DGK inhibitor and checkpoint antagonist combination

Abstract

Combination of DGK inhibitors and checkpoint antagonists The present disclosure provides inhibitors of diacylglycerol kinase (DGK) and methods for treating diseases that may benefit from stimulating the immune system (e.g., cancer and infectious diseases), comprising 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 encompass numerous genetic and epigenetic variations and produce neoantigens that the immune system may recognize (Sjoblom et al. (2006) Science 314:268-74). The adaptive immune system, composed of T and B lymphocytes, possesses the potential to exhibit potent anti-cancer activity, exhibiting broad capabilities and high specificity to respond to diverse tumor antigens. Furthermore, this immune system has considerable flexibility and memory components. By effectively utilizing all these characteristics of the adaptive immune system, immunotherapy becomes a distinctive approach among all cancer treatments. However, although an endogenous immune response to cancer has been observed in preclinical models and patients, this response is ineffective, and established cancers are recognized as "self," exhibiting resistance to the immune system. This acceptance allows tumors to actively overcome anti-tumor immunity by utilizing multiple different mechanisms. These mechanisms include signaling from dysfunctional T cells (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 reduce the intensity of the adaptive immune response, protect normal tissue from secondary damage by tumors, and 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 mediated the cessation of T cell function via the TCR signaling pathway. Therefore, DGK is expected to function as an intracellular checkpoint, and inhibiting DGK is expected to enhance T cell signaling pathways and T cell activation. Supporting this is evidence that knockout mouse models of either DGKα or DGKζ exhibit hyperreactive T cell phenotypes 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 extracted from human renal cell carcinoma patients have been observed to overexpress DGKα, which inhibits 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). [Overview of the project]

[0004] This application provides a method for treating a disease or disorder, characterized by administering an inhibitor of DGKα, DGKζ, or both DGKα and DGKζ (e.g., a compound of formula (I) or (II), for example, a compound selected from compounds 1 to 34, or a pharmaceutically acceptable salt thereof) to a subject in combination with a PD1 / PD-L1-binding antagonist and / or a CTLA4 antagonist. Examples of diseases or disorders for which effects can be obtained by stimulating the immune system include, for example, cancer and infectious diseases. The 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), for example, a compound selected from compounds 1 to 34, or a pharmaceutically acceptable salt thereof) for use in the manufacture of a pharmaceutical for treating a disease or disorder for which effects can be obtained by stimulating the immune system (e.g., cancer and infectious diseases). Herein, the inhibitor is administered in combination with a PD1 / PD-L1-binding antagonist and / or a CTLA4 antagonist. The present invention relates to inhibitors of DGKα, DGKζ, or both DGKα and DGKζ (e.g., compounds of formula (I) or (II), for example, compounds selected from compounds 1 to 34 or pharmaceutically acceptable salts thereof) for manufacturing pharmaceuticals for treating diseases or disorders (e.g., cancer and infectious diseases) in which the effect can be obtained by stimulating the immune system. The inhibitor is administered in combination with a PD1 / PD-L1 antagonist and a CTLA4 antagonist.

[0005] Furthermore, a PD1 / PD-L1 antagonist is used for the manufacture of pharmaceuticals for treating diseases or disorders (e.g., cancer and infectious diseases) in which the effects can be obtained by stimulating the immune system. Here, 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), for example, a compound selected from compounds 1 to 34, or a pharmaceutically acceptable salt thereof) and / or an antagonist of CTLA4. The present invention uses a PD1 / PD-L1 antagonist for the manufacture of pharmaceuticals for treating diseases or disorders (e.g., cancer and infectious diseases) in which the effects can be obtained by stimulating the immune system. Here, 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), for example, a compound selected from compounds 1 to 34, or a pharmaceutically acceptable salt thereof) and an antagonist of CTLA4.

[0006] Furthermore, the present invention uses CTLA4 antagonists for the manufacture of pharmaceuticals for the treatment of diseases or disorders (e.g., cancer and infectious diseases) in which the effects can be obtained by stimulating the immune system. Herein, 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), for example, a compound selected from compounds 1 to 34, or a pharmaceutically acceptable salt thereof) and / or a PD1 / PD-L1 conjugated antagonist. The present invention uses CTLA4 antagonists for the manufacture of pharmaceuticals for the treatment of diseases or disorders (e.g., cancer and infectious diseases) in which the effects can be obtained by stimulating the immune system. Herein, 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), for example, a compound selected from compounds 1 to 34, or a pharmaceutically acceptable salt thereof) and a PD1 / PD-L1 conjugated antagonist.

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

[0008] The above and other features of the new treatment method will be described in more detail as they are disclosed. [Brief explanation of the drawing]

[0009] [Figure 1A-1B] Figures 1A and 1B show that T cells incubated with higher concentrations of DGKi and nivolumab (A) or ipilimumab (B) in the same assay showed increased IFN-γ secretion compared to the MLR assay in the absence of nivolumab or ipilimumab. [Figure 2A-H] Figures 2A-H show that tumor growth is suppressed when anti-PD-1 antibody, anti-CTLA4 antibody, and DGKi are combined, compared to mice treated with anti-PD-1 antibody and anti-CTLA4 antibody alone. Figures 2A-H show the tumor size over time after transplantation of mouse B16 melanoma cells into mice, respectively, treated with Vehicle alone (Figure 2A), anti-PD-1 antibody alone (Figure 2B), anti-PD-1 antibody and anti-CTLA4 antibody (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 antibody, and anti-CTLA4 antibody (Figure 2G). Figure 2H shows the mean tumor size after transplantation 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 treatment with a combination of a DGK inhibitor and an anti-PD-1 antibody and / or anti-CTLA4 antibody 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 from OT1 CD8 T cells incubated with increased antigen levels 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 antigens 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 and recombinant peptides (1000 ng / mL), indicating that DGK inhibition promotes T cell response even with low-affinity tumor antigens. [Figure 5A-5B] Figures 5A and 5B show that inhibition of DGK activates human CTL effector function and enhances tumor-killing activity. Figure 5A shows the amount of IFN-γ secreted from T cells incubated with peptides as the concentration of DGKi is increased. Figure 5B shows that tumor cells with increased levels of the congener peptide exhibit enhanced tumor-killing activity after incubation for 3 days. [Figure 6A-6B] Figures 6A and 6B demonstrate that DGKi can restore the reduced T cell effector function caused by decreased B2M levels. Figure 6A shows the amount of β2 microglobulin in CRISPR knockout HCT116 cells with B2M. Figure 6B shows that DGKi increases IFN-γ levels. [Figure 7] Figure 7 shows the relationship between tumor volume and days after tumor cell transplantation in the CT26 animal model. It shows that in mice treated with DGKi compound 16 and anti-PD-1 antibody, with or without the presence of CD8 depletion antibody, mice treated with CD8 depletion antibody showed less tumor reduction. [Figure 8]Figure 8 shows the relationship between tumor volume and the number of days after tumor cell transplantation in the CT26 animal model. It shows that in mice treated with DGKi compound 16 and anti-PD-1 antibody, with or without the presence of CD4 depletion antibody, the mice treated with CD4 depletion antibody showed a greater reduction in tumor size. [Figure 9] Figure 9 shows the relationship between tumor volume and the number of days after tumor cell transplantation in the CT26 animal model. It shows that in mice treated with DGKi compound 16 and anti-PD-1 antibody, with or without the presence of NK cell depletion antibody, the mice treated with NK cell depletion antibody showed less tumor reduction. [Figure 10] Figure 10 shows that complete tumor regression (CR) is possible in the MC38 tumor model with a combination of DGKi and either anti-PD-1 or anti-CTLA4. Tumor volume for each animal group is shown after treatment with the vehicle (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, or anti-CTLA4 alone can delay 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 resulted in complete regression in 70% of test mice. [Figure 11]Figure 11 shows that in both the MC38 and CT26 animal models, adding DGKi to anti-PD-1 therapy enables complete tumor regression (CR), and that cured animals in these groups develop sufficient immunological memory and reject re-transplanted tumors. Tumor volumes 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) are shown. In the MC38 and CT26 models, DGKi, due to its potent effect in combination with anti-PD-1, resulted in 100% or 60% complete tumor regression, respectively. To evaluate immunological memory in cured animals, ten times the number of tumor cells used in the initial transplant were re-transplanted into mice, tumor volume was measured, and its relationship to the number of days post-transplant was shown. In the MC38 cohort (Figure 11D) and the CT26 cohort (Figure 11H), all re-transplanted animals spontaneously rejected the tumors, confirming that combination therapy with DGKi and anti-PD-1 sustains long-term immunological memory. [Figure 12] Figure 12 shows that treatment with anti-PD-1, anti-CTLA4, and three types of DGKi can reduce tumor growth in a 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. [Modes for carrying out the invention]

[0010] The present invention provides a method for treating any disease, disorder, or symptom that is effective by stimulating a proliferative disorder (e.g., cancer) or viral infection, or the more general immune system, as well as any disease, disorder, or symptom that can be prevented, improved or cured by inhibiting DGKα and / or DGKζ enzyme activity, characterized by administering a therapeutically effective amount of an inhibitor of DGKα and / or DGKζ or a pharmaceutically acceptable salt thereof and (i) a PD1 / PD-L1-binding antagonist (e.g., a human PD1 or human PD-L1 antagonist) and / or (ii) a human CTLA4 antagonist to the target subject.

[0011] (definition) The characteristics and usefulness of the treatment method can be more readily understood by those skilled in the art by reading the following detailed description. For clarity, it is understood that certain characteristics of the treatment method described before and after the context of another embodiment may be combined to form a single embodiment. Conversely, for brevity, various characteristics of the treatment method described in the context of a single embodiment may also be combined to form its subcombinations. The embodiments described herein as examples or preferred are intended to be illustrative and not limiting.

[0012] Unless otherwise specified in this specification, singular references may include plurals. For example, "a" and "an" can refer to either "one" or "one or more."

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

[0014] Unless otherwise specified, any atom whose valence is not met is assumed to contain enough hydrogen atoms to satisfy the valence requirement.

[0015] The definitions contained herein supersede any definitions contained in any patent, patent application and / or patent application publication incorporated herein by reference.

[0016] The definitions of various terms used herein are listed below. These definitions apply to the terms used throughout the specification, either individually or as part of a larger group (unless otherwise specified).

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

[0018] In accordance with the conventions used in that field, [ka] The symbol is used in the structural formulas of this specification to represent a bond that is a bonding point of a partial or substituent to the core or skeletal 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 -NH2.

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

[0023] As used herein, the term "alkyl" refers to both branched and straight-chain saturated aliphatic hydrocarbon groups having, for example, 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 is shown in subscript after the symbol "C", the subscript letter more specifically limits the number of carbon atoms that a particular group may contain. For example, "C 1-4 alkyl" means straight-chain and branched-chain alkyl groups having 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 fluoroalkyl" means encompassing C1, C2, C3, and C4 alkyl groups substituted with one or more fluorine atoms. Representative examples of fluoroalkyl groups include, but are not limited to, -CF3 and -CH2CF3.

[0025] The term "cyanoalkyl" encompasses both branched and straight-chain saturated alkyl groups substituted with one or more cyano groups. For example, "cyanoalkyl" includes -CH2CN, -CH2CH2CN, and C 1-4 cyanoalkyl.

[0026] The term "aminoalkyl" encompasses 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.

[0027] The term "hydroxyalkyl" refers to saturated alkyl groups, both branched and linear, that are substituted with one or more hydroxyl groups. For example, "hydroxyalkyl" includes -CH2OH, -CH2CH2OH, and C 1-4 Examples include hydroxyalkyl groups.

[0028] The term "alkenyl" refers to a linear or branched 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 linear or branched alkenyl group having 2 to 6 carbon atoms.

[0029] The term "alkynyl" refers to a linear or branched hydrocarbon radical containing 2 to 12 carbon atoms and at least one carbon-carbon triple bond. An example of such a group is ethynyl. For example, "C 2-6 "Alkynyl" refers to a linear or branched 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 or 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 follows the symbol "C," the subscript more specifically limits the number of carbon atoms that a particular cycloalkyl group may contain. For example, "C3-C6 cycloalkyl" means a cycloalkyl group having 3 to 6 carbon atoms.

[0031] As used herein, the term "alkoxy" refers to an alkyl group that connects to a part of the parent molecule via an oxygen atom, such as a methoxy group (-OCH3). For example, "C 1-3 "Alkoxy" refers to an alkoxy group having 1 to 3 carbon atoms.

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

[0033] The term "alkarenyl" refers to a saturated carbon chain having two attachment points to a core or backbone structure. The alkarenyl group has the structure -(CH2) n -, where n is an integer greater than or equal to 1. Examples of alkarenyl bonds include -CH2CH2-, -CH2CH2CH2- and -(CH2) 2-4 0-

[0034] As used herein, the phrase "pharmaceutically acceptable" means that within the scope of ordinary medical judgment, it is suitable for contact with human and animal tissues without causing excessive toxicity, irritation, allergic reaction, or other problems or complications, and provides a reasonable benefit / risk ratio, referring to compounds, substances, compositions, and / or dosage forms.

[0035] For example, the compound of formula (I) may form a pharmaceutically acceptable salt, which may be used in the methods described herein. Unless otherwise specified, references to a compound are understood to include references to one or more pharmaceutically acceptable salts thereof. The term “salt” refers to a pharmaceutically acceptable acid acid and / or base salt formed by inorganic and / or organic acids and bases. Furthermore, the term “salt” may include zwitterions (intramolecular salts) if, for example, the compound of formula (I) has both a basic moiety (e.g., an amine, pyridine, or imidazole ring) and an acidic moiety (e.g., a carboxylic acid). Medicinically acceptable (i.e., non-toxic and physiologically acceptable) salts are preferably acceptable metal salts and amine salts, for example, 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 the manufacturing process, and are therefore considered herein. For example, a salt of the compound of formula (I) may be formed by reacting the compound of formula (I) with a certain amount of acid or base (e.g., 1 equivalent), thereby precipitating the salt in a solvent, for example, or by subsequently freeze-drying the aqueous solution.

[0036] Examples of acid addition salts include acetates (e.g., acetates prepared from acetic acid or trihaloacetic acid (e.g., trifluoroacetic acid)), adipines, alginates, ascorbicates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphor sulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (prepared from hydrochloric acid), and hydrobromides (prepared from hydrogen bromide). Examples include hydroiodide, maleate (prepared from maleic acid), 2-hydroxyethanesulfonate, lactate, methanesulfonate (prepared from methanesulfonic acid), 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate (prepared from sulfuric acid, for example), sulfonate (for example, those described herein), tartrate, thiocyanate, toluenesulfonate (for example, tosylate), and undecanoate.

[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'-dibenzylethylenediamine, dehydroabiethylamine, N-ethylpiperidine, benzylamine, dicyclohexylamine), or similar pharmaceutically acceptable amines, and amino acid salts (e.g., arginine, lysine). The basic nitrogen-containing group may be quaternized with reagents (e.g., 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, bisulfates, methanesulfons, 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 freeze-drying.

[0039] Furthermore, solvates (e.g., hydrates) of compounds of formula (I), for example, should also be considered to be subject to the methods described herein. The term “solvate” means, for example, a physical bond between a compound of formula (I) and one or more organic or inorganic solvent molecules. This physical bond includes hydrogen bonds. In some cases, solvates can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. “Solvate” includes both the solution phase and the separable solvate. Examples of solvates include hydrates, ethanolates, methanelates, isopropanolates, acetonitrile solvate, and ethyl acetate solvate. Methods of solvation are known in the art.

[0040] Various forms of prodrugs are well known in this field: 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) It is described there.

[0041] In addition, for example, after the compound of formula (I) is prepared, it is isolated, purified, and obtained as a composition containing the compound of formula (I) in an amount of 99% or more (a "substantially pure" composition), and then it is used or formulated as described herein. Such "substantially pure" compounds of formula (I) are also contemplated herein.

[0042] The terms "stable compound" and "stable structure" are intended to mean compounds that are robust enough not to decompose upon isolation to useful purity from a reaction mixture or upon formulation into an effective therapeutic agent. Compounds used herein are contemplated to embody stable compounds.

[0043] The compounds described herein are intended to contain all isotopes of the atoms contained in the compounds of the present invention. Isotopes include atoms having the same atomic number but different mass numbers. Common examples include, but are not limited to, the isotopes of hydrogen including deuterium (D) and tritium (T). Isotopes of carbon include 13 C and 14 C. Compounds labeled with isotopes can generally be produced by conventional techniques known to those of ordinary skill in the art or by methods similar to those described herein, using appropriate isotope-labeled reagents in place of the unlabeled reagents otherwise employed.

[0044] As used herein, "treatment" encompasses any dosing or method of treating a disease in a human and includes inhibiting the progression of a disease or one or more disease symptoms, reducing a disease or the progression of a disease or one or more disease symptoms, halting its progression, partially or wholly alleviating a disease or one or more disease symptoms, or preventing recurrence of one or more symptoms of a disease.

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

[0046] "Inhibitors of DGKα and / or DGKζ" refers to "inhibitors of DGKα and / or DGKζ enzyme activity," and all are 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 non-natural 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 non-natural amino acids (e.g., His tag or specific N-terminal amino acids).

[0047] In this specification, 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 dose" or "therapeutic dose" refers to the amount of drug that is effective in treating a disease or disorder in a subject, for example, the amount that partially or completely relieves one or more symptoms. In some embodiments, the effective dose refers to the amount administered and over the duration required to obtain the desired therapeutic outcome or preventive effect.

[0051] As used herein, the term "cancer" refers to a cell type 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 treatment methods described herein apply 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 tumors, 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" means the proliferation or growth of cells, including cancer cells, to the size or extent of the corresponding cancer.

[0053] The “combined” administration of one or more therapeutic agents includes simultaneous and sequential administration in any order.

[0054] (Treatment method) The present invention provides a method for treating any disease, disorder, or symptom that can be prevented, improved, or cured by inhibiting DGKα and / or DGKζ enzyme activity, as well as any disease, disorder, or symptom that can be prevented, improved, or cured by stimulating the proliferative disorder (e.g., cancer) or viral infection or more general immune system. The method is characterized by administering a therapeutically effective amount of (i) a DGKα and / or DGKζ inhibitor and (ii) a PD1 / PD-L1-binding antagonist (e.g., a human PD1 or human PD-L1 antagonist) and / or a human CTLA4 antagonist to the target subject. This application provides a method for treating any disease, disorder, or symptom that is effective by stimulating the proliferative disorder (e.g., cancer) or viral infection, or the more general immune system, as well as any disease, disorder, or symptom that can be prevented, improved or cured by inhibiting DGKα and / or DGKζ enzyme activity, characterized by administering a therapeutically effective amount of (i) a DGKα and / or DGKζ inhibitor and (ii) a PD1 / PD-L1 binding antagonist (e.g., a human PD1 or human PD-L1 antagonist) to the subject in question. This application provides a method for treating any disease, disorder, or symptom that is effective by stimulating the proliferative disorder (e.g., cancer) or viral infection, or the more general immune system, as well as any disease, disorder, or symptom that can be prevented, improved or cured by inhibiting DGKα and / or DGKζ enzyme activity, characterized by administering a therapeutically effective amount of (i) a DGKα and / or DGKζ inhibitor and a human CTLA4 antagonist to the subject in question. In some embodiments, treatment of proliferative disorders (e.g., cancer) or viral infections, or any disease, disorder or condition that is effective by stimulating the more general immune system, as well as any disease, disorder or condition that can be prevented, improved or cured by inhibiting DGKα and / or DGKζ enzyme activity, comprises administering to the target subject in question a therapeutically effective amount of (i) a DGKα and / or DGKζ inhibitor and (ii) a PD1 / PD-L1 binding antagonist (e.g., a human PD1 or human PD-L1 antagonist) and a human CTLA4 antagonist.

[0055] (i) inhibitors of DGKα and / or DGKζ and (ii) PD1 / PD-L1-binding antagonists (e.g., human PD1 or human PD-L1 antagonists) and / or human CTLA4 antagonists may be administered simultaneously or sequentially. For example, in some embodiments, a method for treating cancer or a disease that can be treated by promoting an immune response may involve first administering a DGKα and / or DGKζ inhibitor to the target subject, and then subsequently (e.g., 6 hours, 12 hours, 24 hours, 2 days, 3 days, or later) administering a PD1 / PD-L1-binding antagonist (e.g., human PD1 or human PD-L1 antagonist) and / or human CTLA4 antagonist. For example, a cancer treatment method may treat cancer or a disease that can be treated by promoting an immune response, and such method may include first administering a PD1 / PD-L1-binding antagonist (e.g., a human PD1 or human PD-L1 antagonist) and / or a human CTLA4 antagonist to the target, and then subsequently (e.g., after 6 hours, 12 hours, 24 hours, 2 days, 3 days, or thereafter) administering a DGKα and / or DGKζ inhibitor. For example, a cancer treatment method may first administer a DGKα and / or DGKζ inhibitor, and then subsequently (e.g., after 6 hours, 12 hours, 24 hours, 2 days, 3 days, or thereafter) simultaneously administering a PD1 / PD-L1-binding antagonist (e.g., a human PD1 or human PD-L1 antagonist) and a human CTLA4 antagonist. For example, in cancer treatment methods, a DGKα and / or DGKζ inhibitor may be administered first, followed later (e.g., 6 hours, 12 hours, 24 hours, 2 days, 3 days, or thereafter) by simultaneously administering a PD1 / PD-L1-binding antagonist (e.g., a human PD1 or human PD-L1 antagonist) and a human CTLA4 antagonist.For example, in cancer treatment methods, a PD1 / PD-L1 antagonist (e.g., a human PD1 or human PD-L1 antagonist) and a human CTLA4 antagonist may be administered simultaneously first, followed by the administration of DGKα and / or DGKζ inhibitors at subsequent times (e.g., 6 hours, 12 hours, 24 hours, 2 days, 3 days, or thereafter).

[0056] The methods described herein may be used in the treatment of cancer. Examples of cancer include advanced cancer, metastatic cancer, solid tumors, advanced solid tumors, hematological malignancies, cancers refractory to checkpoint inhibitors (or checkpoint antagonists), or the above cancers that have progressed after treatment with checkpoint inhibitors.

[0057] Examples of cancers 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, kidney 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, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer (and (Malignant tumors), gastric cancer, germ cell tumors, pediatric sarcomas, sinus natural killer tumors, melanoma (e.g., metastatic malignant melanoma, e.g., cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary central nervous system lymphoma, tumor angiogenesis, axis tumors, brain tumors, brainstem gliomas, ptosis Environmental factor cancers such as somatic adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, asbestos-related, virus-related or virus-derived cancers (e.g., human papillomavirus (HPV-related tumors or HPV-derived tumors)) and hematological malignancies (e.g., all leukemias, lymphomas and myelomas, e.g., acute, chronic) resulting from either of the two main hematological cell lineages (i.e., spinal cord cells (which become granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cells (which become B, T, NK cells, and plasma)) Lymphocytic and / or myeloid leukemias, such as acute lymphoblastic 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 chloroplast;Lymphoma (e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL)), B-cell hematological malignancies (e.g., B-cell lymphoma), T-cell lymphoma, lymphoplasmacytic lymphoma, monocyticoid 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, vascular lymphoma, enteropathy-type T-cell lymphoma, mediastinal primary B-cell lymphoma, progenitor 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 malignant lymphoma of the central nervous system, primary exudative lymphoma, B-cell lymphoma, B-lymphoblastic lymphoma Lymphoma (LBL), lymphoid hematopoietic malignancies, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large B-cell lymphoma, precursor B-cell lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary syndrome), and lymphoplasmacytic lymphoma (LPL) / Waldenström Macroglobulinemia; myeloma (e.g., IgG type myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called asymptomatic myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; myeloid hematopoietic malignancies, mesenchymal tumors (fibrosarcoma and rhabdomyosarcoma, etc.); seminomas, malignant teratomas, central and peripheral nervous system tumors (astrocytoma, Schwann cell tumor, etc.); Mesenchymal tumors (fibrosarcoma, rhabdomyosarcoma, and osteosarcoma, etc.); and other tumors (melanoma, xeroderma pigmentosum, keratosacral cell tumor, seminomas, follicular thyroid carcinoma, and malignant teratomas, etc.), lymphoid hematopoietic tumors (T-cell tumors and B-cell tumors, but not limited to T-cell disorders (e.g., pre-T lymphocytic leukemia (T-PLL) (including small cell and cerebral cell types); T-cell macrogranular lymphocyte leukemia (LGL); a / d T-NHL hepatosplenic T-cell lymphoma; peripheral / mature T-cell lymphoma (pleomorphic and immunoblastic T-cell lymphoma); vascular central (nasal) T-cell lymphoma, etc.)); head and neck cancer, kidney cancer, rectal cancer, thyroid cancer;Examples include acute myeloid lymphoma and any combination of the aforementioned cancers. Furthermore, the methods described herein may be used in the treatment of metastatic cancer, unresectable cancer, refractory cancer (e.g., cancer that is unresponsive to conventional immunotherapies such as inhibition of CTLA-4 or PD-1 antibodies), and / or recurrent cancer.

[0058] In some embodiments, the combination therapies described herein are administered to cancer patients who have not responded to or improved adequately to previous treatments (e.g., treatment with immuno-oncological or immunotherapeutic drugs). 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 acquiredly resistant or unresponsive. For example, the combination therapies described herein may be administered to subjects who did not respond or did not respond adequately to initial treatment, or to subjects whose disease has progressed due to treatment with an anti-PD-1 pathway antagonist alone or in combination with another therapy. In other embodiments, the combination therapies described herein are administered to patients who have not previously been treated with immuno-oncological drugs (e.g., PD-1 pathway antagonists).

[0059] Combination therapy may include one or more other treatments (such as radiation therapy, surgery, or chemotherapy).

[0060] Furthermore, the methods described herein may be used to treat patients exposed to specific toxins or pathogens (e.g., patients with infectious diseases). Therefore, this disclosure also envisions methods for treating target infectious diseases characterized by administering the combination therapies described herein. Similar to the applications to tumors described above, the combination therapies may be used alone or as adjuvants in combination with vaccines to stimulate an immune response to pathogens, toxins, and autoantigens. Examples of pathogens for which this therapeutic method may be particularly useful include pathogens for which there are currently no effective vaccines, or pathogens for which conventional vaccines are not entirely effective. These include, but are not limited to, HIV, hepatitis (types A, B, and C), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa. Combination therapies may be useful against established drug-induced infections (e.g., HIV, where the antigen changes during the course of infection).

[0061] Some examples of pathogenic viruses that cause infections treatable by the methods described herein include HIV, hepatitis (types A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenoviruses, influenza viruses, flaviviruses, echoviruses, rhinoviruses, coxsackieviruses, coronaviruses, respiratory syncytial virus (RSV), mumps virus, rotavirus, measles virus, rubella virus, parvovirus, cowpox virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arbovirus encephalitis virus.

[0062] Some examples of pathogenic bacteria that cause infections treatable by the methods described herein include Chlamydia, Rickettsia, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria bacillus, Salmonella, Bacillus, Vibrio cholerae, Neisseria tetanus, Clostridium botulinum, Bacillus anthrax, Plague bacillus, Leptospirae and Lyme disease bacteria.

[0063] Some examples of pathogenic fungi that cause infections treatable by the methods described herein include Candida species (such as Candida albicans, Candida crusei, Candida glabrata, and Candida tropicalis), Cryptococcus neoformans, Aspergillus species (such as Aspergillus fumigatus and Aspergillus niger), Mucor species (such as Mucor, Mucorus humilis, and Rhizopus), Sporotrichii schenkyi, Blastomyces dermatitisis, South American spore-forming fungi, Coccidioides imitis, and Histoplasma capsulatum.

[0064] Some examples of pathogenic parasites that cause infections treatable by the methods described herein include Entamoeba histolytica, Balantidium colonis, Naegleria fowleri, Acanthamoeba species, Giardia giardia, Cryptosporidium species, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brussei, Trypanosoma cruzi, Donovan leishmania, Toxoplasma gondii, and Hookworm brachycephalus.

[0065] In all of the above methods, treatment may be combined with other forms of immunotherapy (e.g., cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy) to prepare for the emergence of potent tumor antigens (see, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123).

[0066] In some embodiments, the method involves using inhibitors of DGKα and / or DGKζ as antagonists of PD1 / PD-L1 binding and / or CTLA4, and CD4 + Drugs that inhibit T cells and / or CD8 + In some embodiments, CD4 + T cell inhibitors and CD8 + Drugs that enhance T cells may be drugs that act locally at the tumor site.

[0067] Examples of DGKα and / or DGKζ enzyme activity inhibitors 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 inhibition level of the enzymes may be measured as described later herein. In some embodiments, the inhibitor of DGKα and / or DGKζ does not significantly inhibit the other DGK enzyme.

[0068] In some embodiments, inhibitors of DGKα and / or DGKζ promote the immune response, for example, by activating T cells. For example, inhibitors of DGKα and / or DGKζ may activate primary T cell signaling by increasing pERK / pPKC signaling, as is evident from the measurements described later herein. In some embodiments, inhibitors of DGKα and / or DGKζ have one or more of the following properties: (i) lowering the antigen stimulation threshold; (ii) activating CTL effector function; and (iii) enhancing tumor-killing activity. When inhibitors of DGKα and / or DGKζ enhance tumor-killing activity, this activity is CD8, as demonstrated, for example, in the CT26 animal model. +T cells may be involved. If inhibitors of DGKα and / or DGKζ enhance tumor-killing activity, this activity may be influenced by NK cells, as demonstrated, for example, in the CT26 animal model. If inhibitors of DGKα and / or DGKζ enhance tumor-killing activity, this activity may be influenced by CD8 cells, as demonstrated, for example, in the CT26 animal model. + T cells and NK cells may be affected. If inhibitors of DGKα and / or DGKζ enhance tumor-killing activity, this activity may be enhanced by a reduction in CD4 cells, for example, in the CT26 animal model. In some embodiments, inhibitors of DGKα and / or DGKζ promote the appearance of AH1+ tetramer antigen in the CT26 animal model. Inhibitors of DGKα and / or DGKζ preferably have one or more of the above properties, or may have all of them. The expression of these properties may be determined, for example, by the implementation assays described in the section labeled “Biological Assays” herein.

[0069] A treatment method for a disease (e.g., cancer) may be characterized by administering a PD1 / PD-L1-binding antagonist and / or a CTLA4 antagonist and a DGKα and / or DGKζ inhibitor to the target, wherein the DGKα and / or DGKζ inhibitor is defined by formula (I): [ka] [In the formula, R1 consists of H, F, Cl, Br, -CN, and 0 to 4 R atoms. 1a C replaced by 1-3 Alkyl, 0-4 R 1a C replaced by 3-4 Cycloalkyl, 0-4 R 1a C replaced by 1-3 Alkoxy, -NR a R a , -S(O) n R e or -P(O)R e R e and; Each R 1aThese are independently F, Cl, -CN, -OH, -OCH3, or -NR a R a and; Each R a H or C 1-3 It is alkyl; Each R e Independently, C 3-4 Cycloalkyl or 0-4 R 1a C replaced by 1-3 It is alkyl; R2 is H, 0 to 4 R 2a C replaced by 1-3 Alkyl or 0-4 R 2a C replaced by 3-4 It is a cycloalkyl; Each R 2a These are independently F, Cl, -CN, -OH, -O(C 1-2 Alkyl), C 3-4 Cycloalkyl, C 3-4 Alkenyl or C 3-4 It is alkinyl; R3 consists of H, F, Cl, Br, -CN, and C. 1-3 Alkyl, C 1-2 Fluoroalkyl, C 3-4 Cycloalkyl, C 3-4 It is 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 It is 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 is cycloalkyl, heterocyclyl, phenyl or heteroaryl, each of which is F, Cl, Br, -CN, -OH, 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 substituted with 0 to 4 substituents independently selected from; or (iii) C 1-4 alkyl substituted with one cyclic group, and the cyclic group is C 3-6Selected from cycloalkyl, heterocyclyl, aryl, and heteroaryl, the cyclic group is 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 Substituting with 0 to 3 substituents independently selected from the cycloalkyl group; or R 4a and R 4b And they bond with the carbon atoms and become one with C 3-6 They form cycloalkyl or 3-6 membered heterocyclines, each with 0-3 R f Replaced by; Each R f These are independently F, Cl, Br, -OH, -CN, and C. 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c Alternatively, it is a cyclic group, and the cyclic group is C 3-6 The cyclic groups are selected from cycloalkyl, 3-6 membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl groups, where each cyclic group is 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 C 1-6 Alkyl or C3-6 They are cycloalkyl groups, with F, Cl, -OH, and C respectively. 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 H or C 1-2 It is alkyl; R d It is phenyl, substituted with 0 to 1 substituent selected from F, Cl, -CN, -CH3 and -OCH3; Each R5 independently has -CN, 0 to 4 R g C replaced by 1-6 Alkyl, 0-4 R g C replaced by 2-4 Alkenyl, 0-4 R g C replaced by 2-4 Alkinyl, 0-4 R g C replaced by 3-4 Cycloalkyl, 0-4 R g Phenyl substituted with, 0-3 R g Oxadiazolyl substituted with, 0-4 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 It is a cycloalkyl group; Each R g These are independently F, Cl, -CN, -OH, and 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. It is a compound of or a pharmaceutically acceptable salt thereof.

[0070] A treatment method for a disease (e.g., cancer) may be characterized by administering a PD1 / PD-L1-binding antagonist and / or a CTLA4 antagonist and a DGKα and / or DGKζ inhibitor to the target, wherein the DGKα and / or DGKζ inhibitor is defined in the formula, R1 is H, F, Cl, Br, -CN, 0 to 4 R 1a C replaced by 1-3 Alkyl, 0-3 R 1a Cyclopropyl substituted with, 0-3 R 1a C replaced by 1-3 Alkoxy, -NR a R a , -S(O) n It is either CH3 or -P(O)(CH3)2; Each R 1a However, independently, they are F, Cl, or -CN; Each R a However, independently, H or C 1-3 It is alkyl; R2 is H or 0 to 2 R 2a C replaced by 1-2 It is alkyl; Each R 2a However, independently, F, Cl, -CN, -OH, -O(C 1-2 Alkyl), cyclopropyl, C 3-4 Alkenyl or C 3-4 It is alkinyl; R3 is H, F, Cl, Br, -CN, C 1-2It is alkyl, -CF3, cyclopropyl, or -NO2; R 4a and R 4b However, they became independent, (I C 1-4 It is 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 These are cycloalkyl, heterocyclyl, phenyl, or heteroaryl compounds, respectively F, Cl, Br, -CN, -OH, and 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, tetrahydropyranil, morphholinyl, thiophenyl, methylpiperidinyl and R d Substituted with 0 to 4 substituents independently selected from; or (iii) C substituted with one cyclic group 1-3 It is alkyl, and the cyclic group is C 3-6 Selected from cycloalkyl, heterocyclyl, phenyl, and heteroaryl, the cyclic group is 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 Substituting with 0 to 3 substituents independently selected from the cycloalkyl group; or R 4a and R 4b And they bond with the carbon atoms and become one with C 3-6 They form cycloalkyl or 3-6 membered heterocyclines, each with 0-3 R f Replaced by; Each R f However, 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 Alternatively, it is a cyclic group, and the cyclic group is C 3-6The cyclic groups are selected from cycloalkyl, 3-6 membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl groups, where each cyclic group is 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 However, C 1-4 Alkyl or C 3-6 They are cycloalkyl groups, with F, Cl, -OH, and C respectively. 1-2 Alkoxy, C 1-2 Substituted with 0 to 4 substituents independently selected from fluoroalkoxy and -CN; and Each R5 independently has -CN, 0 to 4 R g C replaced by 1-5 Alkyl, 0-4 R g C replaced by 2-3 Alkenyl, 0-4 R g C replaced by 2-3 Alkinyl, 0-4 R g C replaced by 3-4 Cycloalkyl, 0-3 R g Phenyl substituted with, 0-3 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 It is a cycloalkyl group. It is a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0071] A treatment method for a disease (e.g., cancer) may be characterized by administering a PD1 / PD-L1-binding antagonist and / or a CTLA4 antagonist and a DGKα and / or DGKζ inhibitor to the target, wherein the DGKα and / or DGKζ inhibitor is defined by formula (I): [ka] [Here, R1 is -CN; R2 is -CH3; R3 is H, F, or -CN; R4 is [ka] [is] A compound having a structure, or a pharmaceutically acceptable salt thereof.

[0072] A treatment method for a disease (e.g., cancer) may be characterized by administering a PD1 / PD-L1 antagonist and / or a CTLA4 antagonist and a DGKα and / or DGKζ inhibitor to the target, wherein the DGKα and / or DGKζ inhibitor is a compound of formula (I) having one of the following structures or formulas (or isomers thereof) or a pharmaceutically acceptable salt thereof. 1-(bis(4-fluorophenyl)methyl)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-4-yl)piperazine-2-carboxylate methyl [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-methylpyridine-2-yl)methyl]-2,5-dimethylpiperazine-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonilicate [ka] ; 4-[(2S,5R)-4-[(4-chlorophenyl)(4-fluorophenyl)methyl]-2,5-dimethylpiperazine-1-yl]-6-methoxy-1-methyl-1,2-dihydro-1,5-naphthyridine-2-one [ka] ; 8-[(2S,5R)-4-{[2-(difluoromethyl)-4-fluorophenyl]methyl}-2,5-dimethylpiperazine-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-dimethylpiperazine-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-dimethylpiperazine-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] ; 8-((3R)-4-((4-chlorophenyl)(5-fluoropyridine-2-yl)methyl)-3-methylpiperazine-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-dimethylpiperazine-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka]

[0073] A treatment method for a disease (e.g., cancer) may be characterized by administering a PD1 / PD-L1 antagonist and / or a CTLA4 antagonist and a DGKα and / or DGKζ inhibitor to the target, wherein the DGKα and / or DGKζ inhibitor is defined by formula (II): [ka] [In the formula, R1 consists of H, F, Cl, Br, -CN, -OH, and 0 to 4 R atoms. 1a C replaced by 1-3 Alkyl, 0-4 R 1a C replaced by 3-4 Cycloalkyl, 0-4 R 1a C replaced by 1-3 Alkoxy, -NR a R a , -S(O) n R e or -P(O)R e R e and; Each R 1a These are independently F, Cl, -CN, -OH, -OCH3, or -NR a R a and; Each R a H or C 1-3 It is alkyl; Each R e Independently, C 3-4 Cycloalkyl or 0-4 R 1a C replaced by 1-3 It is alkyl; R2 is H, 0 to 4 R 2a C replaced by 1-3 Alkyl or 0-4 R 2a C replaced by 3-4 It is a cycloalkyl; Each R 2a These are independently F, Cl, -CN, -OH, -O(C 1-2 Alkyl), C 3-4 Cycloalkyl, C 3-4 Alkenyl or C 3-4 It is alkinyl; 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 It is 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 These are cycloalkyl, 4-10 membered heterocyclyl, phenyl, or 5-10 membered heteroaryl compounds, respectively F, Cl, Br, -CN, -OH, and 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 (Molfolinyl), -(CR x R x ) 1-2 (Difluoromorpholinyl), -(CR x R x ) 1-2 (Dimethylmorpholinyl), -(CR x R x ) 1-2 (Oxazabicyclo[2.2.1]heptanil), (CR x R x ) 1-2 (Oxazaspiro[3.3]heptanil), -(CR x R x ) 1-2 (Methylpiperazinonyl), -(CR x R x ) 1-2 (Acetylpiperazinyl), -(CR x R x ) 1-2 (Piperidinil), -(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 (Molfolinyl), -O(CR x R x ) 0-2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butoxycarbonyl)azetidinyl, triazolyl, tetrahydropyranil, morpholinil, thiophenyl, methylpiperidinyl, dioxolanil, pyrrolidinonil and R d Substituted with 0 to 4 substituents independently selected from; or (iii) C substituted with one cyclic group 1-4 It is alkyl, and the cyclic group is C 3-6 The cyclic group is selected from cycloalkyl, 4-10 membered heterocyclyl, monocyclic or bicyclic aryl, or 5-10 membered heteroaryl, wherein the cyclic group is 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 Substituting with 0 to 3 substituents independently selected from the cycloalkyl group; or R 4a and R 4b And they bond with the carbon atoms and become one with C 3-6 They form cycloalkyl or 3-6 membered heterocyclines, each with 0-3 R f Replaced by; Each R f These are independently F, Cl, Br, -OH, -CN, and C. 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c Alternatively, it is a cyclic group, and the cyclic group is C 3-6 The cyclic groups are selected from cycloalkyl, 3-6 membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl groups, where each cyclic group is 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 C 1-6 Alkyl or C 3-6 They are cycloalkyl groups, with F, Cl, -OH, and C respectively. 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 H or C 1-2 It is alkyl; R dIt is phenyl, substituted with 0 to 1 substituent selected from F, Cl, -CN, -CH3 and -OCH3; Each R5 independently has -CN, 0 to 4 R g C replaced by 1-6 Alkyl, 0-4 R g C replaced by 2-4 Alkenyl, 0-4 R g C replaced by 2-4 Alkinyl, 0-4 R g C replaced by 3-4 Cycloalkyl, 0-4 R g Phenyl substituted with, 0-3 R g Oxadiazolyl substituted with, 0-4 R g Pyridinyl substituted with -(CH2) 1-2 (0 to 4 R g (4-10 member 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 It is a cycloalkyl group; Each R g These are independently F, Cl, -CN, -OH, and 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. It is a compound or a salt thereof.

[0074] A treatment method for a disease (e.g., cancer) may be characterized by administering a PD1 / PD-L1-binding antagonist and / or a CTLA4 antagonist and a DGKα and / or DGKζ inhibitor to the target, wherein the DGKα and / or DGKζ inhibitor is defined in the formula, R1 is H, F, Cl, Br, -CN, -OH, or 0 to 4 R 1a C replaced by 1-3 Alkyl, 0-3 R 1a Cyclopropyl substituted with, 0-3 R 1a C replaced by 1-3 Alkoxy, -NR a R a , -S(O) n It is either CH3 or -P(O)(CH3)2; R2 is H or 0 to 2 R 2a C replaced by 1-2 It is alkyl; Each R 2a However, independently, F, Cl, -CN, -OH, -O(C 1-2 Alkyl), cyclopropyl, C 3-4 Alkenyl or C 3-4 It is alkinyl; R 4a and R 4b However, independently (i) -CN or C 1-4 It is 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 These are cycloalkyl, 4-10 membered heterocyclyl, phenyl, or 5-10 membered heteroaryl compounds, respectively F, Cl, Br, -CN, -OH, and C. 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-2 Bromoalkyl, C 1-2 Cyanoalkyl, C1-2 Hydroxyalkyl, -CH2NR 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 (Molfolinyl), -CR x R x (Difluoromorpholinyl), -CR x R x (Dimethylmorpholinyl), -CR x R x (Oxaazabicyclo[2.2.1]heptanil), -CR x R x (Oxazaspiro[3.3]heptanil), -CR x R x (Methylpiperazinonyl), -CR x Rx (Acetylpiperazinyl), -CR x R x (Piperidinil), -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 (Molfolinyl), -O(CH2) 0-2 (tetrahydropyranyl), -O(CH2) 0-2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butoxycarbonyl)azetidinyl, dioxolanil, pyrrolidinonil, triazolyl, tetrahydropyranil, morpholinil, thiophenyl, methylpiperidinil and R d Substituted with 0 to 4 substituents independently selected from; or (iii) C substituted with one cyclic group 1-3 It is alkyl, and the cyclic group is C 3-6 Selected from cycloalkyl, 4-10 membered heterocyclyl, monocyclic or bicyclic aryl, or 5-10 membered heteroaryl, the cyclic group is 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 Substituting with 0 to 3 substituents independently selected from the cycloalkyl group; or R 4a and R 4b And they bond with the carbon atoms and become one with C 3-6 They form cycloalkyl or 3-6 membered heterocyclines, each with 0-3 R f Replaced by; Each R f However, 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 The cyclic groups are selected from cycloalkyl, 3-6 membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl groups, where each cyclic group is 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 However, C 1-4 Alkyl or C 3-6 They are cycloalkyl groups, with F, Cl, -OH, and C respectively. 1-2 Alkoxy, C 1-2 Substituted with 0 to 4 substituents independently selected from fluoroalkoxy and -CN; Each R5 independently has -CN, 0 to 4 R g C replaced by 1-5 Alkyl, 0-4 R g C replaced by 2-3 Alkenyl, 0-4 R g C replaced by 2-3 Alkinyl, 0-4 R gC replaced by 3-4 Cycloalkyl, 0-3 R g Phenyl substituted with, 0-3 R g Oxadiazolyl substituted with, 0-3 R g Pyridinyl substituted with -(CH2) 1-2 (0 to 4 R g (4-10 member 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 It is a cycloalkyl group; Each R x However, independently, it is H or -CH3; and m is 1, 2, or 3. It is a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0075] A treatment method for a disease (e.g., cancer) may be characterized by administering a PD1 / PD-L1 antagonist and / or a CTLA4 antagonist and a DGKα and / or DGKζ inhibitor to the target, wherein the DGKα and / or DGKζ inhibitor is defined as having m = 2 and one R5 = R 5a And the other R5 is R 5c And equation (III): [ka] [In the formula, R 5ais -CH3 or -CH2CH3; and R 5c [This is -CH3, -CH2CH3, or -CH2CH2CH3] A compound of formula (II) having the structure shown, or a pharmaceutically acceptable salt thereof.

[0076] A treatment method for a disease (e.g., cancer) may be characterized by administering a PD1 / PD-L1 antagonist and / or a CTLA4 antagonist and a DGKα and / or DGKζ inhibitor to the target, wherein the DGKα and / or DGKζ inhibitor is defined by formula (III): [ka] [In the formula, R1 is -CN; R2 is -CH3; R 5a is -CH3 or -CH2CH3; and R 5c [This is -CH3, -CH2CH3, or -CH2CH2CH3] It is a compound of or a pharmaceutically acceptable salt thereof.

[0077] A treatment method for a disease (e.g., cancer) may be characterized by administering a PD1 / PD-L1 antagonist and / or a CTLA4 antagonist and a DGKα and / or DGKζ inhibitor to the target, wherein the DGKα and / or DGKζ inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof having one of the following structures.

[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 above 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-carbonitrin 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-carbonitrin.

[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 above 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-carbonitrin 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-carbonitrin.

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

[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 above 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-carbonitrin 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-carbonitrin.

[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 above 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-carbonitrin 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-carbonitrin.

[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 above 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-carbonitrin 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-carbonitrin.

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

[0085] 4-((2S,5R)-4-((3-cyclopropyl-1,2,4-oxadiazole-5-yl)(4-fluorophenyl)methyl)-2,5-dimethylpiperazine-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] The above compounds include 4-((2S,5R)-4-((R)-(3-cyclopropyl-1,2,4-oxadiazole-5-yl)(4-fluorophenyl)methyl)-2,5-diethylpiperazine-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrin and 4-((2S,5R)-4-((S)-(3-cyclopropyl-1,2,4-oxadiazole-5-yl)(4-fluorophenyl)methyl)-2,5-diethylpiperazine-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrin.

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

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

[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 above 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 above 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-carbonitrin 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-carbonitrin.

[0090] PD1 / PD-L1 binding antagonist The following are examples of PD1 / PD-L1 binding antagonists that can be combined with DGK inhibitors:

[0091] A PD1 / PD-L1-binding antagonist is a human PD1 antagonist or human PD-L1 antagonist that stimulates an immune response by inhibiting a negative checkpoint. The antagonist can be any type of molecule, such as a protein, nucleic acid, or small molecule. In some embodiments, the PD1 / PD-L1-binding antagonist 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 may be used in the manner described herein. Various human monoclonal antibodies that specifically bind to PD-1 and have 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) 1 x 10 -7 (b) binds to human PD-1 with a KD of M or less (determined by surface plasmon resonance using the Biacore biosensor system); (b) substantially does not bind to human CD28, CTLA-4, or ICOS; (c) promotes T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (d) increases interferon-γ production in an MLR assay; (e) increases IL-2 secretion in an MLR assay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) induces immunological memory against a specific antigen; (i) stimulates an antibody response; and (j) inhibits tumor cell proliferation in vivo. Anti-PD-1 antibodies available for use in the methods of this 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 features.

[0093] Other anti-PD-1 monoclonal antibodies include, for example, U.S. publication numbers 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 This is described in 2017 / 133540, and all of its contents are incorporated by reference.

[0094] In some embodiments, the anti-PD-1 antibody is nivolumab (also known as OPDIVO (trademark), 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA (trademark), lambrolizumab, and MK-3475; see WO2008 / 156712), PDR001 (Novartis; see WO 2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012 / 145493), semiprimab (Regeneron; also known as REGN-2810; see WO 2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; also known as tripalimab; Si-Yang Liu et al.) See Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017), cintilimab, BGB-A317 (Beigene; also known as tislerizumab; see WO 2015 / 35606 and US 2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see 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; see 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.The selection is made from the group consisting of 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 one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab is a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively inhibits the suppression of antitumor T cell function by inhibiting the interaction with PD-1 ligands (PD-L1 and PD-L2) (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. Patents 8,354,509 and 8,900,587.

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

[0098] In one embodiment, an antibody that cross-competes with human PD-1 antibody (nivolumab) and binds to human PD-1, or an antibody that binds to the same epitope region, is a monoclonal antibody. In human administration, these cross-competitive antibodies are chimeric antibodies, modified antibodies, or humanized antibodies or human antibodies. Such chimeric, modified, humanized, or human monoclonal antibodies can be manufactured and isolated by methods well known to those skilled in the art.

[0099] The anti-PD-1 antibodies usable in the methods of this disclosure include the antigen-binding sites of the antibodies described above. It has been well demonstrated that the antigen-binding ability of an antibody can be exerted in fragments of a full-length antibody.

[0100] Anti-PD-1 antibodies suitable for use in the disclosed compositions and methods are antibodies 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 composition or method of the present disclosure, the anti-PD-1 "antibody" includes an antigen-binding site or fragment that binds to the PD-1 receptor and exhibits functional properties similar to the functional properties of the whole antibody in inhibitory ligand binding and promotion of the immune system. In some embodiments, the anti-PD-1 antibody or its antigen-binding site cross-competes with nivolumab that binds to human PD-1.

[0101] In one embodiment, a PD1 / PD-L1 conjugated antagonist is a PD-L1 antagonist. Anti-PD-L1 antibodies known to those skilled in the art may be used in the compositions and methods disclosed herein. An example of an anti-PD-L1 antibody useful in the compositions and methods disclosed herein is the antibody 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) 1 x 10 -7 (b) Binds to human PD-1 with a KD of M or less (determined by surface plasmon resonance using the Biacore biosensor system); (b) Promotes T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (c) Increases interferon-γ production in an MLR assay; (d) Increases IL-2 secretion in an MLR assay; (e) Stimulates an antibody response; and (f) Reverses the effect of regulatory T cells on effector T cells and / or dendritic cells. Anti-PD-L1 antibodies available in this 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 features.

[0102] In one embodiment, the anti-PD-L1 antibody is BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Patent No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; also known as TECENTRIQ (trademark registered), MPDL3280A, RG7446; see U.S. 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; BAVENCIO (trademark), also known as 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, for example, WO 2017 / 034916), BGB-A333 (BeiGene; Desai et al., JCO 36 The patient is selected from the group consisting of (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 (trademark registered)). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

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

[0105] In one embodiment, the PD-L1 antibody is avelumab (BAVENCIO (trademark registered)). Avelumab is a human IgG1λ monoclonal anti-PD-L1 antibody.

[0106] Anti-PD-L1 antibodies usable in the methods of this disclosure include isolated antibodies that specifically bind to human PD-L1 and cross-compete with any anti-PD-L1 antibodies of this 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 anti-PD-L1 antibody 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-competitive antibodies to a particular epitope region. Because these cross-competitive antibodies bind to the same epitope region as PD-L1, they are expected to have very similar functional properties to the reference antibody (e.g., atezolizumab and / or avelumab). Cross-competitive antibodies can be readily identified based on their cross-competitive activity with atezolizumab and / or avelumab by standard PD-L1 binding assays such as Biacore analysis, ELISA assay, or flow cytometry (see, e.g., WO 2013 / 173223).

[0107] In some embodiments, an antibody that cross-competes with a human PD-L1 antibody (atezolizumab, durvalumab, and / or avelumab) to bind to human PD-L1, or an antibody that binds to the same epitope region, is a monoclonal antibody. In human administration, these cross-competitive antibodies are chimeric antibodies, modified antibodies, or humanized or human antibodies. Such chimeric, modified, humanized, or human monoclonal antibodies can be manufactured and isolated by methods well known to those skilled in the art.

[0108] The anti-PD-L1 antibodies usable in the methods of this disclosure also include the antigen-binding sites of the above-mentioned antibodies. It has been well demonstrated that the antigen-binding ability of an antibody can be exerted in fragments of a full-length antibody.

[0109] A suitable anti-PD-L1 antibody for use in the methods of this disclosure is an antibody that binds to PD-L1 with high specificity and affinity, inhibits PD-1 binding, and inhibits the immunosuppressive effects of the PD-1 signaling pathway. In any method of this disclosure, the anti-PD-L1 "antibody" includes an antigen-binding site or fragment that binds to PD-L1 and exhibits functional properties similar to the functional properties of the whole antibody in inhibiting receptor binding and promoting the immune system. In some embodiments, the anti-PD-L1 antibody or its antigen-binding site cross-competes with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1.

[0110] The anti-PD-L1 antibody useful in this disclosure may be an antibody that cross-competes with any PD-L1 antibody that specifically binds to PD-L1 (e.g., durvalumab, avelumab, or atezolizumab) (e.g., an antibody that binds to the same epitope as durvalumab, avelumab, or atezolizumab). In one embodiment, the anti-PD-L1 antibody is durvalumab. In another embodiment, the anti-PD-L1 antibody is avelumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab.

[0111] CTLA4 Antagonist The following are examples of CTLA4 antagonists that can be combined with DGK inhibitors:

[0112] A CTLA-4 antagonist is a human CTLA-4 antagonist that stimulates an immune response by inhibiting a negative checkpoint. Antagonists can be various types of molecules, such as proteins, nucleic acids, or small molecules. In some embodiments, a 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 may 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. Since the interaction between CTLA-4 and B7 transmits a signal that inactivates T cells having a CTLA-4 receptor, inhibiting this interaction effectively induces, enhances, and prolongs T cell activation. This can thereby induce, enhance, or prolong an immune response.

[0114] A human monoclonal antibody that specifically binds to CTLA-4 with high affinity is disclosed in U.S. Patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies are described, for example, in U.S. Patents Nos. 5,977,318, 6,051,227, 6,682,736 and 7,034,121 and International Publication Numbers WO 2012 / 122444, WO 2007 / 113648, WO 2016 / 196237 and WO 2000 / 037504, all of which are incorporated by reference. The anti-CTLA-4 human monoclonal antibody disclosed in U.S. Patent No. 6,984,720 has been demonstrated to exhibit one or more of the following characteristics: (a) an equilibrium association constant (Ka) of at least approximately 10 7 M -1 or about 10 9 M -1 or about 10 10 M -1 ~10 11 M -1 (b) It specifically binds to human CTLA-4 with the above binding affinity (determined by Biacore analysis); (b) it has a kinetic association constant (ka) of at least approximately 10 3 , about 10 4 or about 10 5 m -1 s -1 (c) The kinetic dissociation constant (kd) is at least about 10 3 , about 10 4 or about 10 5 m -1 s -1(d) inhibits the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies useful in this 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 features.

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

[0116] In certain embodiments, the CTLA-4 antibody used in the method of the present disclosure is ipilimumab. Ipilimumab is a fully human IgG1 monoclonal antibody that activates T cells by inhibiting CTLA-4 from binding to B7 ligand, thereby 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] The anti-CTLA-4 antibodies usable in the methods of this disclosure include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with any anti-CTLA-4 antibodies of this 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 anti-CTLA-4 antibody 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-competitive antibodies to a particular epitope region. Because these cross-competitive antibodies bind to the same epitope region as CTLA-4, they are expected to have very similar functional properties to the reference antibody (e.g., ipilimumab and / or tremelimumab). Cross-competitive antibodies can be readily identified based on their cross-competitive activity with ipilimumab and / or tremelimumab by standard CTLA-4 binding assays such as Biacore analysis, ELISA assay, or flow cytometry (see, e.g., WO 2013 / 173223).

[0121] In some embodiments, an antibody that cross-competes with a human CTLA-4 antibody (ipilimumab and / or tremelimumab) and binds to human CTLA-4, or an antibody that binds to the same epitope region, is a monoclonal antibody. In human administration, these cross-competitive antibodies are chimeric antibodies, modified antibodies, or humanized or human antibodies. Such chimeric, modified, humanized, or human monoclonal antibodies can be manufactured and isolated by methods well known to those skilled in the art.

[0122] The anti-CTLA-4 antibodies usable in the methods of this disclosure also include the antigen-binding sites of the antibodies described above. It has been well demonstrated that the antigen-binding ability of antibodies is exerted in fragments of full-length antibodies.

[0123] An anti-CTLA-4 antibody suitable for use in the methods of this disclosure is an antibody that binds to CTLA-4 with high specificity and affinity, inhibits the activity of CTLA-4, and prevents CTLA-4 from interacting with the human B7 receptor. In any composition or method of this disclosure, the anti-CTLA-4 "antibody" includes an antigen-binding site or fragment that binds to CTLA-4 and exhibits functional properties similar to those of the whole antibody in inhibiting the interaction between CTLA-4 and the human B7 receptor and promoting the immune system. In some embodiments, the anti-CTLA-4 antibody or its antigen-binding site cross-competes with ipilimumab and / or tremelimumab that bind to human CTLA-4.

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

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

[0126] Furthermore, this specification includes a class of pharmaceutical compositions comprising a compound of formula (I) or (II) (selected from compounds 1 to 34) and / or a pharmaceutically acceptable salt thereof; and one or more non-toxic, pharmaceutically acceptable carriers and / or diluents and / or adjuvants (substances collectively referred to herein as “carriers”) and, optionally, other active ingredients. The compound of formula (I) or (II) (selected from compounds 1 to 34) may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to 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 intravascular, intravenous, intraperitoneal, subcutaneous, intramuscular, and intrasternal, in dosage unit formulations containing pharmaceutically acceptable conventional carriers, adjuvants, and vehicles. For example, the pharmaceutical carrier may include a mixture of mannitol or lactose and microcrystalline cellulose. The mixture may contain additives such as a lubricant (e.g., magnesium stearate) and a disintegrant (e.g., crospovidone). The carrier mixture may be filled into gelatin capsules or compressed into tablets. The pharmaceutical composition may be administered, for example, in oral dosage form or by intravenous 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. The pharmaceutical compositions are preferably formulated in dosage units having a specific amount of the active ingredient. For example, the pharmaceutical compositions may be provided as tablets or capsules containing an amount of the active ingredient ranging from about 0.1 to 1000 mg, preferably about 0.25 to 250 mg, and more preferably about 0.5 to 100 mg. An appropriate daily dose for administration to humans or other mammals may be determined using conventional methods, although this may vary considerably depending on the patient's condition and other factors.

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

[0129] Tablets may be manufactured, for example, by mixing at least one compound of formula (I) or (II) (selected from compounds 1 to 34) and / or at least one pharmaceutically acceptable salt with at least one non-toxic and pharmaceutically acceptable additive suitable for the manufacture of tablets. Examples of additives include, but are not limited to, inert diluents (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate, and sodium phosphate), granulators and disintegrants (e.g., microcrystalline cellulose, croscarmellose sodium, corn starch, and alginic acid), binders (e.g., starch, gelatin, polyvinylpyrrolidone, and gum arabic), and lubricants (e.g., magnesium stearate, stearic acid, and talc). Furthermore, tablets may be left uncoated or coated by known techniques to mask the unpleasant taste of the drug or to delay the disintegration and absorption of the active ingredient in the gastrointestinal tract, thereby prolonging the effect of the active ingredient over a longer period. Examples of water-soluble taste-masking materials include, but are not limited to, hydroxypropyl methylcellulose and hydroxypropylcellulose. Examples of time-delaying materials include, but are not limited to, ethylcellulose and cellulose acetate-butyrate.

[0130] Hard gelatin capsules can be manufactured, for example, by mixing at least one compound of formula (I) or (II) (selected from compounds 1 to 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 may be manufactured, for example, by mixing at least one compound of formula (I) or (II) (selected from compounds 1 to 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 oily 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 to 34) and / or at least one pharmaceutically acceptable salt thereof with at least one additive suitable for the preparation of aqueous suspensions. Examples of additives suitable for the preparation of aqueous suspensions include, but are not limited to, suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, alginic acid, polyvinylpyrrolidone, tragacanth gum, and gum arabic), dispersants 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), ethylene oxide and fatty acids and Examples include condensation products with partial esters derived from hexitol (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-hydroxybenzoic acid), at least one coloring agent, at least one flavoring agent, and / or at least one sweetener (but not limited to, e.g., sucrose, saccharin, and aspartame).

[0133] An oily suspension may be prepared, for example, by suspending at least one compound of formula (I) or (II) (selected from compounds 1 to 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). The oily suspension may also contain at least one thickening agent (e.g., beeswax, solid paraffin, and cetyl alcohol). To provide an easily drinkable oily suspension, at least one sweetener already described above and / or at least one flavoring agent may be added to the oily suspension. The oily suspension may further contain at least one preservative (but not limited to, for example, an antioxidant (e.g., butylhydroxyanisole, and α-tocopherol)).

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

[0135] Emulsions of at least one compound of formula (I) or (II) (selected from compounds 1 to 34) and / or at least one pharmaceutically acceptable salt can be prepared, for example, as an oil-in-water emulsion. The oil phase of an emulsion containing a compound of formula (I) or (II) (selected from compounds 1 to 34) may consist of known components in known ways. The oil phase may be provided by, but is not limited to, vegetable oils (e.g., olive oil and peanut oil), mineral oils (e.g., liquid paraffin), and mixtures thereof. The oil phase may consist only of emulsifiers, or it may consist of at least one emulsifier and fats or oils, or mixtures of both fats and oils. Suitable emulsifiers include, but is not limited to, naturally occurring phosphatides (e.g., soy lecithin), esters or partial esters derived from fatty acids and hexitol anhydrides (e.g., sorbitan monooleate), and condensation products of partial esters and ethylene oxides (e.g., polyoxyethylene sorbitan monooleate). Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier acting as a stabilizer. It is also preferable to include both oils and fats. Together, the emulsifier, together with or without the stabilizer, forms a so-called emulsifying wax, and the wax, together with the oils and fats, forms a so-called emulsifying ointment base, which forms the oily dispersion phase of the cream formulation. The emulsion may also include sweeteners, flavoring agents, preservatives, and / or antioxidants. Suitable emulsifiers and emulsion stabilizers for use in formulations used in the therapeutic methods of this invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate, either alone or in combination with wax; or other substances known in the art.

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

[0137] Parenteral formulations 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 carriers or diluents described for use in oral formulations, or by using other suitable dispersants or wetting 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 known and widely practiced in the pharmaceutical field. The active ingredient may also be administered by injection as a composition with a suitable carrier (e.g., physiological saline, dextrose, or water), or with cyclodextrin (i.e., Captisol), a solubilizing cosolvent (i.e., propylene glycol), or a solubilizing micelle (i.e., Tween 80).

[0138] Furthermore, sterile injectable formulations may be sterile injectable solutions or suspensions (e.g., solutions in 1,3-butanediol) in non-toxic, parenterally acceptable diluents or solvents. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and sodium chloride isotonic solutions. In addition, sterile non-volatile oils are conventionally used as solvents or suspension media. For this purpose, any sterile non-volatile oil, including synthetic monoglycerides or diglycerides, may be used. Furthermore, fatty acids such as oleic acid are used as injectable formulations.

[0139] A sterile oil-in-water microemulsion for injection can be produced, for example, by: 1) dissolving at least one compound of formula (I) or (II) (selected from compounds 1 to 34) in an oil phase (e.g., a mixture of soybean oil and lecithin); 2) combining the compound of formula (I) or (II) (selected from compounds 1 to 34) containing the oil phase with a mixture of water and glycerol; and 3) processing the combination to form a microemulsion.

[0140] Sterile aqueous suspensions or sterile oily suspensions may be prepared according to methods known to those skilled in the art. For example, sterile aqueous solutions or sterile aqueous suspensions may be prepared using non-toxic, parenterally acceptable diluents or solvents (e.g., 1,3-butanediol), and sterile oily suspensions may be prepared using sterile, non-toxic, acceptable solvents or suspension media (e.g., sterile non-volatile 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 surfactant (BASF), or other similar polymer delivery matrices)), serum proteins (e.g., human serum albumin), buffering substances (e.g., phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts, or electrolytes (e.g., protamine sulfate) Examples include disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate), polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. Cyclodextrins (e.g., α-, β-, and γ-cyclodextrins) or chemically modified derivatives (e.g., hydroxyalkyl cyclodextrins including 2- and 3-hydroxypropyl cyclodextrins, or other solubilized derivatives) may also be advantageously used to enhance the transport of compounds of the formulas described herein.

[0142] The pharmaceutically active compounds described herein may be processed according to conventional pharmaceutical methods for preparing drugs to be administered to patients (e.g., humans and other mammals). Pharmaceutical compositions may be subjected to conventional pharmaceutical procedures (e.g., sterilization) and / or may contain conventional adjuvants (e.g., preservatives, stabilizers, humectants, emulsifiers, buffers, etc.). Tablets and pills may also be prepared with enteric coatings. Such compositions may also contain adjuvants (e.g., humectants, sweeteners, flavorings, and fragrances).

[0143] The amount of compounds administered to treat a medical condition using the compounds and / or compositions described herein, and the dosage schedule, are influenced by various factors (e.g., age, weight, sex, patient's condition, type of disease, severity of disease, route and frequency of administration, and the specific compound used). Therefore, the dosage schedule may be significantly modified, but it can be determined according to standard methods. The daily dose may be appropriate between about 0.001 and 100 mg / kg body weight, preferably between about 0.0025 and 50 mg / kg body weight, and most preferably between about 0.005 and 10 mg / kg body weight. The daily dose may be administered one to four times a day. Other dosage schedules include weekly and bi-weekly cycles.

[0144] For therapeutic purposes, the active compounds described herein are typically combined with one or more adjuvants appropriate to the intended route of administration. When administered orally, the compounds may be mixed with lactose, sucrose, starch powder, cellulose esters of alkanates, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfate, gelatin, gum arabic, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, and then encapsulated or tableted for convenient administration. Such capsules or tablets may contain a controlled-release formulation and may be provided with the active compound dispersed in hydroxypropyl methylcellulose.

[0145] The pharmaceutical compositions described herein comprise, as appropriate, at least one compound of formula (I) or (II) (selected from compounds 1 to 34) and / or at least one pharmaceutically acceptable salt thereof, and additives selected from any pharmaceutically acceptable carriers, adjuvants, and vehicles. Another composition described herein comprises a compound of formula (I) or (II) (selected from compounds 1 to 34) as described herein, or its prodrug, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0146] In some embodiments, the anti-PD-L1 antibody used in the therapeutic method described herein is administered once every 2, 3, 4, 5, 6, 7, or 8 weeks in a dose ranging from approximately 0.1 mg / kg body weight to approximately 20.0 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg, approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 17 mg / kg, approximately 18 mg / kg, approximately 19 mg / kg, or approximately 20 mg / kg.

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

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

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

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

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

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

[0153] In some embodiments, the anti-CTLA-4 antibody or its antigen-binding site is administered in the maximum effective dose. In some embodiments, the anti-CTLA-4 antibody is administered in the maximum effective dose of approximately 10 to 1000 mg, approximately 10 mg to 900 mg, approximately 10 mg to 800 mg, approximately 10 mg to 700 mg, approximately 10 mg to 600 mg, approximately 10 mg to 500 mg, approximately 100 mg to 1000 mg, approximately 100 mg to 900 mg, approximately 100 mg to 800 mg, approximately 100 mg to 700 mg, approximately 100 mg to 100 mg, approximately 100 mg to 500 mg, approximately 100 mg to 480 mg, or approximately 240 mg to 480 mg. In one embodiment, the anti-CTLA-4 antibody or its antigen-binding site is present in amounts of at least approximately 60 mg, at least approximately 80 mg, at least approximately 100 mg, at least approximately 120 mg, at least approximately 140 mg, at least approximately 160 mg, at least approximately 180 mg, at least approximately 200 mg, at least approximately 220 mg, at least approximately 240 mg, at least approximately 260 mg, at least approximately 280 mg, at least approximately 300 mg, at least approximately 320 mg, at least approximately 340 mg, at least approximately 360 mg, at least approximately 380 mg, at least approximately 400 mg, at least approximately 420 mg, at least approximately 440 mg, at least approximately 460 mg, at least approximately 480 mg, at least approximately 500 mg, and at least approximately 520 mg. The maximum effective dose is administered at least approximately 540 mg, at least approximately 550 mg, at least approximately 560 mg, at least approximately 580 mg, at least approximately 600 mg, at least approximately 620 mg, at least approximately 640 mg, at least approximately 660 mg, at least approximately 680 mg, at least approximately 700 mg, or at least approximately 720 mg. In another embodiment, the anti-CTLA-4 antibody or its antigen-binding site is administered once every approximately 1, 2, 3, 4, 5, 6, 7, or 8 weeks at the maximum effective dose.

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

[0155] (Method of producing compounds) The compounds described herein can be synthesized by many methods available to those skilled in the art of organic chemistry. A general synthesis scheme for producing the compounds described herein is described below. This scheme is illustrative and is not intended to limit the possible techniques that those skilled in the art may use to produce the compounds described herein. Various methods for producing the compounds described herein are obvious to those skilled in the art. Examples of compounds produced by the methods described in the general scheme are shown in the Examples section below. The production of homochiral examples can be carried out by techniques known to those skilled in the art. For example, homochiral compounds can be produced by separating racemic products or diastereomers by chiral phase preparative HPLC. Alternatively, the compounds can be produced by known methods that yield enantiomer-rich or diastereomer-rich products.

[0156] The reactions and techniques described in this section are carried out in solvents suitable for the reagents and substances used and are appropriate for the resulting transformations. Furthermore, in the descriptions of the synthetic methods described below, all presented reaction conditions (including solvent selection, reaction atmosphere, reaction temperature, experimental time, and work-up method) are understood to be selected to be standard conditions for the reaction, and this should be readily apparent to those skilled in the art. It is understood to those skilled in the field of organic synthesis that the functional groups present in various parts of the molecule must be compatible with the presented reagents and reactions. It is readily apparent to those skilled in the art that the substituents suitable for the reaction conditions are thus limited, and if the available substituents are unsuitable, alternatives will be required. The reaction may require a change in the order of the synthetic steps or a decision to select a different particular reaction process in order to obtain the desired compound. Another important consideration in any synthetic route planning in this art is the wise selection of protecting groups to be used to protect the reactive functional groups present in the desired compounds described herein. For experienced experimenters, a highly authoritative publication listing numerous protecting group alternatives is Greene's Protective Groups in Organic Synthesis (Fourth Edition, Wiley & Sons, 2007) by Wuts and Greene.

[0157] (Examples) The following examples illustrate specific and preferred embodiments of the present disclosure and do not limit the scope of the disclosure. Chemical abbreviations and symbols, as well as scientific abbreviations and symbols, have their general and conventional meanings unless otherwise noted. Further abbreviations used in the examples and throughout 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 preparation methods for intermediates or examples are described. Experienced chemists in the field of synthesis may frequently devise preferred alternative preparation methods based on one or more considerations (e.g., shorter reaction times, cheaper starting materials, ease of handling and purification, higher yields, ease of catalysis, avoidance of toxic reagents, availability with specialized equipment, and reduction in the number of steps, etc.). The intention of describing alternative preparation methods is to make the examples of this disclosure easier to prepare. In some cases, some functional groups in the outlined examples and claims may be substituted by biological isometric substitutions known to those skilled in the art (e.g., substituting a carboxylic acid group with a tetrazole or phosphate moiety). Measured with deuterated dimethyl sulfoxide. 1 The 1H NMR data were processed using water suppression. The spectra shown are not corrected for the effect of water suppression. Protons adjacent to the 3.35 ppm water suppression frequency are shown with reduced signal intensity.

[0158] TIFF0007856566000031.tif238164

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

[0160] The assay was performed as follows: Peripheral blood mononuclear cells were isolated from whole blood treated with EDTA by cell isolation (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 treated with GMCSF and IL-4 for 6 days in a 37°C CO2 incubator to differentiate them into dendritic cells (DCs). T cells were seeded at a rate of 100,000 cells / well in 10% FBS RPMI medium in a 96-well round-bottom plate. Allogeneic dendritic cells were added to appropriate wells so that the T cell:immature DC ratio was 10:1. DGKi compound 15, a DGK inhibitor, was diluted in DMSO, then diluted in 10% FBS RPMI medium, and added to appropriate wells of T cells:immature DCs so that the final DMSO concentration was 0.1% at 250 μL (final volume). A mixed lymphocyte reaction was performed in an incubator for 5 days. On day 5, 130 μL of medium was removed, and 10 μL was used for the IFN-γ ELISA assay (BD cat 555142).

[0161] The results shown in Figures 1A and 1B indicate that T cells treated with PD-1 or CTLA-4 inhibitors secrete increased IFN-γ due to DGK inhibition.

[0162] Example 2: In the B16 animal tumor model, DGK inhibition activates a combination of PD-1 antagonist and CTLA4 antagonist. This example demonstrates that administering DGKi simultaneously with a PD-1 antagonist and a CTLA4 antagonist leads to tumor reduction compared to the combination of a PD-1 antagonist and a CTLA4 antagonist. This assay was performed using the B16 tumor model (human melanoma tumor model). Mice were administered anti-PD-1 antibody (mIgG1-D265A monoclonal antibody against mouse PD-1), anti-CTLA4 antibody (mIgG2b monoclonal antibody against mouse CTLA4), Vehicle alone, and / or DGKi, and tumor growth was measured. The results, shown in Figures 2A-G, showed that no significant tumor reduction was observed with individual drugs or combinations of two drugs, but tumor reduction was observed when anti-PD-1 antibody and anti-CTLA4 antibody were combined with DGKi (Figure 2G).

[0163] Example 3: Inhibition of DGK enhances PD-1 inhibitory activity and / or CTLA-4 inhibitory activity in the CT26 animal tumor model. This example demonstrates that in the CT26 model, administration of a DGK inhibitor promotes tumor reduction mediated by anti-PD-1 and / or anti-CTLA4 antibodies. The assay was performed as follows: CT26 cells (mouse colorectal cancer cell line (ATCC)) were cultured in RPMI 1640 medium (Gibco / ThermoFisher Scientific) containing 10% fetal bovine serum (Invitrogen / ThermoFisher Scientific). Female BALB / c mice aged 6-8 weeks were purchased from Envigo. At tumor transplantation (day 0), a suspension of CT26 cells (1x10⁶) was used. 7 The solution (0.1 mL) was subcutaneously injected into the right flank of a mouse. The tumor was reduced to a predetermined volume (~100 mm). 3When the tumor reached the stage of remission (usually about 10 days after transplantation), mice were randomly selected, classified into various control and treatment groups, and administration was initiated. DGKi compound 16 was formulated in 90% PEG400, 5% ethanol, and 5% TPGS, and administered orally at a dose of 10 mL / kg body weight. Anti-CTLA4 (anti-mCTLA4, mIgG2b) and anti-PD1 (mIgG1-D265A monoclonal antibody against mouse PD-1) and isotype controls were diluted in DPBS to a dose of 10 mg / kg. Antibody therapeutics were administered by intraperitoneal infusion (IP) every 4 days in a total dose of three doses (Q4Dx3). Tumor volume was reduced to 0 mm when the tumor completely regressed. 3 ) or 1000mm 3 The mice were measured twice a week with digital calipers until they reached a certain level and were euthanized. 100 μL of blood was collected from each mouse and added to a lithium heparin tube for AH1 tetramer staining. The blood was stained with AH1 tetramer (MBL), anti-Cd3, anti-Cd4, and anti-Cd8 (Biolegend). The samples were lysed with Lyse / Fix buffer (BD), and the samples collected from a CantoX hemocytometer (BD) were analyzed using FlowJo (BD).

[0164] The results shown in Figures 3A-H demonstrate that DGKi activates the tumor volume reduction effects in the CT26 mouse model mediated by (i) PD1 inhibitors; (ii) CTLA-4 inhibitors; and (iii) PD1 inhibitors and CTLA-4 inhibitors. The results shown in Figure 3I demonstrate that DGKi increases the proportion of CD8 cells positive for the AH1+ tetramer tumor antigen. Therefore, this combination therapy resulted in an increased complete remission rate in the CT26 model, which correlated with an increase in AH1+ T cells. The combination of both CTLA4 antagonists and PD1 antagonists with a DGK inhibitor yielded the highest number of complete remissions, with 10 out of 10 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) promotes the T cell response even with low affinity tumor antigens, and (2) reduces the concentration of tumor antigens required for T cell activation. This assay was performed as follows: MC38 cells (mouse 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 product instructions. MC38 cells were pulsed with the peptide (1 μg / mL or specified concentration) for 3 hours, followed by washing to remove the free peptide. OT1 mice (obtained by crossing C57BL / 6 mice, with a TCR that recognizes only MHC class I, and genetically modified to be specific to ovalbumin (OVA(SIINFEKL) or derivatives of the following OVA peptides: A2(SAINFEKL), Q4(SIIQFEKL), T4(SIITFEKL), Q4H7(SIIQFEHL)), but do not recognize the recombinant peptide (FILKSINE)) were purchased from Jackson Laboratory. The binding affinity of these peptides to the TCR is shown in the table below. CD8 T cells were purified from whole splenocytes (StemCells) of OT1 mice, activated using CD3 / CD28 beads (Invitrogen), and then frozen. The frozen activated OT-1 CD8 T cells were thawed during peptide pulses and fixed to plates for 1 hour with DGKi compound 15, a control compound, or DMSO. Protein-pulsed MC38 cells were added to the plates and co-cultured overnight at 37°C. The supernatant was collected, and IL-2 levels were measured using AlphaLISA (PerkinElmer). [Table 1] The results shown in Figures 4A-F indicate that DGKi compound 15 reduces the affinity and antigen concentration necessary for T cells to recognize it as an antigen and activate it.

[0166] Example 5: Inhibition of DGK activates human CTL effector function and enhances tumor-killing activity. This example demonstrates that inhibition of DGK enhances CTL effector function and tumor-killing activity. The assay was performed as follows: HCT116-GFP (human colorectal cancer) cells were purchased from Cellomics. HCT116-GFP cells were pulsed with predetermined concentrations of A2 and B35 peptides (Astarte) for 1 hour, followed by washing. The 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 the HCT116-GFP cells. After 24 hours of co-culture, the supernatant was collected and IFNγ was measured using AlphaLISA (PerkinElmer). GFP images were taken using a fluorescence microscope. The results shown in Figures 5A and 5B indicate that DGKi compound 15 activates human CTL effector function and enhances tumor-killing activity.

[0167] Example 6: Inhibition of DGK can restore the reduced T cell effector function caused by decreased B2M levels. Many human tumors exhibit 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 allows T cells to recognize tumor cells with reduced MHC levels. 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 harvesting, cells were seeded in each well to produce single-cell clones. Clonal cells were stained with B2M (Biolegend) and evaluated by flow cytometry. Clonal cells were then pulsed with 1 mg / mL of 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 shown in Figures 6A and 6B indicate that DGKi compound 15 increases IFN-γ levels from T cells that recognize tumor cells with reduced MHC class I antigens.

[0168] Example 7: Indication of tumor therapeutic activity by DGK inhibition and PD1 antagonists with CD8 + T cells have an effect. This example demonstrates the tumor therapeutic activity of CD8 in the CT26 animal model. + This shows that T cells have an influence. 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). Female BALB / c mice aged 6-8 weeks were purchased from Envigo. At tumor transplantation (day 0), CT26 cell suspension (1x10) was used. 7A dose of (0.1 mL) of the solution (cells / mL) was subcutaneously injected into the right flank of mice. CD8 depletion antibody (2.43, Bio X Cell) was diluted in PBS and administered at a dose of 100 μg per mouse. Administration began on day 1 and continued every 3-4 days until the experiment was completed. The tumor was reduced to a predetermined volume (~100 mm). 3 At this stage (generally about 10 days after transplantation), mice were randomly selected, classified into various control and treatment groups, and administration was initiated. DGKi compound 16 was formulated in 90% PEG400, 5% ethanol, and 5% TPGS, and administered orally at a dose of 10 mL / kg body weight. A total of five doses (Q3Dx5) were administered at 5 mg / kg every three days. 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 therapy was administered by intraperitoneal injection (IP) at a total of three doses (Q4Dx3) every four days. Tumor volume was reduced to 0 mm when the tumor completely regressed. 3 ) or 1000mm 3 We measured the animal twice a week with a digital caliper until it reached a certain point and was euthanized. The results shown in Figure 7 indicate that CT26 mice treated with an anti-PD-1 antagonist and DGKi compound 16 showed a higher CD8 + This indicates that the reduction in the number of cells led to a decrease in the tumor's volume.

[0169] Example 8: Tumor volume reduction induced by DGK inhibition and PD1 antagonists is increased by a decrease in CD4 cells. This example demonstrates that tumor reduction occurs with a combination of a DGK inhibitor and a PD-1 antagonist, and further tumor reduction occurs due to a decrease in CD4 cells. 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). Female BALB / c mice aged 6-8 weeks were purchased from Envigo. At tumor transplantation (day 0), CT26 cell suspension (1x10) was used. 7A dose of (0.1 mL) was subcutaneously injected into the right flank of mice. CD4 depletion antibody (GK1.5, Bio X Cell) was diluted in PBS and administered at a dose of 100 μg per mouse. Administration began on day 1 and continued every 3-4 days until the experiment was completed. The tumor was reduced to a predetermined volume (~100 mm). 3 At approximately 10 days after transplantation (generally), mice were randomly selected, classified into various control and treatment groups, and administration was initiated. DGKi compound 16 was formulated in 90% PEG400, 5% ethanol, and 5% TPGS and administered orally at a dose of 10 mL / kg body weight. A total of five doses (Q3Dx5) were administered at 5 mg / kg every three days. 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 therapy was administered by intraperitoneal injection (IP) at a total of three doses (Q4Dx3) every four days. Tumor volume was reduced to 0 mm when the tumor completely regressed. 3 ) or 1000mm 3 We measured the animal twice a week with a digital caliper until it reached a certain point and was euthanized. The results shown in Figure 8 indicate that MC38 mice treated with an anti-PD-1 antagonist and DGKi compound 16 showed a decrease in CD4 cells, likely due to a reduction in Treg cells. + This indicates that the tumor volume further decreased due to the reduction in cells.

[0170] Example 9: NK cells are required for the activation of DGKi and anti-PD1 antitumor agents. This example demonstrates that NK cells influence the tumor reduction activity induced by DGKi and PD1 antagonists in the CT26 animal model. The assay was basically carried out as described in Examples 6 and 7, but instead of antibodies that bind to CD4 or CD8, anti-asialoGM1 (Life Technologies) was administered at a dose of 50 μg per animal starting on day 4 after tumor transplantation, and continued every 7 days until the end of the experiment. The results shown 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: Combinations of DGKi of formula II with either anti-PD-1 or anti-CTLA4 demonstrate reliable efficacy. This example demonstrates that, in the MC38 animal model, the combination of 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 exhibits potent antitumor activity. The assay was performed as follows: Mouse colon adenocarcinoma tumor cell line MC38 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) containing 10% fetal bovine serum (FBS, Invitrogen) in T75 flasks. The cells were cultured until the cell density reached subconfluence, and then passed through twice a week by simply rinsing with DPBS (Dulbecco's phosphate-buffered saline, Gibco), allowing the cells to stand for several minutes, and then removing them from the flask. Depending on the timing and cell density, the MC38 cell density was passed through to a range of 1:16 to 1:20. For in vivo transplantation, the cells were rinsed with DPBS and then collected in ice-cold HBSS (Hank's equilibrium salt solution, Gibco) in a conical tube (50 mL) on ice. The tube was centrifuged at 1300 rpm for 10 minutes, the supernatant was carefully removed, the precipitate was washed with HBSS, and centrifuged again. The precipitate was suspended in approximately the transplantation volume of HBSS. Cell concentration was measured using Moxi-Z (Orflo), and the final concentration was adjusted using HBSS. Cell viability was measured using Countess II (Life Technologies) by trypan blue exclusion method. Female C57Bl / 6 mice aged 6-8 weeks were purchased from Charles River Laboratories (Kingston, NY) and acclimatized for 3-7 days. At the time of tumor transplantation (day 0), MC38 cells (8.5 x 10⁶) were transplanted using a tuberculin syringe (1 mL) fitted with a 25 gauge needle. 6The cells (0.1 mL) were subcutaneously injected into mice and transplanted into both the left and right flanks. On day 6 (post-transplant), the tumors reached a predetermined volume (~78 mm²). 3 The tumors grew to ) [size]. Based on the average tumor volume at that point, the animals were classified into various treatment and control groups (1 group n=10). Treatment was started on day 7 (post-transplant), at which point the tumors were ~100 mm. 3 The following was done. A 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 a dose of 0.3 mg / kg. Anti-PD-1 (mIgG1-D265A monoclonal antibody against mouse PD-1), anti-CTLA4 (mIgG2b monoclonal antibody against mouse CTLA4), and corresponding isotype controls (InVivoPlus mouse IgG1, clone MOPC-21 and InVivoMab mouse IgG2b, clone MPC-11 (the respective anti-PD-1 and anti-CTLA4 isotype controls purchased from Bio X Cell (West Lebanon, NH))) were diluted in DPBS to a dose of 10 mg / kg. Antibody therapies were administered by intraperitoneal infusion (IP) in a total of three doses (Q4Dx3) every four days. Tumor volume is when the tumor completely regresses (0 mm) 3 ) or 1000mm 3 We measured the animal twice a week with a digital caliper until it reached a certain point and was euthanized. The results shown in Figure 10 indicate that while no significant activity was observed with DGKi alone (Figures 10B-D), potent antitumor activity was observed when DGKi was combined with an anti-PD-1 antibody or an anti-CTLA4 antibody (Figures 10E and F, respectively).

[0172] Example 11: In both MC38 and CT26 animal models, the combination of the compound of formula II and the anti-PD-1 antibody exhibited potent antitumor activity and sustained immunological memory. This example demonstrates that in both MC38 and CT26 animal models, the combination of Example DGKi of Formula II, selected from the group of compounds 17-34, and anti-PD-1 exhibits potent antitumor activity, enabling complete regression and sustained immunological memory. The studies were conducted as follows: The MC38 animal model study was performed as described in Example 10. The CT26 animal model study was performed as described in Example 3. DGKi and anti-PD-1 were prepared (similar to Example 10) and administered as described in Example 10. Animals cured by this treatment method showed no change in tumor volume even after a 10-fold tumor volume doubling time (TVDT, 10 x 4.2 days = 42 days). Cells at 10 times the initial cell concentration were subcutaneously transplanted into the right flank of these animals, and the secondary response of T cells was evaluated by measuring twice a week for more than 42 days. The results shown in Figures 11A-H demonstrate that the combination of DGKi (formula II) and the anti-PD-1 antibody leads to potent antitumor activity in animal models. Furthermore, in the MC38 and CT26 models, 100% of transplanted tumor cells were rejected during re-transplantation (Figures 11D and H).

[0173] Example 12: In the B16F10 animal model, the combination of compound II with anti-PD-1 and anti-CTLA4 showed more potent activity compared to the combination of compound II with anti-PD-1 or anti-CTLA4. This example is B16F10 (melanoma / MHCI). lo In animal models, we demonstrate that combinations of three antibodies—DGKi of formula II (selected from the group of compounds 17-34), an anti-PD-1 antibody, and an anti-CTLA4 antibody—produce antitumor activity, and that this activity is more potent than that of two antibodies. The animal model studies were conducted as follows: Mouse melanoma cell line B16F10 was cultured in Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Invitrogen) in T75 flasks. Cells were cultured until subconfluent, and passage was performed twice a week by simply rinsing the flask with DPBS (Dulbecco's phosphate-buffered saline, Gibco), then with trypsin (0.25% trypsin, Gibco), allowing the cells to stand for several minutes before removing them from the flask. Depending on the timing and cell density, passage was performed so that the density of B16F10 cells was in the range of 1:18 to 1:20. For in vivo transplantation, cells were trypsinized as described above and then collected in ice-cold HBSS (Hank's equilibrium salt solution, Gibco) in a conical tube (50 mL) on ice. The tubes were centrifuged at 1300 rpm for 10 minutes, the supernatant was carefully removed, the precipitate was washed with HBSS, and centrifuged again. The precipitate was resuspended in approximately the transplant volume of HBSS. Cell concentration was measured using Moxi-Z (Orflo), and the final concentration was adjusted with HBSS. Cell viability was measured using Countess II (Life Technologies) by trypan blue exclusion method. Female C57Bl / 6 mice aged 6-8 weeks were purchased from Charles River Laboratories (Raleigh, NC) and acclimatized for 3-7 days. At the time of tumor transplantation (day 0), B16F10 cells (1x10) were transplanted using a tuberculin syringe (1 mL) fitted with a 25 gauge needle. 7 The cells (0.1 mL) were subcutaneously injected into mice and transplanted into the right flank. The tumor was then divided into a predetermined volume (~50 mm²). 3Treatment was initiated on day 8 (post-transplant) when the tumor had grown to 0 mm. Based on the average tumor volume at that time, the animals were classified into various treatment and control groups (1 group n=10). Formula II DGKi, selected from a group of 17-34 compounds, 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 a rate of 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 (similar to Example 10). Antibody therapies were administered by intraperitoneal infusion (IP) in three doses (Q4Dx3) every four days. Tumor volume was measured until the tumor completely regressed (0 mm). 3 ) or 1000mm 3 We measured the animal twice a week with a digital caliper until it reached a certain point and was euthanized. The results shown in Figures 12A-F demonstrate that in the B16F10 animal model, the combination of three treatments resulted in a better response compared to the combination of two treatments.

[0174] Example 13: Synthesis of a DGK inhibitor

[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-naphthyridine-4-yl)piperazine-2-carboxylate methyl [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-dimethylpiperazine-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-dimethylpiperazine-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-dimethylpiperazine-1-yl]-6-methoxy-1-methyl-1,2-dihydro-1,5-naphthyridine-2-one [ka]

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

[0186] Intermediate 1 6-Cyano-3-(N-methylacetamide)Ethyl Picolinate [ka] To a pale yellow, stirred solution of ethyl 3-(N-methylacetamide)-1-(1-oxydanyl)-1l4-pyridine-2-carboxylate (50 g, 210 mmol) / DCM (500 mL), trimethylsilyl cyanide (39.4 mL, 294 mmol) was added at room temperature. The mixture was stirred for 10 minutes and cooled to -10°C. Next, benzoyl chloride (34.1 mL, 294 mmol) was added over 15 minutes using a dropping funnel (50 mL), followed by the slow addition of TEA (41.0 mL, 294 mmol) over 20 minutes using a dropping funnel (50 mL). 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 organic solutions were combined and washed with saline solution (2 x 250 mL), then dried over Na2SO4, concentrated, and a pale yellow crude product was obtained. The crude product was purified using an ISCO (trademark registered) normal-phase RediSep silica column (eluent: EA / petroleum ether). The product was isolated (65-70% EA / petroleum ether), the fraction was concentrated, and ethyl 6-cyano-3-(N-methylacetamide)picolinate was obtained as a pale brown liquid (43 g, 83% yield). LC-MS: m / z=248.0(M+H); rt 1.255 min; LC-MS method: Column-KINETEX-XB-C18 (75x3mm; 2.6μm); Mobile phase A: 10mM ammonium formate aqueous solution:acetonitrile (98:2); Mobile phase B: 10mM ammonium formate aqueous solution:acetonitrile (2:98); Gradient: Elute with 20-100% B over 4 minutes (flow rate 1.0 mL / min), then elute with 100% B for 0.6 minutes (flow rate 1.5 mL / min). Subsequently elute with a gradient of 100-20% B over 0.1 minutes (flow rate 1.5 mL / min).

[0187] Intermediate 2 8-Hydroxy-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile TIFF0007856566000045.tif26356-Cyano-3-(N-methylacetamide) ethyl picolinate (0.9 g, 3.64 mmol) / tetrahydrofuran (10 mL) was stirred, and KHMDS (4.80 mL, 4.37 mmol) was added over 10 minutes at -78°C. The reaction mixture was stirred for 15 minutes, slowly warmed to room temperature over 30 minutes, and then stirred for a further 90 minutes. 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. This mixture was stirred for 15 minutes to form a solid, which was filtered through a Buchner funnel to obtain 8-hydroxy-5-methyl-6-oxo-5,6-dihydro-1,5-naphthirizine-2-carbonitrile (550 mg, 75% yield) as a brown solid. LC-MS: m / z=202.0(M+H); rt 0.361 min; LC-MS method: Column-KINETEX-XB-C18 (75x3 mm; 2.6 μm); Mobile phase A: 10 mM ammonium formate aqueous solution:acetonitrile (98:2); Mobile phase B: 10 mM ammonium formate aqueous solution:acetonitrile (2:98); Gradient: Elution over 4 minutes from 20 to 100% B at a flow rate of 1.0 mL / min, then elution over 0.6 minutes at 100% B (flow rate 1.5 mL / min). Subsequently, elution was performed over 0.1 minutes using a 100-20% B gradient (flow rate 1.5 mL / min).

[0188] Intermediate 3 8-Chloro-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] POCl3 (1.53 mL, 16.4 mmol) was added to a stirred solution of 8-hydroxy-5-methyl-6-oxo-5,6-dihydro-1,5-naphthiridine-2-carbonitrile (0.55 g, 2.73 mmol) / acetonitrile (10 mL). This mixture was heated at 85°C for 5 minutes and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure to obtain 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 x 100 mL). The organic layers were dried together over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 8-chloro-5-methyl-6-oxo-5,6-dihydro-1,5-naphthiridine-2-carbonitrile (0.25 g, 29.1% yield) as a brown solid. LC-MS: m / z=220.2(M+H); rt 1.528 min; LC-MS method: Column-KINETEX-XB-C18 (75x3mm; 2.6μm); Mobile phase A: 10mM ammonium formate aqueous solution:acetonitrile (98:2); Mobile phase B: 10mM ammonium formate aqueous solution:acetonitrile (2:98); Gradient: Elute over 4 minutes with 20-100% B (flow rate 1.0 mL / min), then elute over 0.6 minutes with 100% B (flow rate 1.5 mL / min). Subsequently elute over 0.1 minutes with a gradient of 100-20% B (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. Iodoacetonitrile (7 mL, 16.7 g, 68.3 mmol) was added dropwise to this mixture while vigorously stirring to obtain a yellowish-brown precipitate. The cold bath was removed and the reaction mixture was stirred overnight at room temperature. The flask was placed in a sonicator and the aggregated solid was crushed. The reaction mixture was stirred for a further 4 hours, the solid was collected by filtration and vacuum-dried to obtain (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] 6-Cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-4-yltrifluoromethanesulfonic acid (65 g, 195 mmol) and tert-butyl(2R,5S)-2,5-dimethylpiperazine-1-carboxylate (43.9 g, 205 mmol) / acetonitrile (1.3 L) were mixed with DIPEA (0.102 L, 585 mmol). The solution was stirred at 80°C for 6 hours, the solvent was removed, and the resulting crude residue was subjected to 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-naphthyridine-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 for 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-naphthyridine-4-yl)-2,5-dimethylpiperazine-1-carboxylate (30 g, 75 mmol) / ethyl acetate (1000 mL), HCl (4 M dioxane solution, 189 mL, 755 mmol) was added at 0°C, and the mixture was stirred for 6 hours while returning to room temperature. LC / MS analysis showed that at 0.60 RT, approximately 90% of the product mass was present, and at 0.44 RT, approximately 4% of the amide by-product mass (consistent with the nitrile hydrolysate) was shown together. This reaction mixture was diluted with methyl t-butyl ether (MTBE, 2000 mL), stirred for 15 minutes, 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 approximately 8 using a 10% sodium bicarbonate aqueous solution. The organic layers were extracted with DCM (5 x 250 mL), washed together with water (2 x 300 mL), dried over sodium sulfate, and concentrated to obtain 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 (75 x 3 mm; 2.6 μm), flow rate 1 mL / min; gradient time 4 min; 20%~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] To two drum vials containing 192 mg (0.780 mmol) of 8-hydroxy-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyrizine-2-carbonitrile, a stirring bar and acetonitrile (3.1 mL) were added. Next, 0.272 mL (1.560 mmol) of DIEA was added to the suspension, and the mixture was stirred for 1-2 minutes until it became a homogeneous yellow solution. To this reaction mixture, phosphoryl chloride (0.131 mL, 1.404 mmol) was added. Under a nitrogen atmosphere, a bubbler tube filled with 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 volatile substances were removed under reduced pressure using a rotary evaporator. The reaction residue was dissolved in ethyl acetate, poured into a beaker containing ice (~10 mL), and then transferred to a separatory funnel. The aqueous phase was extracted with ethyl acetate. The extracted organic layers were washed sequentially with 1.5 M K2HPO4, saturated sodium bicarbonate aqueous solution, and saline solution. The organic extract was dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure to obtain 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: Elute from 2% to 98% B for 1.5 minutes, then elute with 98% B for 0.5 minutes; 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 ¹H 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] 6-Cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-4-yltrifluoromethanesulfonic acid (65 g, 195 mmol) and tert-butyl(2R,5S)-2,5-dimethylpiperazine-1-carboxylate (43.9 g, 205 mmol) / acetonitrile (1.3 L) were mixed with DIPEA (0.102 L, 585 mmol). The solution was stirred at 80°C for 6 hours, the solvent was removed, and the resulting crude residue was chromatographed on silica gel (product Rf: 0.4; 100% ethyl acetate). The product obtained was tert-butyl(2R,5S)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-4-yl)-2,5-dimethylpiperazine-1-carboxylate (75 g, 189 mmol, 97% yield). 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%~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-naphthyridine-4-yl)-2,5-dimethylpiperazine-1-carboxylate (30 g, 75 mmol) / ethyl acetate (1000 mL), HCl (4 M dioxane solution, 189 mL, 755 mmol) was added at 0°C and stirred for 6 hours, allowing it to return to room temperature. LC / MS analysis showed that at 0.60 RT, approximately 90% of the product mass was present, and at 0.44 RT, approximately 4% of the amide by-product mass (consistent with the nitrile hydrolysate) was present. This reaction mixture was diluted with methyl t-butyl ether (MTBE, 2000 mL), stirred for 15 minutes, 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 approximately 8 using a 10% sodium bicarbonate aqueous solution. The organic layers were extracted with DCM (5 x 250 mL), washed together with water (2 x 300 mL), dried over sodium sulfate, and concentrated to obtain 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 (75x3mm; 2.6μm), flow rate 1mL / min; gradient time 4 min; 20%~100% B; detection wavelength: 254nm (Solvent A: 98% water: 2% acetonitrile (10mM ammonium formate); Solvent B: 2% water: 98% acetonitrile (10mM 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); 13¹³C 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] Method for synthesizing 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-naphthyridine-4-yltrifluoromethanesulfonic acid (80 mg, 0.194 mmol) / acetonitrile (5 mL), 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) were added. This mixture was stirred overnight at 85°C. 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 resulting crude residue was purified by silica gel column chromatography (24 g flash column, elution solvent: 50-80% ethyl acetate / petroleum ether). The fraction was concentrated under reduced pressure to obtain 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 method of DGKi compound 2 1-(bis(4-fluorophenyl)methyl)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-4-yl)piperazine-2-carboxylate methyl [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) / DMA (1 mL) and t-butanol (4 mL), 1-(bis(4-fluorophenyl)methyl)piperazine-2-carboxylate methyl TFA salt (40 mg, 0.087 mmol) and cesium carbonate (85 mg, 0.261 mmol) were added under a nitrogen atmosphere, followed by 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 an oil bath at 70°C, and the oil bath temperature was 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 obtain a brown viscous substance. The crude substance was purified by preparative HPLC (conditions: column: Sunfire C18, 19x150mm, particle size: 5μm; mobile phase A: 10mM ammonium acetate (adjusted to pH 4.5 with acetic acid); mobile phase B: acetonitrile; gradient: elution over 15 minutes from 30-100% B, then elution over 5 minutes at 100% B; flow rate: 17mL / min). The fractions containing the product were combined and dried in a centrifugal evaporator to obtain 1-(bis(4-fluorophenyl)methyl)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-4-yl)piperazine-2-carboxylate methyl (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 (50 x 2.1 mm, 2.5 μm; flow rate 1.1 mL / min; gradient time 3 min; temperature: 50°C; solvent: 0% B ~ 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 method 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-naphthyridine-4-yltrifluoromethanesulfonic acid (100 mg, 0.243 mmol) / acetonitrile (8 mL), DIPEA (0.127 mL, 0.728 mmol) and the HCl salt of (R)-1-(bis(4-fluorophenyl)methyl)-2-methylpiperazine (82 mg, 0.243 mmol) were added. The reaction system 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 obtain a brown viscous substance. The obtained crude compound was purified using ISCO (trademark registered) (12g silica gel column; 60-67% ethyl acetate / petroleum ether) to obtain (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 substance. LCMS: m / z = 566.0 (M + 2H); rt 2.23 min; LCMS method: Column: AQUITY UPLC BEH C18 (3.0 x 50 mM) 1.7 μm; Mobile phase A: Buffer: Acetonitrile (95:5); Mobile phase B: Buffer: Acetonitrile (5:95), Buffer: 10 mM ammonium acetate; Gradient: Elute over 2.0 min at 20-100% B, then elute over 0.2 min at 100% B; Flow rate 0.7 mL / min

[0196] Synthesis method 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) / NMP (5 mL), zinc (2.085 mg, 0.032 mmol) and zinc cyanide (37.4 mg, 0.319 mmol) were added under a nitrogen atmosphere. The nitrogen was purged for 3 minutes, and dppf (5.30 mg, 9.57 μmol) and Pd2(dba)3 (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 obtain a brown viscous substance. 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 aqueous solution; Mobile phase B: Acetonitrile; Gradient: Elute with 40-60% B for 3.0 minutes (flow rate 17 mL / min), then elute with 60-100% B for 17 minutes (flow rate 17 mL / min)). The fractions containing the product were combined and concentrated under high vacuum. The sample was then diluted (EtOH / H2O, 1:3) and freeze-dried overnight to obtain (R)-8-(4-(bis(4-fluorophenyl)methyl)-3-methylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthiridine-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 (75 x 3 mm; 2.6 μm); Mobile phase A: 10 mM ammonium formate aqueous solution:acetonitrile (98:2); Mobile phase B: 10 mM ammonium formate aqueous solution:acetonitrile (2:98); Gradient: Elute over 4 minutes with 20-100% B (flow rate 1.0 mL / min), then elute over 0.6 minutes with 100% B (flow rate 1.5 mL / min), and finally elute over 0.1 minutes with a gradient of 100-20% B (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 method of DGKi compound 5 8-[(2S,5R)-4-[(4-fluorophenyl)(phenyl)methyl]-2,5-dimethylpiperazine-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] In a two-drum reaction vessel, a mixture of 8-((2S,5R)-2,5-dimethylpiperazin-1-yl)-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitride·TFA (41.1 mg, 100 μmol), (4-fluorophenyl)(phenyl)methanol (28.3 mg, 140 μmol), and (cyanomethyl)trimethylphosphonium iodide (48.6 mg, 200 μmol) / acetonitrile (200 μl) was added and sealed. Hünig base (75 μL, 429 μmol) was added and the mixture was heated at 110°C for 2 hours. This reaction mixture was directly injected into a 12 g silica gel column and eluted with 20-100% ethyl acetate / hexane to obtain Example 182 as a diastereomer 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, 200mm x 19mm, 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: elute at 47%B for 0 minutes, elute at 47-87%B for 20 minutes, then elute at 100%B for 4 minutes; flow rate: 20mL / min; column temperature: 25℃). The fractions were collected by determining the MS signal. The fractions containing the product were combined and dried in a centrifugal evaporator to obtain 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] Synthesis method for DGKi compounds 6 and 7 8-[(2S,5R)-4-[(4-fluorophenyl)(phenyl)methyl]-2,5-dimethylpiperazine-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] The diastereomers of Example 5 were separated using chiral solid-phase chromatography (column: Chiralpak OJ-H, 21x250mm; 5μ, mobile phase: 90%CO2 / 10% methanol, flow rate: 45mL / min, detection wavelength: 225nm, injection details: 500μL (15mg dissolved in 1mL methanol / acetonitrile)). The first eluted diastereomer of Example 6 (66.4 mg) was isolated in a yield of 20.2%. Analytical LC / MS was used to determine the final purity. Injection 1 conditions: Column: Waters XBridge C18, 2.1mm x 50mm, particle size: 1.7μm; Mobile phase A: 5:95 acetonitrile:water (containing 10mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10mM ammonium acetate); Temperature: 50℃; Gradient: Elute over 3 minutes from 0%B to 100%B, then elute over 0.50 minutes at 100%B; Flow rate: 1mL / min; Detection: MS and UV (220nm); Injection 1 results: Purity: 100.0%; Observed mass: 482.1; Retention time: 2.49 min; Injection 2 conditions: Column: Waters XBridge C18, 2.1mm x 50mm, particle size: 1.7μm; 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: Elute over 3 minutes from 0%B to 100%B, then elute over 0.50 minutes at 100%B; 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 min The second eluted diastereomer (Example 7, 71.7 mg) was isolated in a yield of 21.9%. 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: Elute from 0% B to 100% B over 3 minutes, then elute at 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 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 0.1% trifluoroacetic acid); Mobile phase B: 95:5 Acetonitrile:Water (containing 0.1% trifluoroacetic acid); Temperature: 50°C; Gradient: Elute over 3 minutes from 0%B to 100%B, then elute over 0.50 minutes at 100%B; 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 min

[0199] Synthesis method of DGKi compound 8 4-[(2S,5R)-4-[(4-chlorophenyl)(4-fluorophenyl)methyl]-2,5-dimethylpiperazine-1-yl]-6-methoxy-1-methyl-1,2-dihydro-1,5-naphthyridine-2-one [ka] 4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-6-methoxy-1-methyl-1,5-naphthyridine-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) / acetonitrile (3 mL), and the reaction mixture was heated overnight at 55°C. LC / MS confirmed that 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: elution at 42% B for 0 minutes, then elution at 42-82% B for 25 minutes, followed by elution at 100% B for 5 minutes; flow rate: 20 mL / min; column temperature: 25°C). The fractions were collected based on MS signal analysis, and the fractions containing the product 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 method of DGKi compound 9 8-[(2S,5R)-4-{[2-(difluoromethyl)-4-fluorophenyl]methyl}-2,5-dimethylpiperazine-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), 2-(difluoromethyl)-4-fluorobenzaldehyde (16.86 mg, 0.097 mmol) was added and the mixture was stirred at room temperature for 1 hour. Sodium cyanoborohydride (15.22 mg, 0.242 mmol) was added, and the mixture was stirred overnight at room temperature. LC / MS analysis showed that the reaction was complete. The crude material was purified by preparative LC / MS (conditions: column: XBridge C18, 200mm x 19mm, 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: elution at 31%B for 0 minutes, then elution at 31-71%B for 25 minutes, followed by elution at 100%B for 5 minutes; flow rate: 20mL / min; column temperature: 25°C). The fractions were collected by MS and UV signal analysis. The fractions containing the product 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, 2.1mm x 50mm, particle size: 1.7μm; 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℃; Gradient: Elute over 3 minutes from 0%B to 100%B, then elute over 0.50 minutes at 100%B; Flow rate: 1mL / min; Detection: MS and UV (220nm); Injection 1 results: Purity: 100.0%; Observed mass: 456.08; Retention time: 1.39 min; Injection 2 conditions: Column: Waters XBridge C18, 2.1mm x 50mm, particle size: 1.7μm; Mobile phase A: 5:95 Acetonitrile:Water (containing 10mM ammonium acetate); Mobile phase B: 95:5 Acetonitrile:Water (containing 10mM ammonium acetate); Temperature: 50℃; Gradient: Elute over 3 minutes from 0%B to 100%B, then elute over 0.50 minutes at 100%B; Flow rate: 1 mL / min; Detection: MS and UV (220nm); Injection 2 results: Purity: 100.0%; Observed mass: 456.07; Retention time: 2.22 min; Elute over 3 minutes from 0%B to 100%B, then elute over 0.50 minutes at 100%B; Flow rate: 1 mL / min; Detection: MS and UV (220nm); Injection 2 results: Purity: 100.0%; Observed mass: 456.07; Retention time: 2.22 min.

[0201] Method for synthesizing DGKi compound 10 8-[(2S,5R)-4-[(4-fluorophenyl)(4-methylphenyl)methyl]-2,5-dimethylpiperazine-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] 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) / acetonitrile (0.3 mL) were mixed with Hünig base (0.105 mL, 0.600 mmol). The reaction mixture was stirred at 110°C for 2 hours, 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 Hünig base (0.058 mL, 0.300 mmol). The reaction mixture was stirred at 110°C for a further 2 hours. The resulting crude reaction mixture was directly injected into Si-RediSep Rf (12 g) and flash chromatography was performed (20-100% ethyl acetate / hexane). The fractions containing the product were combined and vacuum-dried. The obtained substance was further purified by preparative LC / MS (conditions: column: XBridge C18, 200mm x 19mm, particle size: 5μm; 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: elution at 20% B for 0 minutes, then elution at 20-60% B for 25 minutes, followed by elution at 100% B for 5 minutes; flow rate: 20 mL / min; column temperature: 25°C). The fractions were determined by MS and UV signaling and recovered. The fractions containing the product were combined and dried using a centrifugal evaporator. The yield of the diastereomer product TFA salt was 47.1 mg. The diastereomer product was split 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: 100mL / min; column temperature: 25℃). The title compound was recovered at the second elution peak (>91% DEA, theoretical molecular weight 495.602). Analytical LC / MS was used to determine the final purity. Injection 1 conditions: Column: Waters XBridge C18, 2.1mm x 50mm, particle size: 1.7μm; Mobile phase A: 5:95 acetonitrile:water (containing 10mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10mM ammonium acetate); Temperature: 50℃; Gradient: Elute over 3 minutes from 0%B to 100%B, then elute over 0.50 minutes at 100%B; Flow rate: 1mL / min; Detection: MS and UV (220nm); Injection 1 results: Purity: 97.6%; Observed mass: 496.26; Retention time: 2.52 min; Injection 2 conditions: Column: Waters XBridge C18, 2.1mm x 50mm, particle size: 1.7μm; 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: Elute over 3 minutes from 0%B to 100%B, then elute over 0.50 minutes at 100%B; 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 min

[0202] Synthesis method of DGKi compound 11 8-[(2S,5R)-4-[1-(2,6-difluorophenyl)ethyl]-2,5-dimethylpiperazine-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] 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-dicarbonitride·TFA (34.0 mg, 60% by weight, 0.05 mmol) / acetonitrile (0.3 mL) was mixed with Hünig base (0.052 mL, 0.300 mmol) and stirred at 55°C for 2 hours. LC-MS showed that all components were converted 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: elution at 37% B for 0 minutes, then elution at 37-77% B for 20 minutes, followed by elution at 100% B for 5 minutes; flow rate: 20 mL / min; column temperature: 25 °C), and the fractions were recovered by MS and UV signal analysis. The fractions containing the product were combined and dried in a centrifugal evaporator to obtain 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, 2.1mm x 50mm, particle size: 1.7μm; Mobile phase A: 5:95 acetonitrile:water (containing 10mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10mM ammonium acetate); Temperature: 50℃; Gradient: Elute from 0%B to 100%B over 3 minutes, then elute at 100%B for 0.50 minutes; Flow rate: 1mL / min; Detection: MS and UV (220nm); Injection 1 results: Purity: 100.0%; Observed mass: 438.14; Retention time: 2.36 min; Injection 2 conditions: Column: Waters XBridge C18, 2.1mm x 50mm, particle size: 1.7μm; 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: Elute over 3 minutes from 0%B to 100%B, then elute over 0.50 minutes at 100%B; 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] Synthesis methods for DGKi compounds 12-14 8-((2S,5R)-4-(1-(2,4-difluorophenyl)propyl)-2,5-dimethylpiperazine-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 (29.7 mg, 0.1 mmol) and 1-(1-bromopropyl)-2,4-difluorobenzene (25.9 mg, 0.110 mmol) / acetonitrile (0.3 mL) was mixed with Hünig base (87 μL, 0.500 mmol) and stirred on a hot plate at 55°C for 16 hours. The crude material was purified by preparative LC / MS (conditions: column: XBridge C18, 200mm x 19mm, particle size: 5μm; 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: elution with 3% B for 0 minutes, then elution with 3-43% B for 25 minutes, followed by elution with 100% B for 5 minutes; flow rate: 20 mL / min; column temperature: 25°C). The fractions were determined by MS and UV signaling and recovered. The fractions containing the product were combined and dried using a centrifugal evaporator (stereochemistry: diastereomer mixture). A mixture of synthetic diastereomers of DGKi compound 12 was further separated and divided 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: 100 mL / min; detection wavelength: 220 nm). DGKi compound 13 (isomer 1) was recovered as the first elution peak (95% de; stereochemistry: homochiral). DGKi compound 14 (isomer 2) was recovered as the second elution peak (95% de; stereochemistry: homochiral).

[0204] Synthesis method 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 spurged under 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-naphthyridine-2(1H)-one (21.38 mg, 0.037 mmol) were added to one drum vial. Spurged DMF (0.3 mL) was added, the mixture was capped under a nitrogen atmosphere, and immersed in a 50°C oil bath for 15 minutes. Zinc cyanide (2.86 mg, 0.024 mmol) was added, the mixture was capped under a nitrogen atmosphere, and immersed in a 50°C oil bath for 3 hours. LC / MS analysis showed that 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: elution at 50-90% B for 15 minutes, then elution at 100% B for 5 minutes; flow rate: 20mL / min), and the fractions containing the product were combined and dried in a centrifugal evaporator. The title compound (11.4 mg) was isolated with a yield of 59.7%. Another synthesis method: 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 the addition of Hünig base (0.990 mL, 5.67 mmol), and the mixture was stirred overnight at room temperature. LC / MS analysis indicated that the reaction was complete. The crude product was filtered and purified by preparative HPLC (acetonitrile aqueous solution (containing ammonium acetate as buffer)) to obtain 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, 2.1x50mm, particle size: 1.7μm; Mobile phase A: 5:95 acetonitrile:water (containing 10mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10mM ammonium acetate); Temperature: 50℃; Gradient: Elute from 0 to 100% B for 3 minutes, then elute at 100% B for 0.75 minutes; Flow rate: 1.0 mL / min; Detection: UV (220nm); Injection 2 conditions: Column: Waters Acquity UPLC BEH C18, 2.1x50mm, particle size: 1.7μm; 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: Elute from 0-100% B for 3 minutes, then elute at 100% B for 0.75 minutes; Flow rate: 1.0 mL / min; Detection: UV (220 nm); Injection 1 result: Purity: 100%; Observed mass: 517.0; Retention time: 2.4 minutes; Injection 2 result: Purity: 98.4%; Observed mass: 517.0; Retention time: 1.7 minutes; 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 ¹³C 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 method of DGKi compound 16 8-[(2S,5R)-4-[bis(4-methylphenyl)methyl]-2,5-dimethylpiperazine-1-yl]-5-methyl-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile [ka] 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-carbonitride·TFA (72.4 mg, 54% wt, 0.095 mmol) / acetonitrile (0.3 mL) was mixed with Hünig base (0.10 mL, 0.57 mmol) and stirred at 110°C for 2 hours. The crude material was purified by preparative LC / MS (conditions: column: XBridge C18, 200mm x 19mm, 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: elution at 55%B for 0 minutes, then elution at 55-95%B for 20 minutes, followed by elution at 100%B for 4 minutes; flow rate: 20mL / min; column temperature: 25℃), and the fractions were recovered by MS and UV signal analysis. The fractions containing the product were combined and dried in a centrifugal evaporator to obtain 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, 2.1mm x 50mm, particle size: 1.7μm; Mobile phase A: 5:95 acetonitrile:water (containing 10mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10mM ammonium acetate); Temperature: 50℃; Gradient: Elute over 3 minutes from 0%B to 100%B, then elute over 0.75 minutes at 100%B; Flow rate: 1mL / min; Detection: MS and UV (220nm); Injection 1 results: Purity: 100.0%; Observed mass: 492.21; Retention time: 2.77 min; Injection 2 conditions: Column: Waters XBridge C18, 2.1mm x 50mm, particle size: 1.7μm; 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: Elute over 3 minutes from 0%B to 100%B, then elute over 0.75 minutes at 100%B; 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 min. 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-carbonitride·TFA (0.12 g, 0.27 mmol) / acetonitrile (10 mL), 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) were added, and the mixture was heated at 85 °C for 16 hours. The reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure to obtain 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: Elute over 18 minutes from 0 to 100% B, then elute over 5 minutes at 100% B; Flow rate: 17 mL / min], the fractions were concentrated under reduced pressure, and lyophilized with EtOH / H2O (1:5) to obtain 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 (50 x 2.1) 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 (50 x 2.1) 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-carbonitride·TFA (70 mg, 0.22 mmol) / acetonitrile (2 mL), 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) were added at room temperature, and the mixture was heated at 85°C for 16 hours. 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 obtain the crude product. This was purified by preparative HPLC [HPLC method: Column: Sunfire C18 (150 mm x 19.2 mm ID, 5 μm), mobile phase A = 10 mM ammonium acetate aqueous solution, mobile phase B = acetonitrile, flow rate: 19 mL / min], the fraction was concentrated under reduced pressure, diluted with EtOH / H2O (1:5), and lyophilized to obtain 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 (50 x 2.1 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)pyridine-2-yl)methyl)-2-methylpiperazine-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-carbonitride·TFA (0.5 g, 1.17 mmol) / acetonitrile (10 mL), DIPEA (1.02 mL, 5.86 mmol) was added, followed by 2-(bromo(4-fluorophenyl)methyl)-5-(trifluoromethyl)pyridine (0.78 mg, 2.35 mmol), and the mixture was heated at 80°C for 3 hours. The reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure to obtain the crude product. This was purified by preparative HPLC (HPLC method: Column: INERTSIL ODS 21.2X250mm, 5μm; Mobile phase A: 0.1% TFA aqueous solution; Mobile phase B: Acetonitrile; Gradient: Elute with 30-80% B for 14 minutes, then elute with 100% B for 5 minutes; Flow rate: 17 mL / min), the fractions were concentrated under reduced pressure, and lyophilized with EtOH / H2O (1:5) to obtain 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 x 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 (75 x 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%) 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)(pyridine-2-yl)methyl)-5-ethyl-2-methylpiperazine-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-carbonitride (100 mg, 0.32 mmol) / acetonitrile (5 mL), DIPEA (0.3 mL, 1.60 mmol) was added, followed by 2-(bromo(4-chlorophenyl)methyl)pyridine (181 mg, 0.64 mmol), and the mixture was heated at 80°C for 3 hours. The reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure to obtain 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: elution at 90% B, then 100% B for 5 minutes; flow rate: 18 mL / min), the fraction was concentrated under reduced pressure, and lyophilized with EtOH / H2O (1:5) to obtain 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 x 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-oxadiazole-5-yl)(4-fluorophenyl)methyl)-2,5-dimethylpiperazine-1-yl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-6-carbonitrile [ka] 2-((2R,5S)-4-(6-cyano-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyrimidine-4-yl)-2,5-diethylpiperazine-1-yl)-2-(4-fluorophenyl)acetic acid (0.045 g, 0.09 mmol) and N-hydroxycyclopropanecarboximamide (9.4 mg, 0.09 mmol) / DMF (2 mL) were stirred together. BOP (0.01 g, 0.23 mmol) and triethylamine (0.04 mL, 0.23 mmol) were added 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 obtain the crude product. This was purified by preparative HPLC (chiral separation method: column: DAD-1-Cellulose-2 (250 x 4.6 mM), 5 μl; 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 (50 x 2.1) 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)pyridine-2-yl)methyl)-2,5-dimethylpiperazine-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-carbonitride (1 g, 3.35 mmol) / acetonitrile (10 mL), DIPEA (5.9 mL, 33.5 mmol), followed by 2-(bromo(4-fluorophenyl)methyl)-5-(trifluoromethyl)pyridine (2.24 g, 6.70 mmol), was added and heated at 80°C for 4 hours. The reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure to obtain the crude product. This was purified by preparative HPLC (HPLC method: Column: Sunfire C18, 150x19mm ID, 5μm; Mobile phase A: 0.1% TFA aqueous solution; Mobile phase B: Acetonitrile:MeOH (1:1); Gradient: Elute from 50-100% B for 20 minutes, then elute with 100% B for 5 minutes; Flow rate: 19 mL / min), the fractions were concentrated under reduced pressure, and lyophilized with EtOH / H2O (1:5) to obtain compounds 27 and 28. Compound 27: 110 mg, yield 6%; LCMS: m / z = 552.2 (M + H); rt 3.09 min; (LCMS method: Column: Column-Kinetex XB-C18 (75 x 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: 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, yield 8%; LCMS: m / z = 552.2 (M + H); rt 3.09 min; (LCMS method: Column: Column-Kinetex XB-C18 (75 x 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%) 1 ¹H NMR (400MHz, DMSO-d6) δ ppm 8.91 (s, 1H), 8.27-8.16 (m, 2H), 7.99 (d, J=9.0Hz, 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 in the solvent peak.]

[0212] DGKi compounds 29 and 30 4-((2S,5R)-4-(1-(4-(cyclopropylmethoxy)-2-fluorophenyl)propyl)-2,5-diethylpiperazine-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-carbonitride·HCl (200 mg, 0.55 mmol) / acetonitrile (5 mL), 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) were added, and the mixture was heated at 80 °C for 16 hours and then cooled to room temperature. 1-(1-chloropropyl)-4-(cyclopropylmethoxy)-2-fluorobenzene (268 mg, 1.102 mmol) was added again, and heating was continued for another 16 hours. The reaction mixture was cooled, the solvent was removed under reduced pressure, and the resulting residue was dissolved in ethyl acetate (10 x 20 mL). The organic layer was washed with saline solution, dried over Na2SO4, and concentrated under reduced pressure to obtain the crude product. This was purified by preparative HPLC (HPLC method: column: EXRS (20x250mm, 5μm), mobile phase A: 10mM ammonium acetate aqueous solution, mobile phase AB: acetonitrile, flow rate: 20mL / min). Fraction 1 was concentrated under reduced pressure, the product was diluted with EtOH / H2O (1:5), and lyophilized to obtain compound 29 (35 mg, 11.6% yield). LCMS: m / z, 533.4 [M+H] + rt 1.57 min; LC-MS method: Column: KINETIX XB C18 (75x3mm, 2.6μm); Mobile phase A: 10mM ammonium acetate aqueous solution (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-carbonitride·HCl (0.4 g, 1.1 mmol) / acetonitrile (10 mL), 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) were added, and the mixture was heated at 85°C for 16 hours. The reaction mixture was filtered through Celite, washed with ethyl acetate, and the filtrate was concentrated under reduced pressure to obtain the crude compound. This was purified by preparative HPLC [HPLC method: Column: YMC ExRS (250 mm x 21.2 mm, 5 μm); Mobile phase A = 10 mM ammonium acetate aqueous solution (pH 4.5); Mobile phase B = acetonitrile; Gradient: Elute with 80% B for 2 minutes, then elute with 100% B for 16 minutes; Flow rate: 19 mL / min] to obtain 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 x 2.1 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; 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]pyrimidine-2(1H)-one (0.1 g, 0.19 mmol) / DMF (2 mL), 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) was added at room temperature under an argon atmosphere. The mixture was heated overnight at 90 °C, diluted with ethyl acetate (50 mL), filtered through Celite (trademark registered), and further washed with ethyl acetate (2 x 50 mL). The filtrate was washed with water (50 mL) and saline solution, dried over Na2SO4, and concentrated under reduced pressure to obtain the crude product. This was purified by preparative HPLC (HPLC method: column: YMC EXRS (250 x 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 obtain 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 (50 x 2.1 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℃); 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 (50 x 2.1 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 assay) The pharmacological properties of the compounds described herein can be confirmed by numerous biological assays.

[0216] 1. In vitro DGK inhibition assay The reactions for DGKα and DGKζ were carried out using either extruded liposomes (LIPGLO assay for DGKα and DGKζ) or a detergent / lipid micelle substrate (DGKα and DGKζ assay). These 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)). The detergent / lipid micelle substrate reaction also included octyl β-D-glucopyranoside (50 mM). The lipid substrate concentrations in the detergent / lipid micelle reaction solution were 11 mM PS and 1 mM DAG. In the extruded liposome reaction solution, the lipid substrate concentrations were 2 mM PS, 0.25 mM DAG, and 2.75 mM PC. These reactions were carried out in ATP (150 μm). The enzyme concentrations of DGKα and DGKζ were 5 nM. The compound inhibition experiment was performed as follows: 50 nL droplets of each test compound dissolved in DMSO (11 points of each compound, obtained by serially diluting 3-fold from the highest concentration of 10 mM) were transferred to the wells of a white 1536-well plate (Corning 3725). A 2-fold dilution of the final reaction concentration enzyme / substrate solution (5 mL) was prepared by mixing 2.5 mL of a 4-fold dilution of enzyme solution (DGKα or DGKζ (20 nM) / assay buffer (preparation method described below)) and either a 4-fold dilution of liposome solution or a 4-fold dilution of detergent / lipid micelle solution (composition described below), and incubated at room temperature for 10 minutes. Next, a 2-fold dilution of enzyme / substrate solution (1 μL) was added to the well containing the test compound, and ATP (1 μL, 300 μM) was added to initiate the reaction. This reaction was continued for 1 hour, after which 2 μL of Glo reagent (Promega V9101) was added and incubated for 40 minutes. Next, kinase detection reagent (4 μL) was added and incubated for 30 minutes. Luminescence was measured using a microplate reader (EnVision). The inhibition rate was calculated from the ATP conversion rate obtained, with inhibition from the control reaction without the enzyme set to 100% and inhibition from the reaction with only the vehicle set to 0%. The test compound was evaluated at 11 concentrations, and IC50 was calculated. 50 We made that decision.

[0217] Preparation of 4x diluted detergent / lipid micelles A detergent / lipid micelle was mixed with phosphatidylserine (15 g, Avanti 840035P) and diacylglycerol (1 g, 800811O) in a round-bottom flask (2 L), and dissolved in chloroform (150 mL) to prepare the solution. The chloroform was removed under high vacuum using a rotary evaporator. The resulting colorless, viscous oily substance was vigorously mixed and resuspended in 400 mL of MOPS (50 mM, pH 7.5), NaCl (100 mM), NaF (20 mM), MgCl2 (10 mM), CaCl2 (1 μm), DTT (1 mM), and octyl glucoside (200 mM) solution. This lipid / detergent solution was divided into 5 mL portions and stored at -80°C.

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

[0219] Baculovirus expression in human DGKα and DGKζ Baculovirus samples of human DGK-α-TVMV-His-pFBgate and human DGK-ζ-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 DGKα and DGKζ expression were SEQ ID NO1 and NO3, respectively. Baculovirus amplification was performed using infected f9 cells with a virus / cell ratio of 1:1500, and the cells were grown at 27°C for 65 hours after gene transfer. The expression of each protein was scaled up in a Cellbag 50L WAVE bioreactor system 20 / 50 (GE Healthcare Bioscience). 2x10 cells were seeded in ESF921 insect cell medium (Expression System). 6 Sf9 cells at a concentration of cells / mL (12L, Expression System, Davis, CA) were infected with a virus stock solution containing virus at a ratio of 1:200, and the cells were grown at 27°C for 66-68 hours after infection. The infected cell culture medium was obtained by centrifugation using a SORVALL® RC12BP centrifuge (2000 rpm, 20 minutes at 4°C). The pelletized cells were stored at -70°C until purified.

[0220] Purification of human DGKα and DGKζ Full-length human DGKα and DGKζ, each containing a TVMV-cleavable C-terminal Hex-His tag sequence (SEQ ID NO2 and NO4, respectively), were expressed and prepared using the method described above. These were then purified from Sf9 baculovirus-infected insect cell paste. The cells were lysed using a nitrogen disruptor (Parr Instruments) with nitrogen cavitation, and the lysate was clarified by centrifugation. The clarified lysate was purified to ~90% homogeneity using a 3-step column chromatography procedure on an AKTA Purifier Plus system. The 3-step column chromatography used 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 depending on 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 using pre-activated CD4 T cells and Raji cells in a volume of 4 μL. Prior to the assay, CD4 T cells were pre-activated 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 Staphylococcus 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 pre-activated CD4 T cells (2 μL, final density: 6000 cells / well) and SEB-treated Raji cells (2 μL, 2000 cells / well). After incubation for 24 hours in a 37°C / 5% CO2 incubator, IL-2 detection reagent (4 μL) was added to the assay plate (Cisbio, #64IL2PEC). This assay plate was measured with an Envision reader. To evaluate the cytotoxicity of the compound, 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 measured with an Envision reader. 50% effective concentration (IC) 50 The ) was calculated using the four-variable logistic equation: y = A + ((BA) / (1 + ((C / x)^D))). Here, A and B represent the minimum and maximum % activation or inhibition rates, respectively, and C is 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 at 37°C for 2 hours, after which the cell count was measured. A 384-well tissue culture plate was coated overnight at 4°C with anti-human CD3 (20 μL, 0.1 μg / mL in plain RPMI), removed from the plate, and CD8 T cells (20k / 40 μL) were added to each well along with soluble anti-human CD28 (0.5 μg / mL). The test compound was dispensed into the cell plate immediately after cell fixation. After incubation at 37°C for 72 hours, CellTiter-Glo reagent (10 μL, Promega catalog number G7570) was added to each well. The plate was vigorously shaken for 5 minutes and incubated at room temperature for a further 15 minutes, after which CD8 T cell proliferation was measured using Envision. In the analysis, CD8 T cell signaling stimulated with anti-CD3 (0.1 μg / mL) and anti-CD28 (0.5 μg / mL) was in the background. 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, 3 μM) was used to define the range of 100% inhibition, and EC 50 This was used to normalize 50% of the data.

[0223] 4. DGK AP1 Reporter Assay The Jurkat AP1 luciferase reporter was used with the Cignal Lenti AP1 reporter (luc) kit (SABiosciences (CLS-011L)). The test compound was transferred from an 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 destination plate. Cells were transferred to a clean conical tube (50 mL) to prepare cell suspensions (40 mL, 20 mL diluted twice). 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 1.35 x 10⁶ 6 The cell suspension was adjusted to a concentration of cells / mL. This cell suspension was manually added to a 384-well TC plate containing the test compound, 30 μL / well, using a multichannel pipette, and then 4.0 x 10 4 Cells were placed in each well. This cell plate was incubated at 37°C under 5% CO2 for 20 minutes. During incubation, αCD3 (3 μL, 1.3 mg / mL) was mixed with culture 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 culture medium (0.5 mL) [final concentration = 4 μg / mL]. After 20 minutes, culture medium (10 μL) was added to all wells in the first vertical column (wells in columns A-M horizontally), and αCD3 (10 μL, 4 μg / mL) / well was added to columns N-P horizontally in the first vertical column for reference. Then, αCD3 (10 μL, 0.4 μg / mL) / well was added using a multichannel pipette. αCD3-stimulated + / - compound treated cells were incubated at 37°C and 5% CO2 for 6 hours. During incubation, the Steady-Glo (Promega E2520) reagent was slowly thawed to ambient temperature. Next, the Steady-Glo reagent was added using a Multidrop Combi dispenser (20 μL / well). Bubbles were removed by centrifugation (2000 rpm, ambient temperature, 10 seconds). These cells were incubated at room temperature for 5 minutes. The luminescence (RLU) was measured according to the luminescence protocol using an Envision plate reader instrument, and the samples were analyzed. The data were analyzed after 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-naphthiridine-2-carbonitrile).

[0224] 5. Mouse cytotoxic T lymphocyte assay Antigen-specific cytotoxic T cell (CTL) assays were developed to functionally evaluate the DGKα and DGKζ inhibitory activity and enhance effector T cell-mediated tumor cell killing activity. CD8 isolated from OT-1 transgenic mice. + T cells recognize MC38, an antigen-expressing cell that exhibits the ovalbumin-derived peptide SIINFEKL. By recognizing the same antigen, OT-1 antigen-specific CD8 + T cell cytotoxic activity is initiated. Functional CTL cells were prepared as follows: OT-1 splenocytes from 8-12 week old mice were isolated and grown in the presence of SIINFEKL peptide (1 μg / mL) and mIL2 (10 U / mL). After 3 days, fresh medium containing mIL2 (U / mL) was added. On day 5 of growth, 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) at 37°C for 3 hours. These cells were washed three times with fresh medium to remove excess peptide. Finally, in a 96-well plate (U-bottom), CTL cells pre-treated with a DGK inhibitor for 1 hour were mixed with antigen-loaded MC38 tumor cells (ratio 1:10). These cells were then centrifuged at 700 rpm for 5 minutes and incubated overnight at 37°C. After 24 hours, the supernatant was collected to analyze IFN-γ cytokine levels using AlphaLisa (purchased from Perkin Elmer).

[0225] 6. PHA proliferation assay Frozen stock solution 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). The test compound was transferred to each well of the 384-well plate, and the treated blast cells were incubated for 72 hours at 37°C and 5% CO2 in tissue medium containing human IL-2 (20 ng / mL). Growth rates were 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. Inhibition rates were calculated by comparing the values ​​between IL-2 stimulation (0% inhibition) and unstimulated control (100% inhibition). The determined inhibitory concentration (IC) was calculated. 50 The results were calculated based on 50% inhibition by doubling dilution between IL2 stimulation and no stimulation.

[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, after which the cell count was performed. A 384-well tissue culture plate was coated with anti-human CD3 / PBS (0.05 μg / mL, 20 μL) overnight at 4°C. After removing the plate, 40,000 cells / CD8 T cell (40 μL) and soluble anti-human CD28 (0.1 μg / mL) were added to each well. After cell fixation, the test compounds were immediately transferred to the cell plates using an Echo liquid handler. After incubation at 37°C for 20 hours, the supernatant (3 μL / well) was transferred to a new 384-well white assay plate, and cytokine levels were measured. Interferon-γ (IFN-γ) was quantified using the AlphaLISA kit (Cat#AL217) as described in the manufacturer's (Perkin Elmer) instructions. The values ​​obtained from each well were converted to IFN-γ concentration (pg / mL). EC of the test compound 50 The values ​​were determined using anti-CD3 (0.05 μg / mL) and anti-CD28 (0.1 μg / mL) as baseline, and with Example 40 (3 μM) and the co-stimulation of 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 the number of cells was counted. + T cells were added to a 384-well tissue culture plate in AIM-V medium at a concentration of 20,000 cells / well. One compound was added to each well, followed by the addition of bead-immobilized anti-human CD3 and anti-CD28 mAb at a final concentration of 0.3 μg / mL. The 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). The lysate (5 μL / well) was transferred to a new 384-well white assay plate, and pERK activity was measured. EC of compounds 50The activity levels were determined by setting anti-CD3 and anti-CD28 as baseline, and co-stimulation with anti-CD3 and anti-CD28 using 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) and anti-CD3 and anti-CD28 as 100% activity.

[0228] 9. Human whole blood IFN-γ assay Human venous whole blood (22.5 μL / well) obtained from healthy donors was pre-treated with the test compound in a humidified incubator of 95% air / 5% CO2 at 37°C for 1 hour. The blood was stimulated with anti-human CD3 (2.5 μL) and anti-CD2 8 mAb (final concentration 1 μg / mL) at 37°C for 24 hours each. IFN-γ in the supernatant was measured using the AlphaLISA kit (Cat#AL217). EC of compounds 50 The activity levels were determined by setting anti-CD3 and anti-CD28 as baseline, and co-stimulation with anti-CD3 and anti-CD28 using 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) and anti-CD3 and anti-CD28 as 100% activity.

[0229] [Table 2] [Table 3]

[0230] Table A shows the in vitro DGK inhibitory IC257 assays, measured by DGKα and DGKζ liposome (LIPGLO) assays. 50 The activity levels are listed. The compounds described herein have inhibitory activity on one or both of the DGKα and DGKζ enzymes, and therefore can be used to treat diseases associated with inhibition of DGKα and DGKζ activity. [Sequence Listing 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 (Sequence ID: 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 (Sequence ID: 2)

[0233] Nucleotide sequence of hDGKζ-(M1-A928)-transcription 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 TCTGTAAGCT 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)-transcription 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 (Sequence ID: 4)

Claims

1. A pharmaceutical composition for treating cancer in a subject, comprising a DGKα and / or DGKζ inhibitor, a PD1 / PD-L1-binding antagonist, and a CTLA4 antagonist, wherein the PD1 / PD-L1-binding antagonist is an antibody that binds to human PD1 or PD-L1, the CTLA4 antagonist is an antibody that binds to human CTLA4, and the DGKα and / or DGKζ inhibitor has the following structure: 【Chemistry 1】 【Chemistry 2】 A pharmaceutical composition which is a compound or a salt thereof having [a certain property].

2. A pharmaceutical composition for treating cancer in a subject, comprising a DGKα and / or DGKζ inhibitor, a PD1 / PD-L1-binding antagonist, and a CTLA4 antagonist, wherein the PD1 / PD-L1-binding antagonist is an antibody that binds to human PD1 or PD-L1, the CTLA4 antagonist is an antibody that binds to human CTLA4, and the DGKα and / or DGKζ inhibitor is 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, 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, 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, or 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, A pharmaceutical composition selected from the pharmaceutically acceptable salts thereof.

3. The pharmaceutical composition according to claim 1 or 2, wherein the antibody that binds to human PD1 is nivolumab or pembrolizumab.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody that binds to human PD-L1 is atezolizumab.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the antibody that binds to human CTLA4 is ipilimumab.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the cancer is a solid tumor or a hematological (liquid) tumor.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the cancer is selected from the group consisting of 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).

8. A pharmaceutical composition according to any one of claims 1 to 7, characterized in that it is used in combination with one or more other cancer treatments.

9. The pharmaceutical composition according to claim 8, wherein one or more other cancer treatments include radiation therapy, surgery, chemotherapy, or administration of a biopharmaceutical.

10. The pharmaceutical composition according to claim 8, wherein one or more other cancer treatments are the administration of a biopharmaceutical, the biopharmaceutical being an agent that stimulates the immune system.

11. The pharmaceutical composition according to any one of claims 1 to 7, characterized in that the subject is not being treated with other cancer treatments while being treated with a DGKα and / or DGKζ inhibitor, a PD1 / PD-L1-binding antagonist, and / or a CTLA4 antagonist.

12. The pharmaceutical composition according to any one of claims 1 to 11, characterized in that the subject has not been treated with a PD1 / PD-L1 antagonist or a CTLA4 antagonist prior to administration of a DGKα and / or DGKζ inhibitor, a PD1 / PD-L1 antagonist, and / or a CTLA4 antagonist.

13. A pharmaceutical composition according to any one of claims 1 to 11, wherein the subject is resistant to or refractory to treatment with checkpoint inhibitor antagonists (e.g., PD1 / PD-L1 binding antagonists and / or CTLA4 antagonists).

14. The pharmaceutical composition according to any one of claims 1 to 2 and 6 to 13, wherein the PD1 / PD-L1 binding antagonist is nivolumab or a variant thereof, and the CTLA4 antagonist is ipilimumab or a variant thereof.

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