Pyrimidopyrimidone compound and pharmaceutical composition containing the same

Abstract

The present invention relates to a pyrimidopyrimidone compound that inhibits PD-L1 expression and a pharmaceutical composition comprising the compound as an active ingredient. The pyrimidopyrimidone compound of the present invention can be effectively used to treat or prevent cancer.

Description

Technical Field

[0001] The present invention relates to a pyrimidopyrimidone compound and a pharmaceutical composition comprising the same, and more particularly to a pyrimidopyrimidone compound having the effect of inhibiting PD-L1 expression and a pharmaceutical composition comprising the same as an active ingredient. Background Art

[0002] PD-1 (Programmed Death-1, CD279) is a receptor protein on T cells. It is known that PD-1 inhibits the activation signal from the T cell receptor when it binds to its ligand PD-L1 (Programmed Death-Ligand 1, CD274) or PD-L2 (Programmed Death-Ligand 2, CD273). That is, when the PD-1 receptor protein of the T cell binds to the ligand PD-L1 / 2, the functional activity of the T cell (including T cell proliferation, cytokine secretion and cytolytic activity) is reduced, thereby protecting normal cells. The interaction between PD-1 and PD-L1 / 2 downregulates the immune response in infection, tumor or autoimmune diseases (Keir ME, Butte MJ, Freeman GJ et al., PD-1and its ligands in tolerance and immunity. Annu. Rev. Immunol. 2008; 26).

[0003] Blocking the PD-1 / PD-L1 interaction using antibodies against PD-1 and PD-L1 has been shown to restore and enhance T cell activation in many systems. Combination PD-1 / PD-L1 inhibition using monoclonal antibodies is known to be effective in patients with cancers such as melanoma, non-small cell lung cancer, and renal cancer.

[0004] In a study on regulating PD-L1 expression and immune anticancer response in cancer cells by targeting αvβ3-integrin, it was found that transplantation of tumor cells lacking β3-integrin significantly reduced the growth of primary tumors, a result attributed to the reduced expression of PD-L1. Therefore, inhibition of β3-integrin can improve the efficacy of immune anticancer drugs against melanoma (Proc Natl Acad Sci USA., October 1, 2019, 116(40), 20141-20150). It is known that retinoblastoma protein RB, as a tumor suppressor, shows anticancer efficacy by inhibiting PD-L1 expression through inhibiting NF-kB activity. In addition, in a melanoma model, curcumin and apigenin showed melanoma cell growth inhibitory effects, and in particular, apigenin was found to significantly inhibit PD-L1 expression induced by IFN-γ. In addition, it is known that PD-L1 expression induced by IFN-γ was reduced by 40% to 80% in the lung cancer cell line A549 pretreated with EGCG and green tea extract (GTE). At the same time, in a kinase inhibitor library screening study that can enhance the immune anticancer effect against melanoma, regorafenib was identified as the most potent drug among approximately 20 drugs that reduced cell surface PD-L1 expression levels by more than 50%. When used in combination with IFN-γ or immune checkpoint blockers (ICB) in cells and in vivo, this drug can strongly promote anti-tumor efficacy, strongly inhibit JAK1 / 2-STAT1 and MAPK signaling, and then reduce IFN-γ-induced PD-L1 and IDO1 expression (Clin Cancer Res., July 15, 2019, 25(14), 4530-4541).

[0005] As protein kinase inhibitors, 3,4-dihydropyrimido[4,5-d]pyrimidin-2-one derivatives are known as inhibitors of Abl, BCR-Abl, Bmx, c-Raf, Csk, Fes, FGFR, Flt3, Ikk, IR, JNK, Lck, Mkk, PKC, PKD, Rsk, SAPK, Syk, Trk, RTK, Src, EGFR, IGF, Mek, Ros, and Tie2 kinase activity, but their effects on PD-L1 are not mentioned. 3-Phenyl-dihydropyrimido[4,5-d]pyrimidinone derivatives are also known to exhibit inhibitory activity against Src kinase at 1 to 5 nM (IC50), but their effects on PD-L1 are also not mentioned (International Patent Publication No. WO 2005 / 011597).

[0006] Therefore, it is necessary to develop compounds with PD-L1 expression inhibitory activity to obtain anti-tumor efficacy. Summary of the Invention

[0007]

Technical Issues

[0008] The object of the present invention is to provide a compound of formula (I) or a pharmaceutically acceptable salt thereof that has the effect of inhibiting PD-L1 expression.

[0009] Another object of the present invention is to provide a pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0010]

Technical solution

[0011] One aspect of the present invention relates to a pyrimidopyrimidone compound of the following formula (I) or a pharmaceutically acceptable salt thereof.

[0012]

[0013] in,

[0014] R1 is C5-C 15 Aryl, C5-C 15 Heteroaryl, C3-C 10 Cycloalkyl or C3-C 10 heterocycloalkyl;

[0015] R2 is hydrogen, C1-C4 alkyl or C1-C4 alkoxy;

[0016] R3 is hydrogen, halogen, C1-C4 alkyl or C1-C4 alkoxy; and

[0017] R4 is unsubstituted C5-C 15 Aryl or C5-C 15 Heteroaryl or C1-C4 alkyl, C1-C4 haloalkyl or C5-C 15 Heteroaryl-substituted C5-C 15 Aryl or C5-C 15 Heteroaryl.

[0018] As used herein, the term "C5-C 15 The term "aryl" includes both aromatic groups and partially reduced derivatives thereof. Aromatic groups are monocyclic or condensed ring types consisting of 5 to 15 carbon atoms. Representative examples of aryl include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, and the like.

[0019] As used herein, the term "C5-C 15 "Heteroaryl" includes both heteroaromatic groups and partially reduced derivatives thereof. Heteroaromatic groups are monocyclic or fused ring types consisting of 5 to 15 carbon atoms and contain one or more oxygen, sulfur or nitrogen. Representative examples of heteroaryl groups are not limited to pyridyl, furyl, thienyl, indolyl, quinolinyl, imidazolinyl, Azolyl, dihydro Azolyl, thiazolyl, etc.

[0020] As used herein, the term "C3-C 10 "Cycloalkyl" refers to a simple or fused cyclic hydrocarbon having 3 to 10 carbon atoms, examples of which include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0021] As used herein, the term "C3-C 10 The term "heterocycloalkyl" refers to a functional group in which one or more ring carbon atoms of a simple or condensed cyclic hydrocarbon having 3 to 10 carbon atoms is substituted by oxygen, sulfur or nitrogen, and examples include, but are not limited to, thiazolidinyl, oxirane and the like.

[0022] As used herein, the term "C1-C4 alkyl" refers to a straight or branched monovalent hydrocarbon having 1 to 4 carbon atoms, examples of which include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and the like.

[0023] As used herein, the term "C1-C4 alkoxy" refers to a straight or branched chain alkoxy group having 1 to 4 carbon atoms, and examples include, but are not limited to, methoxy, ethoxy, n-propoxy, and the like.

[0024] As used herein, the term "C1-C4 haloalkyl" refers to a straight or branched hydrocarbon having 1 to 4 carbon atoms substituted by one or more halogens selected from fluorine, chlorine, bromine and iodine, examples of which include but are not limited to trifluoromethyl, trichloromethyl, trifluoroethyl and the like.

[0025] In one embodiment of the present invention, the compound has formula (I)

[0026] in,

[0027] R1 is unsubstituted phenyl or phenyl substituted by halogen or hydroxy;

[0028] R2 is a C1-C4 alkyl group;

[0029] R3 is hydrogen, halogen, C1-C4 alkyl or C1-C4 alkoxy; and

[0030] R4 is unsubstituted phenyl, C1-C4 alkyl, C1-C4 haloalkyl or alkyldihydrogen phenyl substituted with oxazolyl, or alkylthiazolyl.

[0031] As used herein, the term "alkyldihydro "Azolyl" refers to a dihydrogen group substituted by one or more C1-C4 alkyl groups. Azole groups, examples of which are not limited to dimethyldihydro Azolyl, etc.

[0032] As used herein, the term "alkylthiazolyl" refers to a thiazolyl group substituted with one or more C1-C4 alkyl groups, and examples include, but are not limited to, tert-butylthiazolyl and the like.

[0033] In one embodiment of the present invention, the compound has formula (I)

[0034] in,

[0035] R1 is phenyl, fluorophenyl or hydroxyphenyl;

[0036] R2 is methyl;

[0037] R3 is hydrogen, chloro, bromo, methyl or methoxy; and

[0038] R4 is trifluoromethylphenyl, tert-butylphenyl, dimethyldihydro oxazolylphenyl or tert-butylthiazolyl.

[0039] The pharmaceutically acceptable salts of the present invention may include both non-toxic inorganic and organic acid salts, examples of which include hydrochloride, phosphate, sulfate, nitrate, tartrate, methanesulfonate, p-toluenesulfonate, acetate, trifluoroacetate, citrate, maleate, succinate, oxalate, benzoate, fumarate, mandelate, propionate, lactate, glycolate, gluconate, galacturonate, glutamate, glutarate, glucuronate, aspartate, ascorbate, carbonate, vanillate, hydroiodide, malate, malonate, and the like.

[0040] Representative compounds according to the present invention are selected from the group consisting of the following.

[0041] N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-1);

[0042] 3-(tert-Butyl)-N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-2);

[0043] N-(6-methoxy-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-3);

[0044] 4-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-4);

[0045] 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-5);

[0046] 3-(tert-Butyl)-N-(6-methoxy-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-6);

[0047] 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methoxy-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-7);

[0048] N-(6-chloro-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-8);

[0049] 3-(tert-Butyl)-N-(6-chloro-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-9);

[0050] 2-(tert-butyl)-N-(6-chloro-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)thiazole-4-carboxamide (I-10); and

[0051] N-(6-chloro-5-(7-((4-hydroxyphenyl)amino)-1-methyl-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-11).

[0052] A method for preparing a compound of formula (I) according to the present invention is depicted in the following reaction scheme 1. However, the reaction schemes exemplified in the following reaction schemes represent only typical methods used in the present invention. The order of operations, reagents, reaction conditions, etc. can be varied without limitation.

[0053] [Reaction Scheme 1]

[0054]

[0055] In Reaction Scheme 1, R1, R2, R3 and R4 are as defined above in Formula (I), and R5 is halogen or oxo (=O).

[0056] In one embodiment of the present invention, R5 is chloro, bromo, iodo or oxo (=0), in particular chloro or oxo.

[0057] Reaction Scheme 1 shows a four-step process for preparing a compound of formula (I) using a compound of formula (II) and a compound of formula (III) as starting materials.

[0058] In step 1, a compound of formula (II) is reacted with a compound of formula (III) to obtain a compound of formula (IV).

[0059] In this case, when R5 is a halogen, an amination reaction is carried out in the presence of an organic base to obtain a compound of formula (IV). When R5 is oxo (=O), a reducing agent is added to obtain a compound of formula (IV) through a reductive amination reaction. As the organic base, triethylamine, N,N-diisopropylethylamine (DIPEA) and the like can be used, while as the reducing agent, sodium borohydride (NaBH4), sodium cyanoborohydride (NaCNBH3), sodium triacetoxyborohydride (Na(CH3COO)3BH) and the like can be used.

[0060] As the reaction solvent, acetonitrile, tetrahydrofuran, 1,4-dihydrofuran, The reaction mixture may be selected from the group consisting of oxane, chloroform, dichloromethane, 1,2-dichloroethane, dimethylformamide, dimethylacetamide, methanol, ethanol, etc. The reaction temperature is preferably -10 to 25°C.

[0061] In step 2, triphosgene is added to the compound of formula (IV) to perform a heterocyclic urea formation reaction to obtain a compound of formula (V). In this case, the heterocyclic urea formation reaction can be performed in the presence of an organic base. As the organic base, triethylamine, N,N-diisopropylethylamine (DIPEA), etc. can be used.

[0062] As the reaction solvent, acetonitrile, tetrahydrofuran, 1,4-dihydrofuran, Alkane, etc., and the reaction temperature is preferably -10-100°C.

[0063] In step 3, the sulfide group of the compound of formula (V) is oxidized to obtain a sulfone compound of formula (VI) having a sulfone group.

[0064] The oxidation reaction can be carried out using potassium monopersulfate (oxone), meta-chloroperbenzoic acid (mPBA), or the like as an oxidizing agent.

[0065] As the reaction solvent, dichloromethane, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, 1,4-dichloroethane, Alkane, water, etc. are used, and the reaction temperature is preferably -10 to 100°C.

[0066] In step 4, the compound of formula (I) is obtained by subjecting the compound of formula (VI) to a substitution reaction with an amine compound of formula (VII).

[0067] As the reaction solvent, acetonitrile, tetrahydrofuran, 1,4-dihydrofuran, In the substitution reaction, an inorganic base or an organic base such as triethylamine, N,N-diisopropylethylamine, and pyridine may be used together, or an organic acid such as trifluoroacetic acid may be used together. The reaction temperature is preferably 50-150°C, more preferably 80-100°C.

[0068] The compound of formula (II) and the compound of formula (III) can be prepared according to known methods or can be obtained as commercially available products.

[0069] For example, methods for preparing compounds of formula (II) and formula (III) are depicted in the following reaction schemes 2 to 4, respectively. However, those methods shown in the following reaction schemes only represent typical methods used in the present invention. The order of operations, reagents, reaction conditions, etc. can be changed without limitation.

[0070] [Reaction Scheme 2]

[0071]

[0072] In Reaction Scheme 2, R2 is as defined above in Formula (I), and R5 is as defined above in Reaction Scheme 1.

[0073] As shown in Reaction Scheme 2, the compound of formula (II) can be prepared by reacting ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate with an amine compound of formula (XIII) to obtain a compound of formula (VIII); subjecting the compound of formula (VIII) to a reduction reaction using a reducing agent such as lithium aluminum hydride (LiAlH4) to obtain a compound of formula (IX); and halogenating the alcohol group of the compound of formula (IX) using thionyl chloride (SOCl2) or the like or forming an aldehyde by oxidation using manganese dioxide (MnO2) or the like.

[0074] [Reaction Scheme 3]

[0075]

[0076] In Reaction Scheme 3, R3 and R4 are as defined above in Formula (I), and Y is halogen or hydroxy.

[0077] In one embodiment of the present invention, Y is chlorine, bromine, iodine or hydroxy, in particular chlorine or hydroxy.

[0078] As shown in Reaction Scheme 3, the compound of formula (III) can be prepared by reacting the compound of formula (X) and the compound of formula (XI) in the presence of an organic base (such as DIPEA or a coupling agent) to obtain the compound of formula (XII), and reducing the nitro group of the compound of formula (XIII) to an amine group. In this case, the reduction reaction can be carried out by using a combination of potassium tert-butoxide and bis(pinacolato)diboron, or by using hydrogen gas on a palladium-carbon catalyst.

[0079] [Reaction Scheme 4]

[0080]

[0081] In Reaction Scheme 4, R3 and R4 are as defined above in Formula (I), and Y is as defined above in Reaction Scheme 3.

[0082] Alternatively, the compound of formula (III) can be prepared by reacting a diamine-pyridine compound of formula (X') with a compound of formula (XI) in the presence of an organic base (eg, DIPEA or a coupling agent), as shown in Reaction Scheme 4 above.

[0083] The compound of formula I or a pharmaceutically acceptable salt thereof according to the present invention exhibits excellent PD-L1 expression inhibitory activity (Experimental Example 1).

[0084] One aspect of the present invention relates to a pharmaceutical composition for inhibiting programmed death-ligand 1 (PD-L1), comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, and in particular, to a pharmaceutical composition for preventing or treating cancer.

[0085] In one embodiment of the present invention, the compound of formula (I) or a pharmaceutically acceptable salt thereof activates immune cells by inhibiting PD-L1 expression, thereby inducing anti-cancer effects, and is therefore effectively used to treat colon cancer, lung cancer, breast cancer, gastric cancer, cervical cancer, bladder cancer, blood cancer or non-Hodgkin's lymphoma.

[0086] The pharmaceutical composition according to the present invention can be administered orally, such as by ingestion or inhalation; or administered parenterally, such as by injection, deposition, implantation or suppository. Injection can be, for example, intravenous injection, subcutaneous injection, intramuscular injection or intraperitoneal injection. Depending on the route of administration, the pharmaceutical composition of the present invention can be formulated as tablets, capsules, granules, fine granules (fine subtilae), powders, sublingual tablets, suppositories, ointments, injection solutions, emulsions, suspensions, syrups, aerosols, etc. The above-mentioned various forms of the pharmaceutical composition of the present invention can be prepared in a manner known in the art using a pharmaceutically acceptable carrier commonly used for each form. The example of a pharmaceutically acceptable carrier includes excipients, binders, disintegrants, lubricants, preservatives, antioxidants, isotonic agents, buffers, coating agents, sweeteners, solvents, alkalis, dispersants, wetting agents, suspending agents, stabilizers, colorants, etc.

[0087] The pharmaceutical composition according to the present invention contains, depending on its form, 0.01% to 95% by weight of the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0088] The specific dosage of the pharmaceutical composition of the present invention may vary depending on the attributes of the mammal, including the mammal's human nature, weight, sex, severity of the disease, doctor's diagnosis, etc. For oral use, preferably 0.01 mg to 50 mg of the active ingredient is administered per kilogram of body weight per day, while for parenteral use, 0.01 mg to 10 mg of the active ingredient is administered per kilogram of body weight per day. The total daily dose may be administered in one or more doses, depending on the severity of the disease, doctor's diagnosis, etc.

[0089] Beneficial effects

[0090] The compound of the present invention can be used in a pharmaceutical composition for treating or preventing cancer because it has the effect of inhibiting PD-L1 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 is a graph showing changes in tumor volume over time after administration of exemplary compounds to mice bearing B16F10 cell-induced cancer.

[0092] Figure 2 is a graph showing the weight of tumors excised from mice bearing B16F10 cell-induced cancer after administration of exemplary compounds to the mice.

[0093] Figure 3 These are the results of measuring the degree of inhibition of PD-L1 expression in tumors excised from mice bearing B16F10 cell-induced cancer by administering the exemplary compounds to the mice, using Western blotting.

[0094] Figure 4 The results are shown in Figure 2, which show the degree of inhibition of PD-L1 expression measured by Western blotting after treating PD-L1-overexpressing cells with the exemplary compounds. DETAILED DESCRIPTION

[0095] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

[0096] Preparation Example 1: Preparation of compound of formula (II)

[0097] Preparation Example 1-1: 5-(Chloromethyl)-N-methyl-2-(methylthio)pyrimidin-4-amine (II-1)

[0098]

[0099] Ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate (10.00 g, 42.98 mmol) was dissolved in 100 mL of tetrahydrofuran, triethylamine (7.37 mL, 52.86 mmol) and 40% methylamine solution (3.54 g, 45.55 mmol) were added thereto, and the mixture was stirred at 20-25° C. for 16 hours. After the reaction was completed, the precipitated salt was filtered, the solvent was removed under reduced pressure, and the concentrated residue was extracted with saturated sodium bicarbonate solution and ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give ethyl 4-(methylamino)-2-(methylthio)pyrimidine-5-carboxylate (VIII-1, 8.82 g, 90%).

[0100] 1 H NMR (400MHz, CDCl3): δ1.37(t,3H),2.55(s,3H),3.09(d,3H),4.33(q,2H),8.18(bs,1H),8.61(s,1H)

[0101] The above-obtained 4-(methylamino)-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester (VIII-1) (8.80 g, 38.72 mmol) was dissolved in 200 mL of anhydrous tetrahydrofuran and cooled to 0° C. 1 M lithium aluminum hydride (116.16 mL, 116.16 mmol) was slowly added dropwise and stirred at 20-25° C. for 2 hours. When the reaction was complete, 5 mL of water, 15 mL of 15% sodium hydroxide solution, and 15 mL of water were slowly added dropwise to the reaction mixture, followed by termination of the reaction and stirring for 1 hour. After filtering the white precipitate, the filtrate was extracted twice with ethyl acetate, and the organic layers were combined and extracted with salt water. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give (4-(methylamino)-2-(methylthio)pyrimidine-5-yl)methanol (IX-1, 6.00 g, 84%).

[0102] 1 H NMR (400MHz, CDCl3): δ2.53(s,3H),3.05(d,3H),4.40(s,2H),5.22(m,1H),6.95(br,1H),7.96(s,1H)

[0103] The (4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methanol (IX-1) (5.72 g, 30.88 mmol) obtained above was dissolved in 240 mL of anhydrous tetrahydrofuran and cooled to 0° C. Then, thionyl chloride (5.59 mL, 77.19 mmol) was slowly added dropwise, and the mixture was stirred at 70° C. for 4 hours. The resulting white solid was filtered to obtain 7.20 g (100%) of the title compound.

[0104] Preparation Example 1-2: 4-(Methylamino)-2-(methylthio)pyrimidine-5-carbaldehyde (II-2)

[0105]

[0106] To (4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methanol (IX-1) (0.74 g, 4.00 mmol) was added 15 mL of dichloromethane, followed by the addition of manganese dioxide (3.47 g, 39.95 mmol), and the mixture was stirred at 20-25° C. for 20 hours. After completion of the reaction, the reaction mixture was filtered through celite, and the solvent was removed under reduced pressure to obtain 0.67 g (92%) of the title compound.

[0107] 1 H NMR (400MHz, CDCl3): δ9.70(s,1H),8.56(bs,1H),8.30(s,1H),3.12(d,3H),2.57(s,3H)

[0108] ES-MS m / z:184.18[M+H] +

[0109] Preparation Example 2: Preparation of compound of formula (III)

[0110] Preparation Example 2-1: N-(5-amino-6-methylpyridin-3-yl)-3-(trifluoromethyl)benzamide (III-1)

[0111]

[0112] After 6-methyl-5-nitropyridine-3-amine (X-1) (1.00 g, 6.53 mmol) was dissolved in 50 mL of dichloromethane, the mixture was cooled to 0 ° C. 3-(trifluoromethyl)benzoyl chloride (XI-1) (0.97 mL, 6.52 mmol) and N, N-diisopropylethylamine (3.33 mL, 19.59 mmol) were added to the reaction solution and stirred at 20-25 ° C for 1 hour. After the reaction was completed, dichloromethane, distilled water and brine were used to extract. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the concentrated residue was purified by column chromatography to obtain N-(6-methyl-5-nitropyridine-3-yl)-3-(trifluoromethyl)benzamide (XII-1) (1.76 g, 83%).

[0113] The N-(6-methyl-5-nitropyridine-3-yl)-3-(trifluoromethyl)benzamide (XII-1) (1.16 g, 3.57 mmol) obtained above was dissolved in 50 ml of methanol, 0.32 g of 10% palladium on carbon was added, and stirred for 3 hours under a hydrogen atmosphere. After completion of the reaction, the reaction mixture was filtered through celite, washed with methanol, and then concentrated under reduced pressure to obtain 1.04 g (99%) of the title compound.

[0114] 1 H NMR (400MHz, CDCl3): δ2.55(m,3H),4.07(br,2H),6.71(d,1H),7.01(s,1H),7.29(d,1H),7.45(d,1H),7.65(t,1H),7.85(s,1H),8.02(d,1H)

[0115] Preparation Example 2-2: N-(5-amino-6-methylpyridin-3-yl)-3-(tert-butyl)benzamide (III-2)

[0116]

[0117] 3-(tert-Butyl)benzoic acid (0.6 g, 3.37 mmol) was suspended in 20 mL of dichloromethane, and then oxalyl chloride (0.58 mL, 6.73 mmol) and 1-2 drops of a catalytic amount of dimethylformamide were added, followed by stirring at room temperature for 3 hours. After completion of the reaction, the reaction solvent was evaporated under reduced pressure to give 3-(tert-Butyl)benzoyl chloride (XI-2) (0.67 g, quantitative) without purification.

[0118] After 6-methyl-5-nitropyridine-3-amine (X-1) (0.90 g, 5.88 mmol) was dissolved in 50 mL of dichloromethane, 3-(tert-butyl)benzoyl chloride (XI-2) (1.15 g, 5.88 mmol) and N,N-diisopropylethylamine (3.07 mL, 17.63 mmol) obtained above were added and stirred at 20-25 ° C for 1 hour. After the reaction was completed, dichloromethane, distilled water and saline were used to extract. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the concentrated residue was purified by column chromatography to obtain 3-(tert-butyl)-N-(6-methyl-5-nitropyridine-3-yl)benzamide (XII-2) (1.39 g, 76%).

[0119] ES-MS m / z:314.25[M+H] +

[0120] The above-obtained 3-(tert-butyl)-N-(6-methyl-5-nitropyridine-3-yl)benzamide (XII-2) (1.39 g, 4.44 mmol) was dissolved in 40 ml of methanol, 0.32 g of 10% palladium on carbon was added, and stirred for 3 hours under a hydrogen atmosphere. After the reaction was completed, the reaction mixture was filtered through diatomaceous earth, washed with methanol, and then concentrated under reduced pressure to obtain the title compound (III-2) (1.18 g, 94%).

[0121] 1 H NMR (400MHz, CDCl3): δ1.34(s,9H),2.55(m,3H),4.10(br,2H),6.71(s,1H), 7.21(d,1H),7.32(d,1H),7.45(d,1H),7.65(t,1H),7.85(s,1H),8.02(d,1H)

[0122] ES-MS m / z:239.31[M+H] +

[0123] Preparation Example 2-3: N-(5-amino-6-methoxypyridin-3-yl)-3-(trifluoromethyl)benzamide (III-3)

[0124]

[0125] After 2-methoxy-3,5-dinitropyridine (0.55 g, 2.76 mmol) was dissolved in 20 mL of ethanol, 6 N hydrochloric acid (4.00 mL) and iron (1.30 g) were added and refluxed for 1 hour. After the reaction was completed, the reaction mixture was cooled to 20-25 ° C, neutralized with 5 M sodium hydroxide solution, and filtered through diatomaceous earth. The filtered solution was concentrated under reduced pressure, dissolved in ethyl acetate, extracted once with water, and washed with brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 2-methoxypyridine-3,5-diamine (X'-1) (0.21 g, 55%).

[0126] 1 H NMR (400MHz, CDCl3): δ3.23(s,3H),3.90(br,4H),6.46(s,1H),6.69(s,1H)

[0127] ES-MS m / z:140.11[M+H] +

[0128] The above-obtained 2-methoxypyridine-3,5-diamine (X'-1) (0.21 g, 1.51 mmol) was dissolved in 30 mL of acetone and cooled to 0 ° C., and triethylamine (0.23 g, 2.27 mmol) and 3-(trifluoromethyl)benzoyl chloride (XI-1) (0.25 g, 1.21 mmol) were added and stirred for 20 minutes. After the reaction was completed, the solvent was evaporated and extracted with ethyl acetate, distilled water and brine in sequence. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain 0.17 g (45%) of the title compound.

[0129] 1 H NMR (400MHz, CDCl3): δ3.23(s,3H),3.90(br,2H),6.59(s,1H),6.78(dd,1H),7.38(d,1H),7.65(t,1H),7.68(s,1H),7.82(d,1H),8.10(s,1H)

[0130] ES-MS m / z:312.10[M+H] +

[0131] Preparation Example 2-4: N-(5-amino-6-methylpyridin-3-yl)-4-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)benzamide (III-4)

[0132]

[0133] 6-Methyl-5-nitropyridin-3-amine (X-1) (1.00 g, 6.52 mmol) was dissolved in 50 mL of dichloromethane and cooled to 0°C, and N,N-diisopropylethylamine (3.41 mL, 19.59 mmol) and 4-(4,4-dimethyl-4,5-dihydro- 4-(4,4-dimethyl-4,5-dihydro-4-nitropropene)-2 ... oxazol-2-yl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide (XII-6) (1.99 g, 86%).

[0134] ES-MS m / z:355.25[M+H] +

[0135] The 4-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide (XII-6) (1.95 g, 5.50 mmol) was dissolved in 50 mL of methanol, 0.30 g of 10% palladium on carbon was added, and the mixture was stirred under a hydrogen atmosphere for 5 hours. Then, 1.64 g (92%) of the title compound was obtained in the same manner as in Preparation Example 2-1.

[0136] ES-MS m / z:325.25[M+H] +

[0137] Preparation Example 2-5: N-(5-amino-6-methylpyridin-3-yl)-3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)benzamide (III-5)

[0138]

[0139] 6-Methyl-5-nitropyridin-3-amine (X-1) (1.00 g, 6.52 mmol) was dissolved in 60 mL of tetrahydrofuran and cooled to 0°C, and N,N-diisopropylethylamine (3.41 mL, 19.59 mmol) and 3-(4,4-dimethyl-4,5-dihydro- 4-dimethyl-4,5-dihydro-3-nitropropene was added to the 4-nitro-2-nitropropene (X-2-yl) benzoyl chloride (XI-4) (1.55 g, 6.52 mmol) and stirred for 2 hours. After the reaction was completed, dichloromethane, distilled water and salt water were used to extract. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the concentrated residue was purified by column chromatography to obtain 3-(4,4-dimethyl-4,5-dihydro-3-nitropropene) benzoyl chloride (XI-4) (1.55 g, 6.52 mmol). oxazol-2-yl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide (XII-7) (1.39 g, 60%).

[0140] The above obtained 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide (XII-7) (1.39 g, 3.92 mmol) was dissolved in 30 mL of methanol, 0.30 g of 10% palladium on carbon was added, and the mixture was stirred for 3 hours under a hydrogen atmosphere. Then, 1.17 g (92%) of the title compound was obtained in the same manner as in Preparation Example 2-1.

[0141] ES-MS m / z:325.16[M+H] +

[0142] Preparation Example 2-6: N-(5-amino-6-methoxypyridin-3-yl)-3-(tert-butyl)benzamide (III-6)

[0143]

[0144] 2-Methoxypyridine-3,5-diamine (X'-1) (0.44 g, 3.18 mmol) was dissolved in 40 mL of acetone and cooled to 0 ° C. Triethylamine (0.48 g, 4.77 mmol) and 3-(tert-butyl)benzoyl chloride (XI-2) (0.50 g, 2.54 mmol) were added and stirred for 2 hours. After the reaction was completed, the solvent was evaporated and extracted with ethyl acetate, distilled water and brine in sequence. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 0.34 g (45%) of the title compound.

[0145] ES-MS m / z:300.28[M+H] +

[0146] Preparation Example 2-7: N-(5-amino-6-methoxypyridin-3-yl)-3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)benzamide (III-7)

[0147]

[0148] 2-Methoxypyridine-3,5-diamine (X'-1) (0.40 g, 2.87 mmol) was dissolved in 40 ml of acetone and cooled to 0°C, and triethylamine (0.44 g, 4.31 mmol) and 3-(4,4-dimethyl-4,5-dihydro- The reaction mixture was stirred for 2 hours. After the reaction was complete, the solvent was evaporated and the mixture was extracted with ethyl acetate, distilled water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 0.34 g (43%) of the title compound.

[0149] ES-MS m / z:341.36[M+H] +

[0150] Preparation Example 2-8: N-(5-amino-6-chloropyridin-3-yl)-3-(trifluoromethyl)benzamide trifluoroacetate (III-8)

[0151]

[0152] By (5-amino-2-chloropyridin-3-yl) tert-butyl carbamate (1.20g, 4.92mmol) is dissolved in 60mL dichloromethane and cooled to 0 ℃, N, N-diisopropylethylamine (2.51mL, 14.77mmol) and 3-(trifluoromethyl) benzoyl chloride (XI-1) (1.13g, 5.42mmol) are added and stirred at 20-25 ℃ for 4 hours. After the reaction is completed, dichloromethane, distilled water and saturated sodium chloride solution are used to extract. The organic layer is dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue is purified by column chromatography to obtain 1.59g (2-chloro-5-(3-(trifluoromethyl) benzamido) pyridin-3-yl tert-butyl carbamate (78%).

[0153] 1 H NMR (400MHz, CDCl3): δ1.53(s,9H),7.06(br,1H),7.64(m,1H),7.84(m,1H),8.10(m,3H),8.58(d,1H),8.78(d,1H).

[0154] 1.50 g (3.61 mmol) of tert-butyl (2-chloro-5-(3-(trifluoromethyl)benzamido)pyridin-3-ylcarbamate obtained above was dissolved in dichloromethane:trifluoroacetic acid (1:1) and stirred at 20-25° C. for 5 hours. After completion of the reaction, the reaction solvent was evaporated under reduced pressure to give 1.10 g (71%) of the title compound trifluoroacetate.

[0155] 1 H NMR (400MHz, DMSO-d6): δ5.69(br,2H),5.76(br,1H),7.73(d,1H),7.79(t,1H),7.97(m,2H),8.26(m,2H),10.53(S,1H).

[0156] Preparation Example 2-9: N-(5-amino-6-chloropyridin-3-yl)-3-(tert-butyl)benzamide trifluoroacetate (III-9)

[0157]

[0158] After dissolving tert-butyl (5-amino-2-chloropyridin-3-yl)carbamate (0.84 g, 3.45 mmol) in 50 mL of dichloromethane and cooling to 0° C., N,N-diisopropylethylamine (1.76 mL, 10.34 mmol) and 3-(tert-butyl)benzoyl chloride (XI-2) (0.68 g, 3.45 mmol) were added, and the mixture was stirred at 20-25° C. for 5 hours. Then, tert-butyl (5-(3-(tert-butyl)benzamido)-2-chloropyridin-3-yl)carbamate (0.90 g, 65%) was obtained in the same manner as in Preparation Example 2-8.

[0159] The above-obtained tert-butyl (5-(3-(tert-butyl)benzamido)-2-chloropyridin-3-yl)carbamate (0.88 g, 2.18 mmol) was dissolved in dichloromethane:trifluoroacetic acid (1:1), and 0.88 g of the title compound trifluoroacetate was quantitatively obtained in the same manner as in Preparation Example 2-8.

[0160] 1 H NMR (400MHz, DMSO-d6): δ1.38(s,9H),5.69(br,2H),5.76(br,1H),7.73(d,1H),7.79(t,1H),7.97(m,2H),8.26(m,2H),10.53(S,1H).

[0161] Preparation Example 2-10: N-(5-amino-6-chloropyridin-3-yl)-2-(tert-butyl)thiazole-4-carboxamide trifluoroacetate (III-10)

[0162]

[0163] After dissolving tert-butyl (5-amino-2-chloropyridin-3-yl)carbamate (0.84 g, 3.45 mmol) in 50 mL of dichloromethane and cooling to 0° C., N,N-diisopropylethylamine (1.76 mL, 10.34 mmol) and 2-(tert-butyl)thiazole-4-carbonyl chloride (XI-5) (0.70 g, 3.45 mmol) were added, and the mixture was stirred at 20-25° C. for 5 hours. In the same manner as in Preparation Example 2-8, tert-butyl (5-(2-(tert-butyl)thiazole-4-carboxamido)-2-chloropyridin-3-yl)carbamate (0.94 g, 67%) was obtained.

[0164] The above-obtained tert-butyl (5-(2-(tert-butyl)thiazole-4-carboxamido)-2-chloropyridin-3-yl)carbamate (0.92 g, 2.24 mmol) was dissolved in dichloromethane:trifluoroacetic acid (1:1), and 0.92 g of the title compound trifluoroacetate was quantitatively obtained in the same manner as in Preparation Example 2-8.

[0165] Preparation Example 3: Preparation of compound of formula (IV)

[0166] Preparation Example 3-1: N-(6-methyl-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)-3-(trifluoromethyl)benzamide (IV-1)

[0167]

[0168] After the compound (III-1) (0.60g 2.03mmol) prepared in Preparation Example 2-1 was dissolved in 50mL of acetonitrile, N,N-diisopropylethylamine (1.06mL, 6.10mmol) was added at 0°C. The compound (II-1) (0.40g, 1.63mmol) prepared in Preparation Example 1-1 was dissolved in 30mL of acetonitrile and slowly added dropwise to the reaction solution, which was then stirred at 20-25°C for 1 hour. The reaction solvent was evaporated under reduced pressure, and extracted with ethyl acetate, water and brine in sequence. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated residue was purified by column chromatography to obtain 0.70g (70%) of the title compound.

[0169] 1 H NMR (400MHz, CDCl3): δ2.55(s,3H),3.05(d,3H),4.16(s,2H),5.57(m,1H),6.88(dd,1H),7.2 9(d,1H),7.43(d,1H),7.65(t,1H),7.82-7.85(m,2H),7.93(s,1H),8.06(d,1H),8.12(s,1H)

[0170] ES-MS m / z:463.03[M+H] +

[0171] Preparation Example 3-2: 3-(tert-Butyl)-N-(6-methyl-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-2)

[0172]

[0173] Using compound (III-2) (0.90 g, 3.18 mmol) prepared in Preparation Example 2-2 instead of compound (III-1) and compound (II-1) (0.76 g, 3.16 mmol) prepared in Preparation Example 1-1, 0.80 g (56%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0174] 1 H NMR (400MHz, CDCl3): δ1.34(s,9H),2.47(s,6H),2.55(s,3H),4.16(s,2H),4.32(m, 2H),6.76(dd,1H),7.36(m,1H),7.54(m,1H),7.75(m,2H),7.98(m,1H),8.25(m,1H)

[0175] ES-MS m / z:451.14[M+H] +

[0176] Preparation Example 3-3: 3-(tert-Butyl)-N-(6-methyl-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-2)

[0177]

[0178] Instead of compound (III-1), compound (III-2) (2.08 g, 7.33 mmol) prepared in Preparation Example 2-2 was dissolved in 50 mL of methanol, acetic acid (0.88 mL, 15.28 mmol) and compound (II-2) (1.12 g, 6.11 mmol) obtained in Preparation Example 1-2 were added and stirred for 15 minutes. After sodium cyanoborohydride (2.30 g, 36.68 mmol) was added to the reaction solution, the temperature was raised to 45 ° C and stirred for 18 hours. After the reaction was completed, the mixture was cooled to 20-25 ° C, the reaction solvent was evaporated under reduced pressure, and ethyl acetate and saturated sodium bicarbonate solution were used for extraction. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated residue was purified by column chromatography to obtain 1.10 g (40%) of the title compound.

[0179] ES-MS m / z:451.14[M+H] +

[0180] Preparation Example 3-4: N-(4-methoxy-3-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)-3-(trifluoromethyl)benzamide (IV-3)

[0181]

[0182] Using compound (III-3) (0.17 g, 0.69 mmol) prepared in Preparation Example 2-3 instead of compound (III-1), 0.10 g (30%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0183] 1 H NMR (400MHz, CDCl3): δ2.55(s,3H),2.87(d,3H),4.01(s,3H),4.25(s,2H),5.60 (dd,2H),6.88(dd,1H),7.12(d,1H),7.42-7.89(m,4H),7.93(s,1H),8.12(s,1H)

[0184] ES-MS m / z:527.97[M+H] +

[0185] Preparation Example 3-5: 4-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-4)

[0186]

[0187] Using compound (III-4) (1.00 g, 3.08 mmol) prepared in Preparation Example 2-4 instead of compound (III-1), 0.45 g (30%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0188] ES-MS m / z:492.54[M+H] +

[0189] Preparation Example 3-6: 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-5)

[0190]

[0191] Using compound (III-5) (1.00 g, 3.08 mmol) prepared in Preparation Example 2-5 instead of compound (III-1), 0.40 g (30%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0192] ES-MS m / z:492.54[M+H] +

[0193] Preparation Example 3-7: 3-(tert-Butyl)-N-(6-methoxy-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-6)

[0194]

[0195] Using compound (III-6) (0.34 g, 1.14 mmol) prepared in Preparation Example 2-6 instead of compound (III-1), 0.18 g (51%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0196] ES-MS m / z:467.61[M+H] +

[0197] Preparation Example 3-8: 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methoxy-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-7)

[0198]

[0199] Using compound (III-7) (0.34 g, 1.00 mmol) prepared in Preparation Example 2-7 instead of compound (III-1), 0.15 g (45%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0200] ES-MS m / z:508.55[M+H] +

[0201] Preparation Example 3-9: N-(6-chloro-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)-3-(trifluoromethyl)benzamide (IV-8)

[0202]

[0203] Using compound (III-8) (1.15 g, 1.99 mmol) prepared in Preparation Example 2-8 instead of compound (III-1), 0.26 g (41%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0204] Preparation Example 3-10: 3-(tert-Butyl)-N-(6-chloro-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-9)

[0205]

[0206] Using compound (III-9) (1.17 g, 2.91 mmol) prepared in Preparation Example 2-9 instead of compound (III-1), 0.28 g (41%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0207] Preparation Example 3-11: 2-(tert-Butyl)-N-(6-chloro-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)thiazole-4-carboxamide (IV-10)

[0208]

[0209] Using compound (III-10) (0.67 g, 3.33 mmol) prepared in Preparation Example 2-10 instead of compound (III-1), 0.32 g (41%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0210] 1H NMR (400MHz, CDCl3): δ9.24(s,1H),8.07(m,1H),7.95(s,1H),7.87(d,1H),5.45(br,1H),4.15-4.19(m,2H),3.07(d,2H),2.55(s,3H),1.47(s,9H).

[0211] Preparation Example 4: Preparation of compound of formula (V)

[0212] Preparation Example 4-1: N-(6-methyl-5-(1-methyl-7-(methylthio)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (V-1)

[0213]

[0214] The compound (IV-1) prepared in Preparation Example 3-1 (0.70 g, 1.51 mmol) was dissolved in 60 mL of 1,4-dimethoxybenzyl alcohol. After the reaction was completed, N,N-diisopropylethylamine (0.78 mL, 4.54 mmol) and triphosgene (0.63 g, 2.12 mmol) were added at 0°C and stirred at 100°C for 3 hours. After the reaction was completed, the mixture was extracted with saturated sodium bicarbonate solution and ethyl acetate, and the organic layer was washed with brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the concentrated residue was purified by column chromatography to obtain 0.37 g (50%) of the title compound.

[0215] 1 H NMR (400MHz, CDCl3): δ2.55(s,3H),2.63(s,3H),3.65(d,3H),5.16(s,2H),6.88(dd,1 H),7.29(d,1H),7.43(d,1H),7.65(t,1H),7.82-7.85(m,2H),7.93(s,1H),8.12(s,1H)

[0216] ES-MS m / z:489.50[M+H] +

[0217] Preparation Example 4-2: 3-(tert-Butyl)-N-(6-methyl-5-(1-methyl-7-(methylthio)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (V-2)

[0218]

[0219] Using compound (IV-2) (0.80 g, 1.78 mmol) prepared in Preparation Example 3-2 instead of compound (IV-1), 0.50 g (60%) of the title compound was obtained in the same manner as in Preparation Example 4-1.

[0220] 1 H NMR (400MHz, CDCl3): δ1.34(s,9H),2.47(d,3H),2.55(d,3H),2.70(d,3H),4.42(d ,2H),6.76(m,1H),7.36(m,1H),7.54(m,1H),7.75(m,2H),7.98(m,1H),8.25(m,1H)

[0221] ES-MS m / z:477.60[M+H] +

[0222] Preparation Example 4-3: N-(6-methoxy-5-(1-methyl-7-(methylthio)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (V-3)

[0223]

[0224] Using compound (IV-3) (0.10 g, 0.21 mmol) prepared in Preparation Example 3-4 instead of compound (IV-1), 0.10 g (95%) of the title compound was obtained in the same manner as in Preparation Example 4-1.

[0225] 1 H NMR (400MHz, CDCl3): δ2.55(s,3H),3.34(d,3H),4.25(s,3H),5.06(s,2H),6 .88(dd,1H),7.31-7.42(m,2H),7.65-7.95(m,4H),8.06(d,1H),8.12(s,1H)

[0226] ES-MS m / z:505.09[M+H] +

[0227] Preparation Example 4-4: 4-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(1-methyl-7-(methylthio))-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (V-4)

[0228]

[0229] Using compound (IV-4) (0.40 g, 0.81 mmol) prepared in Preparation Example 3-5 instead of compound (IV-1), 0.32 g (76%) of the title compound was obtained in the same manner as in Preparation Example 4-1.

[0230] ES-MS m / z:518.09[M+H] +

[0231] Preparation Example 4-5: 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(1-methyl-7-(methylthio)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (V-5)

[0232]

[0233] Using compound (IV-5) (0.40 g, 0.81 mmol) prepared in Preparation Example 3-6 instead of compound (IV-1), 0.35 g (83%) of the title compound was obtained in the same manner as in Preparation Example 4-1.

[0234] ES-MS m / z:518.09[M+H] +

[0235] Preparation Example 4-6: 3-(tert-Butyl)-N-(6-methoxy-5-(1-methyl-7-(methylthio)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (V-6)

[0236]

[0237] Using compound (IV-6) (0.18 g, 0.39 mmol) prepared in Preparation Example 3-7 instead of compound (IV-1), 0.17 g (90%) of the title compound was obtained in the same manner as in Preparation Example 4-1.

[0238] ES-MS m / z:493.62[M+H] +

[0239] Preparation Example 4-7: 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methoxy-5-(1-methyl-7-(methylthio)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (V-7)

[0240]

[0241] Using compound (IV-7) (0.15 g, 0.30 mmol) prepared in Preparation Example 3-8 instead of compound (IV-1), 0.15 g (93%) of the title compound was obtained in the same manner as in Preparation Example 4-1.

[0242] ES-MS m / z:534.65[M+H] +

[0243] Preparation Example 4-8: N-(6-chloro-5-(1-methyl-7-(methylthio)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (V-8)

[0244]

[0245] Using compound (IV-8) (0.27 g, 0.56 mmol) prepared in Preparation Example 3-9 instead of compound (IV-1), 0.27 g (96%) of the title compound was obtained in the same manner as in Preparation Example 4-1.

[0246] Preparation Example 4-9: 3-(tert-Butyl)-N-(6-chloro-5-(1-methyl-7-(methylthio)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (V-9)

[0247]

[0248] Using compound (IV-9) (0.28 g, 0.59 mmol) prepared in Preparation Example 3-10 instead of compound (IV-1), 0.23 g (78%) of the title compound was obtained in the same manner as in Preparation Example 4-1.

[0249] Preparation Example 4-10: 2-(tert-Butyl)-N-(6-chloro-5-(1-methyl-7-(methylthio)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)thiazole-4-carboxamide (V-10)

[0250]

[0251] Using compound (IV-10) (0.31 g, 0.65 mmol) prepared in Preparation Example 3-11 instead of compound (IV-1), 0.25 g (77%) of the title compound was obtained in the same manner as in Preparation Example 4-1.

[0252] Preparation Example 5: Preparation of compound of formula (VI)

[0253] Preparation Example 5-1: N-(6-methyl-5-(1-methyl-7-(methylsulfonyl)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (VI-1)

[0254]

[0255] The compound (V-1) prepared in Preparation Example 4-1 (0.37 g, 0.76 mmol) was dissolved in tetrahydrofuran: distilled water (2: 1, 30 mL), potassium monopersulfate (4.66 g, 7.57 mmol) was added as an oxidant, and the mixture was stirred at 65 ° C for 5 hours. After the reaction was completed, the reaction solvent was evaporated under reduced pressure, washed with ethyl acetate, water and brine in sequence, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated residue was purified by column chromatography to obtain 0.17 g (44%) of the title compound.

[0256] 1 H NMR (400MHz, CDCl3): δ2.70(s,3H),3.38(s,3H),3.40(s,3H),4.42(d,2H),6.76(m,1H),7.36(m,1H),7.48-7.99(m,4H),8.25(m,1H),9.12(s,1H)

[0257] ES-MS m / z:521.60[M+H] +

[0258] Preparation Example 5-2: 3-(tert-Butyl)-N-(6-methyl-5-(1-methyl-7-(methylsulfonyl)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (VI-2)

[0259]

[0260] Using compound (V-2) (0.51 g, 1.07 mmol) prepared in Preparation Example 4-2 instead of compound (V-1), 0.22 g (42%) of the title compound was obtained in the same manner as in Preparation Example 5-1.

[0261] 1H NMR (400MHz, CDCl3): δ1.34(s,9H),2.70(s,3H),3.38(s,3H),3.40(s,3H),4.42(d,2H),6 .76(m,1H),7.36(m,1H),7.54(m,1H),7.75(m,2H),7.98(m,1H),8.25(m,1H),9.15(s,1H)

[0262] ES-MS m / z:509.72[M+H] +

[0263] Preparation Example 5-3: N-(6-methoxy-5-(1-methyl-7-(methylsulfonyl)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (VI-3)

[0264]

[0265] Using compound (V-3) (0.09 g, 0.17 mmol) prepared in Preparation Example 4-3 instead of compound (V-1), 90 mg (97%) of the title compound was obtained in the same manner as in Preparation Example 5-1.

[0266] 1 H NMR (400MHz, CDCl3): δ3.10(s,3H),3.45(s,3H),4.63(s,3H),5.51(s,2H),6 .56(dd,1H),7.13-7.81(m,3H),8.16-8.17(m,2H),8.28(d,1H),9.10(s,1H)

[0267] ES-MS m / z:537.01[M+H] +

[0268] Preparation Example 5-4: 4-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(1-methyl-7-(methylsulfonyl))-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (VI-4)

[0269]

[0270] Using compound (V-4) (0.30 g, 0.58 mmol) prepared in Preparation Example 4-4 instead of compound (V-1), 0.24 g (76%) of the title compound was obtained in the same manner as in Preparation Example 5-1.

[0271] ES-MS m / z:550.25[M+H] +

[0272] Preparation Example 5-5: 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(1-methyl-7-(methylsulfonyl)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (VI-5)

[0273]

[0274] Using compound (V-5) (0.35 g, 0.58 mmol) prepared in Preparation Example 4-5 instead of compound (V-1), 0.24 g (76%) of the title compound was obtained in the same manner as in Preparation Example 5-1.

[0275] ES-MS m / z:550.21[M+H] +

[0276] Preparation Example 5-6: 3-(tert-Butyl)-N-(6-methoxy-5-(1-methyl-7-(methylsulfonyl)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (VI-6)

[0277]

[0278] Using compound (V-6) (0.17 g, 0.35 mmol) prepared in Preparation Example 4-6 instead of compound (V-1), 0.17 g (91%) of the title compound was obtained in the same manner as in Preparation Example 5-1.

[0279] 1 H NMR (400MHz, CDCl3): δ1.36(s,9H),3.37(s,3H),3.90(s,3H),4.82(s,2H),7.41(t, 1H),7.60(d,1H),7.66(d,1H),7.94(s,1H),8.21(dd,1H),8.24(s,1H),8.38(t,1H)

[0280] ES-MS m / z:525.61[M+H] +

[0281] Preparation Example 5-7: 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methoxy-5-(1-methyl-7-(methylsulfonyl)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (VI-7)

[0282]

[0283] Using compound (V-7) (0.15 g, 0.28 mmol) prepared in Preparation Example 4-7 instead of compound (V-1), 0.14 g (89%) of the title compound was obtained in the same manner as in Preparation Example 5-1.

[0284] ES-MS m / z:566.50[M+H] +

[0285] Preparation Example 5-8: N-(6-chloro-5-(1-methyl-7-(methylsulfonyl)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (VI-8)

[0286]

[0287] Using compound (V-8) (0.26 g, 0.51 mmol) prepared in Preparation Example 4-8 instead of compound (V-1), 0.21 g (77%) of the title compound was obtained in the same manner as in Preparation Example 5-1.

[0288] Preparation Example 5-9: 3-(tert-Butyl)-N-(6-chloro-5-(1-methyl-7-(methylsulfonyl)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (VI-9)

[0289]

[0290] Using compound (V-9) (0.23 g, 0.46 mmol) prepared in Preparation Example 4-9 instead of compound (V-1), 0.21 g (86%) of the title compound was obtained in the same manner as in Preparation Example 5-1.

[0291] Preparation Example 5-10: 2-(tert-Butyl)-N-(6-chloro-5-(1-methyl-7-(methylsulfonyl)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)thiazole-4-carboxamide (VI-10)

[0292]

[0293] Using compound (V-10) (0.26 g, 0.52 mmol) prepared in Preparation Example 4-10 instead of compound (V-1), 0.19 g (67%) of the title compound was obtained in the same manner as in Preparation Example 5-1.

[0294] Example: Preparation of compound of formula (I)

[0295] Example 1: N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-1)

[0296]

[0297] The compound (VI-1) prepared in Preparation Example 5-1 (0.17 g, 0.33 mmol) was dissolved in 10 ml of 1,4-dichlorobenzene. To the mixture was added 4 ml of aniline and stirred at 100°C for 3 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated residue was purified by column chromatography to obtain 87 mg (50%) of the title compound.

[0298] 1 H NMR (400MHz, DMSO-d6): δ2.55(s,3H),3.09(s,3H),4.42(d,2H),6.46-7.5 4(m,6H),7.75(m,2H),8.01(m,1H),8.17(m,1H),9.60(s,1H),10.36(s,1H)

[0299] ES-MS m / z:534.62[M+H] +

[0300] Example 2: 3-(tert-butyl)-N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-2)

[0301]

[0302] Using compound (VI-2) (0.21 g, 0.41 mmol) prepared in Preparation Example 5-2 instead of compound (VI-1), 86 mg (40%) of the title compound was obtained in the same manner as in Example 1.

[0303] 1H NMR (400MHz, DMSO-d6): δ1.34(s,9H),2.55(s,3H),3.11(s,3H),4.42(d,2H),6.4 6-7.54(m,9H),7.75(m,1H),7.98(m,1H),8.25(m,1H),9.53(s,1H),10.35(s,1H)

[0304] ES-MS m / z:522.72[M+H] +

[0305] Example 3: N-(6-methoxy-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-3)

[0306]

[0307] Using compound (VI-3) (90 mg, 0.17 mmol) prepared in Preparation Example 5-3 instead of compound (VI-1), 25 mg (27%) of the title compound was obtained in the same manner as in Example 1.

[0308] 1 H NMR(400MHz,DMSO-d6):3.35(s,3H),δ3.90(s,3H),4.63(s,2H),6.95(t,1H),7.29(t,1H),7.70(d,2H),7.7 9(t,2H),8.00(d,1H),8.16-8.17(m,2H),8.28(d,1H),8.33(s,1H),8.51(s,1H),9.60(s,1H),10.67(s,1H)

[0309] ES-MS m / z:550.07[M+H] +

[0310] Example 4: 4-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-4)

[0311]

[0312] Using compound (VI-4) (0.20 g, 0.36 mmol) prepared in Preparation Example 5-4 instead of compound (VI-1), 0.10 g (55%) of the title compound was obtained in the same manner as in Example 1.

[0313] 1 H NMR (400MHz, DMSO-d6): δ1.31(s,6H),2.89(d,3H),3.36(s,3H),4.16(s,2H),4.61(d,1H),4.73(d,1H),7.12-7.23(m,5H ),7.83(t,1H),8.03(d,1H),8.29(d,1H),8.37(s,1H),8.56(d,1H),8.66(s,1H),8.77(s,1H),9.53(s,1H),10.98(s,1H)

[0314] ES-MS m / z:563.72[M+H] +

[0315] Example 5: 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-5)

[0316]

[0317] Using compound (VI-5) (0.23 g, 0.42 mmol) prepared in Preparation Example 5-5 instead of compound (VI-1), 0.14 g (58%) of the title compound was obtained in the same manner as in Example 1.

[0318] 1 H NMR (400MHz, DMSO-d6): δ1.41(s,6H),2.59(d,3H),3.36(s,3H),4.13(s,2H),4.61(d,1H),4.73(d,1H),7.12-7.23(m,5H ),7.81(s,1H),8.03(d,1H),8.29(d,1H),8.37(s,1H),8.56(d,1H),8.66(s,1H),8.77(s,1H),9.53(s,1H),10.98(s,1H)

[0319] ES-MS m / z:563.62[M+H] +

[0320] Example 6: 3-(tert-butyl)-N-(6-methoxy-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-6)

[0321]

[0322] Using compound (VI-6) (0.17 g, 0.32 mmol) prepared in Preparation Example 5-6 instead of compound (VI-1), 83 mg (47%) of the title compound was obtained in the same manner as in Example 1.

[0323] 1 H NMR (400MHz, DMSO-d6): δ1.34(s,9H),3.37(s,3H),3.90(s,3H),4.63(d,2H),6.95(t,1H),7.29(t,1H),7.47(t,1 H),7.64(d,1H),7.76-7.81(m,2H),7.96(t,1H),8.14(d,1H),8.16(s,1H),8.52(d,1H),9.59(s,1H),10.38(s,1H)

[0324] ES-MS m / z:538.64[M+H] +

[0325] Example 7: 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methoxy-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-7)

[0326]

[0327] Using compound (VI-7) (0.14 g, 0.25 mmol) prepared in Preparation Example 5-7 instead of compound (VI-1), 65 mg (44%) of the title compound were obtained in the same manner as in Example 1.

[0328] 1H NMR (400MHz, DMSO-d6): δ1.34(s,9H),3.37(s,3H),3.90(s,3H),4.15(s,2H),4.63(d,1H),4.71(d,1H),6.95(t,1H),7.29(t,1H),7.47( t,1H),7.64(d,1H),7.78-7.85(m,2H),8.01-8.12(m,2H),8.35(t,1H),8.56(d,1H),8.66(s,1H),8.77(s,1H),9.57(s,1H),10.98(s,1H)

[0329] ES-MS m / z:579.65[M+H] +

[0330] Example 8: N-(6-chloro-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-8)

[0331]

[0332] Using compound (VI-8) (0.20 g, 0.37 mmol) prepared in Preparation Example 5-8 instead of compound (VI-1), 92 mg (46%) of the title compound was obtained in the same manner as in Example 1.

[0333] 1 H NMR (400MHz, DMSO-d6): δ3.33(s,3H),4.59-4.74(m,2H),6.91(t,1H),7.25(t,2H),7.72-7.81(m ,3H),7.99(d,1H),8.15(s,1H),8.26(m,2H),8.46(s,1H),8.73(s,1H),9.59(s,1H),10.92(s,1H)

[0334] ES-MS m / z:553.97[M+H] +

[0335] Example 9: 3-(tert-Butyl)-N-(6-chloro-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-9)

[0336]

[0337] Using compound (VI-9) (0.21 g, 0.40 mmol) prepared in Preparation Example 5-9 instead of compound (VI-1), 98.3 mg (46%) of the title compound was obtained in the same manner as in Example 1.

[0338] 1 H NMR (400MHz, DMSO-d6): δ1.31(s,9H),3.33(s,3H),4.59-4.74(m,2H),6.91(t,1H),7.26(t,2H),7.46(t,1H), 7.63(d,1H),7.72-7.79(m,3H),7.79(s,1H),8.15(s,1H),8.44(d,1H),8.75(d,1H),9.58(s,1H),10.65(s,1H)

[0339] ES-MS m / z:542.14[M+H] +

[0340] Example 10: 2-(tert-butyl)-N-(6-chloro-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)thiazole-4-carboxamide (I-10)

[0341]

[0342] Using compound (VI-10) (0.18 g, 0.33 mmol) prepared in Preparation Example 5-10 instead of compound (VI-1), 0.12 g (64%) of the title compound was obtained in the same manner as in Example 1.

[0343] 1 H NMR (400MHz, DMSO-d6): δ1.47(s,9H),3.37(s,3H),4.62-4.77(m,2H),6.95(t,1H),7.27-7.31(t ,2H),7.78(d,2H),8.18(s,1H),8.39(s,1H),8.51(d,1H),8.90(d,1H),9.62(s,1H),10.43(s,1H)

[0344] ES-MS m / z:549.16[M+H] +

[0345] Example 11: N-(6-chloro-5-(7-((4-hydroxyphenyl)amino)-1-methyl-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-11)

[0346]

[0347] The compound (VI-8) (1.00 g, 1.85 mmol) prepared in Preparation Example 5-8 was dissolved in 20 ml of 1,4-dichlorobenzene. The mixture was added to 4-aminophenol (1.00 g, 9.24 mmol) and trifluoroacetic acid (0.71 ml, 9.24 mmol) and stirred at 85° C. for 7 hours. After the reaction was completed, the reaction solvent was removed, methanol was added, and the mixture was refluxed for 2 hours to obtain 0.63 g (60%) of the title compound.

[0348] 1 H NMR (400MHz, DMSO-d6): δ3.33(s,3H),4.58-4.74(m,2H),6.68-6.72(m,2H),7.47-7.50(m,2H),7.81-7.85(m,1H),8 .01-8.03(m,1H),8.11(s,1H),8.28-8.34(m,2H),8.47(d,1H),8.77(d,1H),9.05(s,1H),9.29(s,1H),10.95(s,1H)

[0349] ES-MS m / z:569.16[M+H] +

[0350] Experimental Example 1: Analysis of PD-L1 expression inhibitory activity

[0351] The PD-L1 promoter gene region was inserted into the pGL4.14-[luc2 / Hygro] vector (Promega, Madison, WI) in the following manner to prepare a promoter reporter construct for measuring gene transcription activity.

[0352] After preparing primers containing KpnI and XhoI restriction enzyme sites in PD-L1, PCR (polymerase chain reaction) was performed, and the promoter region DNA was isolated by electrophoresis on agarose gel. The pGL4.14-[luc2 / Hygro] vector containing firefly luciferase as a reporter gene was treated with the same restriction enzymes and separated by agarose gel electrophoresis and gel extraction. The pure isolated PD-L1 promoter gene region and the pGL4.14-[luc2 / Hygro] vector were reacted at 50°C for 1 hour using injection DNA ligase (Takara), and then E. coli was transformed to prepare a promoter reporter construct inserted into the PCSK9 promoter (named pGL4.14-PD-L1).

[0353] To measure the transcriptional activity of the PD-L1 gene promoter, A549 cells were transiently transfected with a PD-L1 promoter reporter construct as follows, and the luciferase activity of the cell extracts was measured.

[0354] DNA-lipofectamine complexes were prepared using 0.1 μg of promoter reporter construct plasmid and lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Opti-MEM medium (Invitrogen) was used during the formation of complexes between DNA and Lipofectamine 2000. 2.5 × 10 5 A549 cells were prepared in a 12-well plate at 100 cells / well (1 ml). The DNA-lipofectamine complex was carefully mixed with the A549 cells and allowed to react in a 37°C incubator for 6 hours. Afterwards, the culture medium was changed to search for transfected cells, and A549 cells transfected with PD-L1 were identified using a luciferase assay. This cell line was designated A549 pGL4.14-PD-L1 and the experiment was performed. IFNγ (50 ng / ml) was used as a PD-L1 inducer.

[0355] PD-L1 expression conditions were established using EGF and IFNγ, which are known to induce PD-L1 in Beas-2B cells (a human normal lung epithelial cell line). At the RNA level, no PD-L1 activation was observed when EGF was treated at concentrations of 25ng / ml, 50ng / ml, and 100ng / ml, while only IFNγ increased PD-L1. In addition, PD-L1 mRNA levels increased 2 hours after treatment with 50ng / ml IFNγ, and finally, the increase in mRNA levels was most pronounced 4 hours after treatment with 50ng / ml IFNγ.

[0356] Similarly, when examining PD-L1 protein levels in Beas-2B cells, we found that IFNγ treatment did not significantly change the cells until 8 hours later, but PD-L1 expression increased after 8 hours. Therefore, after 20 hours of treatment with 50 ng / ml IFNγ, the cells were lysed and used.

[0357] The A549pGL4.14-PD-L1 cell line was treated with the pyrimidopyrimidone derivatives of the examples at concentrations of 0.1 μM and 1.0 μM, respectively, and PD-L1 expression inhibition was evaluated using a photometer. For comparison, the same experiment was performed on GS9973 (Entospletinib), which is known to have PD-L1 expression inhibitory activity.

[0358] The inhibition rate of PD-L1 expression was calculated by the following equation 1.

[0359] [Equation 1]

[0360] PD-L1 expression inhibition rate (%) = 100-[(A / B)×100]

[0361] Here, A is the value when the drug is treated, and B is the value when the drug is not treated.

[0362] The results are shown in Table 1 below.

[0363] [Table 1]

[0364]

[0365]

[0366] As can be seen from Table 1, compared with GS9973 (Entospletinib), the pyrimidopyrimidone compound according to the present invention has excellent PD-L1 expression inhibitory activity, and this PD-L1 expression inhibitory activity is presented in a concentration-dependent manner.

[0367] Experimental Example 2: In vivo efficacy evaluation of PD-L1 expression inhibitors

[0368] Melanoma B16F10 cells were cultured at 1×10 6 A disease model was established by subcutaneously injecting the drug into the neck of 6-week-old male C57BL / 6 mice at a concentration of 10 cells / 100 μl. Following establishment of the disease model, the drug was administered orally once daily for 14 days, as shown in Table 2 below. Tumors were harvested on day 15 of the experiment. Changes in tumor volume and size were measured.

[0369] The results are as follows Figure 1 and Figure 2 As shown in .

[0370] [Table 2]

[0371]

[0372]

[0373] from Figures 1 to 2 As can be seen from the results, compounds I-2, I-3, I-8 and I-9 have tumor growth inhibition abilities ranging from 22% to 59% compared to the control group.

[0374] Experimental Example 3: Western blotting analysis

[0375] Experimental Example 3-1:

[0376] After conducting an in vivo tumor inhibition study using Compound I-8 from Experimental Example 2, Western blotting analysis confirmed the PD-L1 expression inhibitory effect in tumor cells extracted from mice. For comparison, Western blotting analysis also confirmed the efficacy of inhibiting PD-L1 expression in tumor cells extracted from control mice.

[0377] Chemiluminescence (ECL) kit (elpis biotec, EBP-1073) was used with ChemiDoc TM Proteins were identified using an imaging system (Bio-rad). Figure 3 As shown in .

[0378] from Figure 3 As can be seen from the data, when compound I-8 was administered, the expression of PD-L1 was reduced compared with the control group.

[0379] Experimental Example 3-2:

[0380] After treating PD-L1-overexpressing cells with the example compounds, the PD-L1 expression inhibitory effect was confirmed by Western immunoblotting analysis.

[0381] In order to investigate the changes in PD-L1 protein levels, 6×10 5 NCI-H358 (NSCLC) cell line was inoculated with 10 cells / ml. After 24 hours, NCI-H358 (NSCLC) cell line was treated with IFN-γ (20 ng / ml) to overexpress PD-L1, and 24 hours later, the compound was additionally treated at 1 μM and 10 μM, respectively. After 24 hours, the cells were analyzed using a chemiluminescence (ECL) kit (elpisbiotec, EBP-1073) with ChemiDoc TM Proteins were identified using an imaging system (Bio-rad). The results of Western blotting analysis were Figure 4 Shown in.

[0382] from Figure 4 It can be confirmed that the example compounds exhibited an inhibitory effect on PD-L1 expression in a concentration-dependent manner compared with the control group not treated with the drug.

Claims

1. A pyrimidopyrimidone compound of formula (I) or a pharmaceutically acceptable salt thereof: in, R1 is unsubstituted phenyl or phenyl substituted by halogen or hydroxy; R2 is a C1-C4 alkyl group; R3 is hydrogen, halogen, C1-C4 alkyl or C1-C4 alkoxy; and R4 is unsubstituted phenyl, C1-C4 alkyl, C1-C4 haloalkyl or alkyldihydrogen phenyl substituted with oxazolyl, or alkylthiazolyl.

2. The pyrimidopyrimidone compound according to claim 1 or a pharmaceutically acceptable salt thereof, in, R1 is phenyl, fluorophenyl or hydroxyphenyl; R2 is methyl; R3 is hydrogen, chloro, bromo, methyl or methoxy; and R4 is trifluoromethylphenyl, tert-butylphenyl, dimethyldihydro oxazolylphenyl or tert-butylthiazolyl.

3. The pyrimidopyrimidone compound or a pharmaceutically acceptable salt thereof according to claim 1, selected from the group consisting of: N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-1); 3-(tert-Butyl)-N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-2); N-(6-methoxy-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-3); 4-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-4); 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methyl-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-5); 3-(tert-Butyl)-N-(6-methoxy-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-6); 3-(4,4-dimethyl-4,5-dihydro oxazol-2-yl)-N-(6-methoxy-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-7); N-(6-chloro-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-8); 3-(tert-Butyl)-N-(6-chloro-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (I-9); 2-(tert-butyl)-N-(6-chloro-5-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)thiazole-4-carboxamide (I-10); and N-(6-chloro-5-(7-((4-hydroxyphenyl)amino)-1-methyl-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (I-11).

4. A pharmaceutical composition for inhibiting programmed death-ligand 1 (PD-L1), comprising the pyrimidopyrimidone compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 4 , which is used for treating or preventing cancer.

6. The pharmaceutical composition according to claim 5, which is used for treating or preventing colon cancer, lung cancer, breast cancer, stomach cancer, cervical cancer, bladder cancer, blood cancer or non-Hodgkin's lymphoma.

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