9. 9. The method of claim 8, wherein the PD-1 antagonist is nivolumab, pembrolizumab, or any other PD-1 antagonist described herein.

10. The method of any one of claims 1 and 3 to 7, wherein the antagonist of PD1 / PD-L1 binding is a PD-L1 antagonist, for example of human PD-L1.

11. 11. The method of claim 10, wherein the PD-L1 antagonist is atezolizumab or any other PD-L1 antagonist described herein.

12. The method of any one of claims 2 to 11, wherein the CTLA4 antagonist is ipilimumab or any other CTLA4 antagonist described herein.

13. The method of any one of claims 1 to 12, wherein the DGKα and / or DGKζ antagonist activates primary T cell signaling, for example, as evidenced by activation of pERK / pPKC signaling.

14. The method of any one of claims 1 to 13, wherein the inhibitor of DGKα and / or DGKζ lowers the threshold for antigen stimulation and reduces the affinity and / or antigen concentration required for T cell antigen recognition and activation.

15. The method of any one of claims 1 to 14, wherein the inhibitor of DGKα and / or DGKζ activates CTL effector function.

16. The method of any one of claims 1 to 15, wherein the inhibitor of DGKα and / or DGKζ enhances tumor cell killing activity.

17. The antitumor activity of inhibitors of DGKα and / or DGKζ in the CT26 animal model was confirmed by CD8 + The method according to any one of claims 1 to 16, wherein the method is influenced by T cells.

18. The method according to any one of claims 1 to 17, wherein the antitumor activity of an inhibitor of DGKα and / or DGKζ in a CT26 animal model is influenced by NK cells.

19. The method according to any one of claims 1 to 18, wherein the antitumor activity of an inhibitor of DGKα and / or DGKζ in a CT26 animal model is activated by depletion of CD4 cells.

20. The method of any one of claims 1 to 19, wherein an inhibitor of DGKα and / or DGKζ activates the appearance of AH1+ tetramer antigen in a CT26 animal model or restores the reduced T cell effector function due to the reduced amount of B2M.

21. The inhibitor of DGKα and / or DGKζ is represented by the formula (I): 【Chemistry 1】 [In the formula, R 1 is H, F, Cl, Br, -CN, 0 to 4 R 1a C replaced with 1-3 Alkyl, 0 to 4 R 1a C replaced with 3-4 cycloalkyl, 0 to 4 R 1a C replaced with 1-3 Alkoxy, -NR a R a , -S(O) n R e or -P(O)R e R e and; Each R 1a are independently F, Cl, -CN, -OH, -OCH 3 or -NR a R a and; Each R a are independently H or C 1-3 is alkyl; Each R e independently, C 3-4 Cycloalkyl or 0 to 4 R 1a C replaced with 1-3 is alkyl; R 2 is H, 0 to 4 R 2a C replaced with 1-3 Alkyl or 0 to 4 R 2a C replaced with 3-4 is cycloalkyl; Each R 2a are independently F, Cl, -CN, -OH, -O(C 1-2 alkyl), C 3-4 Cycloalkyl, C 3-4 Alkenyl or C 3-4 is alkynyl; R 3 are H, F, Cl, Br, -CN, C 1-3 Alkyl, C 1-2 Fluoroalkyl, C 3-4 Cycloalkyl, C 3-4 Fluorocycloalkyl or -NO 2 and; R 4 is -CH 2 R 4a , -CH 2 CH 2 R 4a , -CH 2 CHR 4a R 4d , -CHR 4a R 4b or -CR 4a R 4b R 4c and; R 4a and R 4b is independently (I C 1-6 Alkyl, F, Cl, -CN, -OH, -OCH 3 , -SCH 3 , C 1-3 Fluoroalkoxy, -NR a R a , -S(O) 2 R e or -NR a S(O) 2 R e substituted with 0 to 4 substituents independently selected from: (ii) C 3-6 cycloalkyl, heterocyclyl, phenyl, or heteroaryl, each of which is F, Cl, Br, -CN, -OH, or C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-4 Hydroxyalkyl, -(CH 2 ) 1-2 O(C 1-3 alkyl), C 1-4 Alkoxy, -O(C 1-4 hydroxyalkyl), -O(CH) 1-3 O(C 1-3 alkyl), C 1-3 Fluoroalkoxy, -O(CH) 1-3 NR c R c , -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -NR c R c , -NR a S(O) 2 (C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl), -P(O)(C 1-3 alkyl) 2 , -S(O) 2 (C 1-3 alkyl), -O(CH 2 ) 1-2 (C 3-6 cycloalkyl), -O(CH 2 ) 1-2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butoxycarbonyl)azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thiophenyl, methylpiperidinyl, and R d or (iii) C substituted with one cyclic group 1-4 alkyl, and the cyclic group is C 3-6 cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the cyclic group is selected from F, Cl, Br, —OH, —CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -NR c R c , -NR a S(O) 2 (C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl) and C 3-6 substituted with 0 to 3 substituents independently selected from cycloalkyl; R 4a and R 4b together with the carbon atoms to which they are bonded, 3-6 cycloalkyl or 3- to 6-membered heterocyclyl, each of which has 0 to 3 R f is replaced by; Each R f are independently F, Cl, Br, -OH, -CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -NR c R c or a cyclic group, the cyclic group being C 3-6 cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being selected from F, Cl, Br, —OH, —CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy and -NR c R c substituted with 0 to 3 substituents independently selected from: R 4c is C 1-6 Alkyl or C 3-6 cycloalkyl, F, Cl, -OH, C 1-2 Alkoxy, C 1-2 substituted with 0 to 4 substituents independently selected from fluoroalkoxy and —CN; R 4d -OCH 3 and; Each R c are independently H or C 1-2 is alkyl; R d is phenyl and is not F, Cl, -CN, -CH 3 and -OCH 3 substituted with 0 to 1 substituents selected from: Each R 5 are independently -CN, 0 to 4 R g C replaced with 1-6 Alkyl, 0 to 4 R g C replaced with 2-4 Alkenyl, 0 to 4 R g C replaced with 2-4 Alkynyl, 0-4 R g C replaced with 3-4 cycloalkyl, 0 to 4 R g phenyl substituted with 0 to 3 R g oxadiazolyl substituted with 0 to 4 R g pyridinyl substituted with -(CH 2 ) 1-2 (0 to 4 R g heterocyclyl substituted with -(CH 2 ) 1-2 NR c C(O)(C 1-4 alkyl), -(CH 2 ) 1-2 NR c C(O)O(C 1-4 alkyl), -(CH 2 ) 1-2 NR c S(O) 2 (C 1-4 alkyl), -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -C(O)O(C 3-4 cycloalkyl), -C(O)NR a R a or -C(O)NR a (C 3-4 cycloalkyl); Each R g are independently F, Cl, -CN, -OH, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -O(CH 2 ) 1-2 O(C 1-2 alkyl) or -NR c R c and; m is 0, 1, 2 or 3; and n is 0, 1 or 2. or a pharmaceutically acceptable salt thereof.

22. Inhibitors of DGKα and / or DGKζ R 1 is H, F, Cl, Br, -CN, 0 to 4 R 1a C replaced with 1-3 Alkyl, 0 to 3 R 1a cyclopropyl substituted with 0-3 R 1a C replaced with 1-3 Alkoxy, -NR a R a , -S(O) n CH 3 or -P(O)(CH 3 ) 2 and; Each R 1a is independently F, Cl, or —CN; Each R a are independently H or C 1-3 is alkyl; R 2 is H or 0-2 R 2a C replaced with 1-2 is alkyl; Each R 2a are independently F, Cl, -CN, -OH, -O(C 1-2 alkyl), cyclopropyl, C 3-4 Alkenyl or C 3-4 is alkynyl; R 3 H, F, Cl, Br, -CN, C 1-2 Alkyl, -CF 3 , cyclopropyl or -NO 2 and; R 4a and R 4b But independently (I C 1-4 Alkyl, F, Cl, -CN, -OH, -OCH 3 , -SCH 3 , C 1-3 Fluoroalkoxy and -NR a R a substituted with 0 to 4 substituents independently selected from: (ii) C 3-6 cycloalkyl, heterocyclyl, phenyl, or heteroaryl, each of which is F, Cl, Br, -CN, -OH, or C 1-6 Alkyl, C 1-3 Fluoroalkyl, -CH 2 OH, -(CH 2 ) 1-2 O(C 1-2 alkyl), C 1-4 Alkoxy, -O(C 1-4 hydroxyalkyl), -O(CH) 1-2 O(C 1-2 alkyl), C 1-3 Fluoroalkoxy, -O(CH) 1-2 NR c R c , -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -NR c R c , -NR a S(O) 2 (C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl), -P(O)(C 1-2 alkyl) 2 , -S(O) 2 (C 1-3 alkyl), -O(CH 2 ) 1-2 (C 3-4 cycloalkyl), -O(CH 2 ) 1-2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butoxycarbonyl)azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thiophenyl, methylpiperidinyl, and R d or (iii) C substituted with one cyclic group 1-3 alkyl, and the cyclic group is C 3-6 cycloalkyl, heterocyclyl, phenyl, and heteroaryl, wherein the cyclic group is selected from F, Cl, Br, —OH, —CN, C 1-3 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy, -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -NR c R c , -NR a S(O) 2 (C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl) and C 3-4 substituted with 0 to 3 substituents independently selected from cycloalkyl; R 4a and R 4b together with the carbon atoms to which they are bonded, 3-6 cycloalkyl or 3- to 6-membered heterocyclyl, each of which has 0 to 3 R f is replaced by; Each R f are independently F, Cl, Br, -OH, -CN, C 1-4 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy, -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -NR c R c or a cyclic group, the cyclic group being C 3-6 cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being selected from F, Cl, Br, —OH, —CN, C 1-4 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy and -NR c R c substituted with 0 to 3 substituents independently selected from: R 4c But C 1-4 Alkyl or C 3-6 cycloalkyl, F, Cl, -OH, C 1-2 Alkoxy, C 1-2 substituted with 0 to 4 substituents independently selected from fluoroalkoxy and —CN; and Each R 5 are independently -CN, 0 to 4 R g C replaced with 1-5 Alkyl, 0 to 4 R g C replaced with 2-3 Alkenyl, 0 to 4 R g C replaced with 2-3 Alkynyl, 0-4 R g C replaced with 3-4 cycloalkyl, 0 to 3 R g phenyl substituted with 0 to 3 R g oxadiazolyl substituted with 0 to 3 R g pyridinyl substituted with -(CH 2 ) 1-2 (0 to 4 R g heterocyclyl substituted with -(CH 2 ) 1-2 NR c C(O)(C 1-4 alkyl), -(CH 2 ) 1-2 NR c C(O)O(C 1-4 alkyl), -(CH 2 ) 1-2 NR c S(O) 2 (C 1-4 alkyl), -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -C(O)O(C 3-4 cycloalkyl), -C(O)NR a R a or -C(O)NR a (C 3-4 cycloalkyl), 22. The method of claim 21, wherein the compound is a compound of formula (I) or a pharmaceutically acceptable salt thereof.

23. An inhibitor of DGKα and / or DGKζ has the following structure: 【change】 [In the formula, R 1 is -CN; R 2 is -CH 3 and; R 3 is H, F or -CN; R 4 teeth, 【Chemistry 2】 is] 23. The method of claim 22, wherein the compound is a compound of formula (I) having the formula:

24. An inhibitor of DGKα and / or DGKζ has the following structure: 【Transformation 3】 【Chemistry 4】 22. The method of claim 21, wherein the compound is a compound of formula (I) having the formula:

25. The inhibitor of DGKα and / or DGKζ is represented by the formula (II): 【Transformation 5】 [In the formula, R 1 is H, F, Cl, Br, -CN, -OH, 0 to 4 R 1a C replaced with 1-3 Alkyl, 0 to 4 R 1a C replaced with 3-4 cycloalkyl, 0 to 4 R 1a C replaced with 1-3 Alkoxy, -NR a R a , -S(O) n R e or -P(O)R e R e and; Each R 1a are independently F, Cl, -CN, -OH, -OCH 3 or -NR a R a and; Each R a are independently H or C 1-3 is alkyl; Each R e independently, C 3-4 Cycloalkyl or 0 to 4 R 1a C replaced with 1-3 is alkyl; R 2 is H, 0 to 4 R 2a C replaced with 1-3 Alkyl or 0 to 4 R 2a C replaced with 3-4 is cycloalkyl; Each R 2a are independently F, Cl, -CN, -OH, -O(C 1-2 alkyl), C 3-4 Cycloalkyl, C 3-4 Alkenyl or C 3-4 is alkynyl; R 4 is -CH 2 R 4a , -CH 2 CH 2 R 4a , -CH 2 CHR 4a R 4d , -CHR 4a R 4b or -CR 4a R 4b R 4c and; R 4a and R 4b is independently (i) -CN or C 1-6 Alkyl, F, Cl, -CN, -OH, -OCH 3 , -SCH 3 , C 1-3 Fluoroalkoxy, -NR a R a , -S(O) 2 R e or -NR a S(O) 2 R e substituted with 0 to 4 substituents independently selected from: (ii) C 3-6 cycloalkyl, 4- to 10-membered heterocyclyl, phenyl, or 5- to 10-membered heteroaryl, each of which is F, Cl, Br, —CN, —OH, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-2 Bromoalkyl, C 1-2 Cyanoalkyl, C 1-4 Hydroxyalkyl, -(CH 2 ) 1-2 O(C 1-3 alkyl), C 1-4 Alkoxy, C 1-3 Fluoroalkoxy, C 1-3 Cyanoalkoxy, -O(C 1-4 hydroxyalkyl), -O(CR x R x ) 1-3 O(C 1-3 alkyl), C 1-3 Fluoroalkoxy, -O(CH 2 ) 1-3 NR c R c , -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -NR c R c , -CH 2 NR a R a , -NR a S(O) 2 (C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -(CR x R x ) 0-2 NR a C(O)O(C 1-4 alkyl), -P(O)(C 1-3 alkyl) 2 , -S(O) 2 (C 1-3 alkyl), -(CR x R x ) 1-2 (C 3-4 cycloalkyl), -(CR x R x ) 1-2 (morpholinyl), -(CR x R x ) 1-2 (difluoromorpholinyl), -(CR x R x ) 1-2 (dimethylmorpholinyl), -(CR x R x ) 1-2 (oxazabicyclo[2.2.1]heptanyl), (CR x R x ) 1-2 (oxazaspiro[3.3]heptanyl), -(CR x R x ) 1-2 (methylpiperazinonyl), -(CR x R x ) 1-2 (acetylpiperazinyl), -(CR x R x ) 1-2 (piperidinyl), -(CR x R x ) 1-2 (difluoropiperidinyl), -(CR x R x ) 1-2 (Methoxypiperidinyl), -(CR x R x ) 1-2 (hydroxypiperidinyl), -O(CR x R x ) 0-2 (C 3-6 cycloalkyl), -O(CR x R x ) 0-2 (methylcyclopropyl), -O(CR x R x ) 0-2 ((ethoxycarbonyl)cyclopropyl), -O(CR x R x ) 0-2 (oxetanyl), -O(CR x R x ) 0-2 (methylazetidinyl), -O(CR x R x ) 0-2 (tetrahydropyranyl), -O(CR x R x ) 1-2 (morpholinyl), -O(CR x R x ) 0-2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butoxycarbonyl)azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thiophenyl, methylpiperidinyl, dioxolanyl, pyrrolidinonyl and R d or (iii) C substituted with one cyclic group 1-4 alkyl, and the cyclic group is C 3-6 cycloalkyl, 4- to 10-membered heterocyclyl, monocyclic or bicyclic aryl or 5- to 10-membered heteroaryl, wherein the cyclic group is selected from F, Cl, Br, —OH, —CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -NR c R c , -NR a S(O) 2 (C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl) and C 3-6 substituted with 0 to 3 substituents independently selected from cycloalkyl; R 4a and R 4b together with the carbon atoms to which they are bonded, 3-6 cycloalkyl or 3- to 6-membered heterocyclyl, each of which has 0 to 3 R f is replaced by; Each R f are independently F, Cl, Br, -OH, -CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -NR c R c or a cyclic group, the cyclic group being C 3-6 cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being selected from F, Cl, Br, —OH, —CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy and -NR c R c substituted with 0 to 3 substituents independently selected from: R 4c is C 1-6 Alkyl or C 3-6 cycloalkyl, F, Cl, -OH, C 1-2 Alkoxy, C 1-2 substituted with 0 to 4 substituents independently selected from fluoroalkoxy and —CN; R 4d -OCH 3 and; Each R c are independently H or C 1-2 is alkyl; R d is phenyl and is not F, Cl, -CN, -CH 3 and -OCH 3 substituted with 0 to 1 substituents selected from: Each R 5 are independently -CN, 0 to 4 R g C replaced with 1-6 Alkyl, 0 to 4 R g C replaced with 2-4 Alkenyl, 0 to 4 R g C replaced with 2-4 Alkynyl, 0-4 R g C replaced with 3-4 cycloalkyl, 0 to 4 R g phenyl substituted with 0 to 3 R g oxadiazolyl substituted with 0 to 4 R g pyridinyl substituted with -(CH 2 ) 1-2 (0 to 4 R g 4- to 10-membered heterocyclyl substituted with -(CH 2 ) 1-2 NR c C(O)(C 1-4 alkyl), -(CH 2 ) 1-2 NR c C(O)O(C 1-4 alkyl), -(CH 2 ) 1-2 NR c S(O) 2 (C 1-4 alkyl), -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -C(O)O(C 3-4 cycloalkyl), -C(O)NR a R a or -C(O)NR a (C 3-4 cycloalkyl); Each R g are independently F, Cl, -CN, -OH, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -O(CH 2 ) 1-2 O(C 1-2 alkyl) or -NR c R c and; m is 0, 1, 2 or 3; and n is 0, 1 or 2. or a salt thereof.

26. The inhibitor of DGKα and / or DGKζ is of the formula R 1 is H, F, Cl, Br, -CN, -OH, 0 to 4 R 1a C replaced with 1-3 Alkyl, 0 to 3 R 1a cyclopropyl substituted with 0-3 R 1a C replaced with 1-3 Alkoxy, -NR a R a , -S(O) n CH 3 or -P(O)(CH 3 ) 2 and; R 2 is H or 0-2 R 2a C replaced with 1-2 is alkyl; Each R 2a are independently F, Cl, -CN, -OH, -O(C 1-2 alkyl), cyclopropyl, C 3-4 Alkenyl or C 3-4 is alkynyl; R 4a and R 4b But independently (i) -CN or C 1-4 Alkyl, F, Cl, -CN, -OH, -OCH 3 , -SCH 3 , C 1-3 Fluoroalkoxy and -NR a R a substituted with 0 to 4 substituents independently selected from: (ii) C 3-6 cycloalkyl, 4- to 10-membered heterocyclyl, phenyl, or 5- to 10-membered heteroaryl, each of which is F, Cl, Br, —CN, —OH, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-2 Bromoalkyl, C 1-2 Cyanoalkyl, C 1-2 Hydroxyalkyl, -CH 2 NR a R a , -(CH 2 ) 1-2 O(C 1-2 alkyl), -(CH 2 ) 1-2 NR x C(O)O(C 1-2 alkyl), C 1-4 Alkoxy, -O(C 1-4 hydroxyalkyl), -O(CR x R x ) 1-2 O(C 1-2 alkyl), C 1-3 Fluoroalkoxy, C 1-3 Cyanoalkoxy, -O(CH 2 ) 1-2 NR c R c , -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -NR c R c , -NR a S(O) 2 (C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl), -P(O)(C 1-2 alkyl) 2 , -S(O) 2 (C 1-3 alkyl), -(CH 2 ) 1-2 (C 3-4 cycloalkyl), -CR x R x (morpholinyl), -CR x R x (difluoromorpholinyl), -CR x R x (dimethylmorpholinyl), -CR x R x (oxazabicyclo[2.2.1]heptanyl), -CR x R x (oxazaspiro[3.3]heptanyl), -CR x R x (methylpiperazinonyl), -CR x R x (acetylpiperazinyl), -CR x R x (piperidinyl), -CR x R x (difluoropiperidinyl), -CR x R x (methoxypiperidinyl), -CR x R x (hydroxypiperidinyl), -O(CH 2 ) 0-2 (C 3-4 cycloalkyl), -O(CH 2 ) 0-2 (methylcyclopropyl), -O(CH 2 ) 0-2 ((ethoxycarbonyl)cyclopropyl), -O(CH 2 ) 0-2 (oxetanyl), -O(CH 2 ) 0-2 (methylazetidinyl), -O(CH 2 ) 1-2 (morpholinyl), -O(CH 2 ) 0-2 (tetrahydropyranyl), -O(CH 2 ) 0-2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butoxycarbonyl)azetidinyl, dioxolanyl, pyrrolidinonyl, triazolyl, tetrahydropyranyl, morpholinyl, thiophenyl, methylpiperidinyl, and R d or (iii) C substituted with one cyclic group 1-3 alkyl, and the cyclic group is C 3-6 cycloalkyl, 4- to 10-membered heterocyclyl, monocyclic or bicyclic aryl or 5- to 10-membered heteroaryl, wherein the cyclic group is selected from F, Cl, Br, —OH, —CN, C 1-3 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy, -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -NR c R c , -NR a S(O) 2 (C 1-3 alkyl), -NR a C(O)(C 1-3 alkyl), -NR a C(O)O(C 1-4 alkyl) and C 3-4 substituted with 0 to 3 substituents independently selected from cycloalkyl; R 4a and R 4b However, together with the carbon atoms that bond to them, 3-6 cycloalkyl or 3- to 6-membered heterocyclyl, each of which has 0 to 3 R f is replaced by; Each R f are independently F, Cl, Br, -OH, -CN, C 1-4 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy, -OCH 2 CH=CH 2 , -OCH 2 C≡CH, -NR c R c or a cyclic group, C 3-6 cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being selected from F, Cl, Br, —OH, —CN, C 1-4 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy and -NR c R c substituted with 0 to 3 substituents independently selected from: R 4c But C 1-4 Alkyl or C 3-6 cycloalkyl, F, Cl, -OH, C 1-2 Alkoxy, C 1-2 substituted with 0 to 4 substituents independently selected from fluoroalkoxy and —CN; Each R 5 are independently -CN, 0 to 4 R g C replaced with 1-5 Alkyl, 0 to 4 R g C replaced with 2-3 Alkenyl, 0 to 4 R g C replaced with 2-3 Alkynyl, 0-4 R g C replaced with 3-4 cycloalkyl, 0 to 3 R g phenyl substituted with 0 to 3 R g oxadiazolyl substituted with 0 to 3 R g pyridinyl substituted with -(CH 2 ) 1-2 (0 to 4 R g 4- to 10-membered heterocyclyl substituted with -(CH 2 ) 1-2 NR c C(O)(C 1-4 alkyl), -(CH 2 ) 1-2 NR c C(O)O(C 1-4 alkyl), -(CH 2 ) 1-2 NR c S(O) 2 (C 1-4 alkyl), -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -C(O)O(C 3-4 cycloalkyl), -C(O)NR a R a or -C(O)NR a (C 3-4 cycloalkyl); Each R x are independently H or -CH 3 and m is 1, 2 or 3; 26. The method of claim 25, wherein the compound is a compound of formula (II) or a pharmaceutically acceptable salt thereof:

27. An inhibitor of DGKα and / or DGKζ has the following structure: 【Transformation 6】 [In the formula, R 1 is -CN; R 2 is -CH 3 and; R 5a is -CH 3 or -CH 2 CH 3 and R 5c is -CH 3 , -CH 2 CH 3 or -CH 2 CH 2 CH 3 is] 27. The method of claim 26, wherein the compound is a compound of formula (II) having the formula:

28. An inhibitor of DGKα and / or DGKζ has the following structure: 【Transformation 7】 【Transformation 8】 26. The method of claim 25, wherein the compound is a compound of formula (II) having the formula:

29. The method of any one of claims 1 to 28, wherein the cancer is a solid tumor or a blood (liquid) tumor.

30. 30. The method of any one of claims 1 to 29, wherein the cancer is selected from the group of cancers described herein.

31. 31. The method according to any one of claims 1 to 30, characterized in that the method comprises one or more other cancer treatments.

32. 32. The method of claim 31, wherein the one or more other cancer treatments include radiation therapy, surgery, chemotherapy, or administration of a biopharmaceutical.

33. 32. The method of claim 31, wherein the one or more other cancer treatments is administration of a biopharmaceutical, and the biopharmaceutical is an agent that stimulates the immune system.

34. The method according to any one of claims 1 to 30, characterized in that no other cancer treatment is performed during treatment with the inhibitor of DGKα and / or DGKζ, the antagonist of PD1 / PD-L1 binding and / or the antagonist of CTLA4.

35. The method of any one of claims 1 to 34, wherein the subject has not been treated with an antagonist of PD1 / PD-L1 binding or an antagonist of CTLA4 prior to administration of the inhibitor of DGKα and / or DGKζ, the antagonist of PD1 / PD-L1 binding and / or the antagonist of CTLA4.

36. The method of claim 35, characterized in that an inhibitor of DGKα and / or DGKζ, an antagonist of PD1 / PD-L1 binding, and an antagonist of CTLA4 are administered to the subject.

37. 35. The method of any one of claims 1 to 34, wherein the subject is resistant or refractory to treatment with a checkpoint inhibitor antagonist (e.g., an antagonist of PD1 / PD-L1 binding and / or an antagonist of CTLA4).

38. The method of claim 37, characterized in that an inhibitor of DGKα and / or DGKζ, an antagonist of PD1 / PD-L1 binding, and an antagonist of CTLA4 are administered to the subject.

39. 26. The method of claim 21 or claim 25, comprising administering to the subject an antagonist of PD1 / PD-L1 binding and an antagonist of CTLA4.

40. 40. The method of any one of claims 1 to 39, comprising administering to the subject an antagonist of PD1 / PD-L1 binding and an antagonist of CTLA4, wherein the antagonist of PD1 / PD-L1 binding is a PD1 / PD-L1 or CTLA4 antagonist described herein or a variant or derivative thereof.

41. 41. The method of claim 40, wherein the antagonist of PD1 / PD-L1 binding is nivolumab or a variant thereof, and the antagonist of CTLA4 is ipilimumab or a variant thereof (e.g., a variant that is less toxic compared to ipilimumab).

42. The method of any one of claims 1 to 3 and 6 to 41, wherein the inhibitor of DGKα and / or DGKζ is an inhibitor of DGKα and DGKζ.

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