PYRIMIDOPYRIMIDINONE COMPOUNDS AND PHARMACEUTICALS CONTAINING THESE COMPOUNDS

Abstract

The invention relates to a pyrimido pyrimidinon compound that inhibits PD-L1 expression and a pharmaceutical composition containing this compound in active form. The pyrimido pyrimidinon compound can be used effectively to treat or prevent cancer.

Description

Pyrimidopyrimidinone compound and pharmaceutical composition containing the same

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

[0002] PD-1 (Programmed death-1, CD279) is a receptor protein on T cells, which is known to inhibit activation signals from the T cell receptor when its ligands PD-L1 (Programmed death-Ligand 1, CD274) or PD-L2 (Programmed death-Ligand 2, CD273) bind to it. That is, when the PD-1 receptor protein on T cells binds to its ligands PD-L1 / 2, the functional activities of T cells, including T cell proliferation, cytokine secretion, and cytolytic activity, are reduced, thereby protecting normal cells. The interaction between PD-1 and PD-L1 / 2 downregulates immune responses in infections, tumors, or autoimmune diseases (Keir M E, Butte M J, Freeman G J, et al. PD-1 and its ligands in tolerance and immunity. Annu. Rev. Immunol. 2008; 26).

[0003] Blockade of PD-1 / PD-L1 interactions using antibodies to PD-1 and PD-L1 has been shown to restore and enhance T cell activation in many systems. Combined PD-1 / PD-L1 blockade using monoclonal antibodies is known for treating cancers such as melanoma, non-small cell lung cancer, and renal cell carcinoma.

[0004] In a study on the regulation of PD-L1 expression and immune-oncological responses in cancer cells by targeting αvβ3-integrin, it was revealed that transplantation of β3-integrin-depleted tumor cells drastically reduced the growth of primary tumors, and this result was due to a decrease in PD-L1 expression, suggesting that inhibition of β3-integrin can increase the efficacy of immune-oncological agents for melanoma (Proc Natl Acad Sci USA., 2019 Oct 1, 116(40), 20141-20150). It is known that retinoblastoma protein RB, known as a tumor suppressor, exhibits anticancer efficacy by suppressing PD-L1 expression through the inhibition of NF-kB activation. In addition, curcumin and apigenin exhibited growth inhibitory effects on melanoma cells in a melanoma model, and in particular, apigenin was found to significantly suppress PD-L1 expression induced by IFN-γ. In addition, it is known that the expression of PD-L1 induced by IFN-γ is reduced by 40 to 80% in the lung cancer cell line A549 when pretreated with EGCG and green tea extract (GTE). Meanwhile, in a library screening study of kinase inhibitors that can increase the immune anticancer effect targeting melanoma, regorafenib was identified as the drug with the most potent effect among about 20 drugs that reduced the level of cell surface PD-L1 expression by more than 50%. The drug is known to strongly promote antitumor efficacy when combined with IFN-γ or immune checkpoint blockers (ICBs) in cells and in vivo, and to strongly inhibit JAK1 / 2-STAT1 and MAPK signaling, followed by a decrease in IFN-γ-induced PD-L1 and IDO1 expression (Clin Cancer Res., 2019 Jul 15, 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 activities, but there is no mention of the effect of these derivatives on PD-L1. In addition, 3-phenyl-dihydropyrimido[4,5-d]pyrimidinone derivatives have an inhibitory effect on Src kinase of 1 to 5 nM (IC 50 ) is known to exhibit inhibitory activity, but its effect on PD-L1 is not mentioned (International Patent Publication No. WO 2005 / 011597).

[0006] Therefore, there is a need for the development of compounds that have PD-L1 expression inhibitory activity for anti-tumor efficacy.

[0007] One object of the present invention is to provide a compound of the following formula I or a pharmaceutically acceptable salt thereof, which exhibits an effect of inhibiting PD-L1 expression.

[0008] Another object of the present invention is to provide a pharmaceutical composition comprising a compound of the above formula I or a pharmaceutically acceptable salt thereof.

[0009] One embodiment of the present invention relates to a pyrimido pyrimidinone compound of the following chemical formula I or a pharmaceutically acceptable salt thereof.

[0010] [Chemical Formula I]

[0011]

[0012] In the above formula,

[0013] R1 is C5-C 15 Aryl group of C5-C 15 Heteroaryl group of C3-C 10Cycloalkyl group or C3-C 10 is a heterocycloalkyl group,

[0014] R2 is hydrogen, a C1-C4 alkyl group, or a C1-C4 alkoxy group,

[0015] R3 is hydrogen, halogen, C1-C4 alkyl group or C1-C4 alkoxy group,

[0016] R4 is a C1-C4 alkyl group, a C1-C4 haloalkyl group, or a C5-C 15 C5-C substituted or unsubstituted with a heteroaryl group 15 Aryl group or C5-C 15 is a heteroaryl group.

[0017]

[0018] C5-C as used herein 15 The aryl group includes both aromatic groups and their partially reduced derivatives. The aromatic group is a simple or fused ring having 5 to 15 carbon atoms. Representative examples of aryl groups include, but are not limited to, phenyl, naphthyl, and tetrahydronaphthyl.

[0019] C5-C as used herein 15 The heteroaryl group includes both heteroaromatic groups and partially reduced derivatives thereof. The heteroaromatic group is a simple or fused ring composed of 5 to 15 carbon atoms and contains at least one oxygen, sulfur, or nitrogen. Representative examples of heteroaryl groups include, but are not limited to, pyridinyl, furanyl, thiophenyl, indolyl, quinolinyl, imidazolinyl, oxazolyl, dihydrooxazolyl, and thiazolyl.

[0020] C3-C as used herein 10The cycloalkyl group refers to a simple or fused cyclic hydrocarbon having 3 to 10 carbon atoms, and examples thereof include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0021] C3-C as used herein 10 The heterocycloalkyl group refers to a functional group in which at least one of the ring carbons of a simple or fused cyclic hydrocarbon composed of 3 to 10 carbon atoms is substituted with oxygen, sulfur or nitrogen, and examples thereof include, but are not limited to, thiazolidinyl and oxiranyl.

[0022] As used herein, the C1-C4 alkyl group means a straight-chain or branched monovalent hydrocarbon having 1 to 4 carbon atoms, and includes, but is not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, etc.

[0023] The C1-C4 alkoxy group used in this specification means a straight-chain or branched alkoxy group consisting of 1 to 4 carbon atoms, and includes, but is not limited to, methoxy, ethoxy, n-propanoxy, etc.

[0024] As used herein, the C1-C4 haloalkyl group means a straight-chain or branched hydrocarbon having 1 to 4 carbon atoms substituted with one or more halogens selected from the group consisting of fluorine, chlorine, bromine, and iodine, and examples thereof include, but are not limited to, trifluoromethyl, trichloromethyl, and trifluoroethyl.

[0025]

[0026] In one embodiment of the present invention, the compound of formula I is

[0027] R1 is phenyl which is optionally substituted with halogen or hydroxy,

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

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

[0030] R4 is a C1-C4 alkyl group, a C1-C4 haloalkyl group, phenyl substituted or unsubstituted with alkyldihydrooxazolyl, or alkylthiazolyl.

[0031]

[0032] As used herein, alkyldihydrooxazolyl refers to a dihydrooxazole group substituted with one or more C1-C4 alkyl groups, and includes, but is not limited to, dimethyldihydrooxazolyl.

[0033] As used herein, alkylthiazolyl refers to a thiazolyl group substituted with one or more C1-C4 alkyl groups, and includes, but is not limited to, t-butylthiazolyl.

[0034]

[0035] In one embodiment of the present invention, the compound of formula I is

[0036] R1 is phenyl, fluorophenyl or hydroxyphenyl,

[0037] R2 is methyl,

[0038] R3 is hydrogen, chloro, bromo, methyl or methoxy,

[0039] R4 is trifluoromethylphenyl, t-butylphenyl, dimethyldihydrooxazolylphenyl or t-butylthiazolyl.

[0040]

[0041] The pharmaceutically acceptable salts herein may include both non-toxic inorganic and organic acid salts, such as hydrochloride, phosphate, sulfate, nitrate, tartrate, methanesulfonate, p-toluenesulfonate, acetate, trifluoroacetate, citrate, maleate, succinate, oxalate, benzoate, tartrate, fumarate, mandelate, propionate, citrate, lactate, glycolate, gluconate, galacturonate, glutamate, glutarate, glucuronate, aspartate, ascorbate, carbonate, vanillate, hydroiodide, malate, malonate, and the like.

[0042]

[0043] Representative compounds among the compounds of the present invention are selected from the following groups.

[0044] 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);

[0045] 3-(t-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);

[0046] 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);

[0047] 4-(4,4-dimethyl-4,5-dihydrooxazol-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);

[0048] 3-(4,4-dimethyl-4,5-dihydrooxazol-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);

[0049] 3-(t-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);

[0050] 3-(4,4-dimethyl-4,5-dihydrooxazol-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);

[0051] 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);

[0052] 3-(t-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);

[0053] 2-(t-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

[0054] 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).

[0055]

[0056] The method for preparing the compound of chemical formula I of the present invention is shown in the following reaction scheme 1. The method described in the following reaction scheme is merely an example of a method typically used in the present invention, and the order of unit operations, reaction reagents, reaction conditions, etc. may be changed at any time depending on the case.

[0057] [Reaction Formula 1]

[0058]

[0059] In the above reaction formula 1, R1, R2, R3 and R4 are as defined in the above chemical formula I, and R5 is halogen or oxo (=O).

[0060] In one embodiment of the present invention, R5 is chloro, bromine, iodine or oxo(=O), particularly chloro or oxo(=O).

[0061]

[0062] The above reaction scheme 1 shows a four-step manufacturing process for manufacturing a compound of chemical formula I using a compound of chemical formula II and a compound of chemical formula III as starting materials.

[0063] In the first step, the compound of chemical formula II and the compound of chemical formula III are reacted to obtain the compound of chemical formula IV.

[0064] At this time, when R5 is halogen, an amination reaction is performed in the presence of an organic base to obtain a compound of chemical formula IV, and when R5 is oxo (=O), a reductive amination reaction is performed by adding a reducing agent to obtain a compound of chemical formula IV. As the organic base, triethylamine, N,N-diisopropylethylamine (DIPEA), etc. can be used, and as the reducing agent, sodium borohydride (sodium borohydride, NaBH4), sodium cyanoborohydride (NaCNBH3), sodium triacetoxyborohydride (Na(CH3COO)3BH), etc. can be used.

[0065] Acetonitrile, tetrahydrofuran, 1,4-dioxane, chloroform, dichloromethane, 1,2-dichloroethane, dimethylformamide, dimethylacetamide, methyl alcohol, ethyl alcohol, etc. can be used as the above reaction solvent, and the reaction temperature is preferably -10 to 25°C.

[0066] In the second step, triphosgene is added to the compound of the above chemical formula IV to undergo a heterocyclic urea formation reaction, thereby obtaining a compound of the above chemical formula V. At this time, the heterocyclic urea formation reaction can be performed in the presence of an organic base. Examples of the organic base that can be used include triethylamine, N,N-diisopropylethylamine (DIPEA), and the like.

[0067] Acetonitrile, tetrahydrofuran, 1,4-dioxane, etc. can be used as the above reaction solvent, and the reaction temperature is preferably -10 to 100°C.

[0068] In the third step, the sulfide group of the compound of the above chemical formula V is oxidized to obtain a sulfone compound of the chemical formula VI having a sulfone group.

[0069] The above oxidation reaction can be performed using oxone, meta-chloroperoxybenzoic acid (mCPBA), etc. as an oxidizing agent.

[0070] Dichloromethane, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, 1,4-dioxane, water, etc. can be used as the above reaction solvent, and the reaction temperature is preferably -10 to 100°C.

[0071] In the fourth step, the compound of the above chemical formula VI is subjected to a substitution reaction with the amine compound of the chemical formula VII to obtain the compound of the chemical formula I.

[0072] Acetonitrile, tetrahydrofuran, 1,4-dioxane, chloroform, dichloromethane, 1,2-dichloroethane, ethyl acetate, dimethylformamide, methyl alcohol, ethyl alcohol, etc. can be used as the above reaction solvent, and when necessary during the substitution reaction, an organic base or an inorganic base such as triethylamine, N,N-diisopropylethylamine, pyridine can be used together, or an organic acid such as trifluoroacetic acid can be used together, and the reaction temperature is 50 to 150°C, more preferably 80°C to 100°C.

[0073]

[0074] The compounds of the above chemical formula II and the compounds of the above chemical formula III can be manufactured and used according to a known method or can be obtained and used as commercially available products.

[0075] For example, the preparation methods of the compounds of the above chemical formula II and the compounds of the above chemical formula III are respectively shown in the following reaction schemes 2 to 4. The methods described in the reaction schemes below are merely examples of methods typically used in the present invention, and the order of unit operations, reaction reagents, reaction conditions, etc. may be changed at any time depending on the case.

[0076] [Reaction Formula 2]

[0077]

[0078] In the above reaction formula 2, R2 is as defined in the above chemical formula I, and R5 is as defined in the above reaction formula 1.

[0079]

[0080] As shown in the above reaction scheme 2, the compound of the chemical formula II can be prepared by reacting ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate with an amine compound of the chemical formula XIII to obtain a compound of the chemical formula VIII, reducing the compound of the chemical formula VIII using a reducing agent such as lithium aluminum hydride (LiAlH4) to obtain a compound of the chemical formula IX, and then halogenating the alcohol group of the compound of the chemical formula IX using thionyl chloride (SOCl2) or the like or oxidizing it using manganese dioxide (MnO2) or the like to form an aldehyde.

[0081]

[0082] [Reaction Formula 3]

[0083]

[0084] In the above reaction formula 3, R3 and R4 are as defined in the above chemical formula I, and Y is halogen or hydroxy.

[0085] In one embodiment of the present invention, Y is chloro, bromine, iodine or hydroxy, particularly chloro or hydroxy.

[0086]

[0087] The compound of the above formula III can be prepared by reacting the compound of the formula X with the compound of the formula XI in the presence of an organic base such as DIPEA or a coupling agent to obtain the compound of the formula XII, as shown in the above reaction scheme 3, and reducing the nitro group of the compound of the formula XII to an amine group. At this time, the reduction reaction can be performed using a combination of potassium t-butoxide and bis(pinacolato)diboron, or using hydrogen gas under a palladium carbon catalyst.

[0088]

[0089] [Reaction Formula 4]

[0090]

[0091] In the above reaction formula 4, R3 and R4 are as defined in the above chemical formula I, and Y is as defined in the above reaction formula 3.

[0092]

[0093] 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 or coupling agent such as DIPEA, as illustrated in Scheme 4 above.

[0094]

[0095] The compound of the above formula I or a pharmaceutically acceptable salt thereof according to the present invention exhibits excellent PD-L1 expression inhibitory activity (Test Example 1).

[0096]

[0097] Accordingly, the present invention relates to a pharmaceutical composition for inhibiting programmed death-ligand 1 (PD-L1), comprising a compound of the above formula I or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier, and specifically to a pharmaceutical composition for treating or preventing cancer.

[0098] In one embodiment of the present invention, the compound of formula I or a pharmaceutically acceptable salt thereof induces an anticancer effect through activation of immune cells by inhibiting the expression of PD-L1, and thus can be usefully used in the treatment of colon cancer, lung cancer, breast cancer, stomach cancer, cervical cancer, bladder cancer, blood cancer, or non-Hodgkin's lymphoma.

[0099] The pharmaceutical composition according to the present invention can be administered orally (e.g., by ingestion or inhalation) or parenterally (e.g., by injection, deposition, implantation, suppository), and the injection can be, for example, intravenous, subcutaneous, intramuscular, or intraperitoneal. The pharmaceutical composition according to the present invention can be formulated into tablets, capsules, granules, fine subtilis, powders, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, sprays, etc., depending on the route of administration. The various forms of the pharmaceutical composition according to the present invention can be prepared by known techniques using pharmaceutically acceptable carriers commonly used in each formulation. Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrating agents, lubricants, preservatives, antioxidants, isotonic agents, buffers, film agents, sweeteners, solubilizers, bases, dispersants, wetting agents, suspending agents, stabilizers, colorants, and the like.

[0100] The pharmaceutical composition according to the present invention comprises about 0.01 to 95 wt% of the compound of the present invention or a pharmaceutically acceptable salt thereof, depending on the form of the pharmaceutical composition.

[0101] The specific dosage of the pharmaceutical composition of the present invention may vary depending on the type, weight, sex, severity of the disease, and the physician's judgment of the mammal, including the human being being treated. Preferably, in the case of oral administration, 0.01 to 50 mg of the active ingredient is administered per kg of body weight per day, and in the case of parenteral administration, 0.01 to 10 mg of the active ingredient is administered per kg of body weight per day. The total daily dosage may be administered all at once or in several divided doses, depending on the severity of the disease, the physician's judgment, and the like.

[0102] Since the compound of the present invention has an effect of inhibiting PD-L1 expression, it can be effectively used in a pharmaceutical composition for treating or preventing cancer.

[0103] Figure 1 is a graph showing the change in tumor volume over time after administration of an example compound to mice induced with cancer using B16F10 cells.

[0104] Figure 2 is a graph showing the weight of tumors extracted from mice after administration of an example compound to mice that had cancer induced by B16F10 cells.

[0105] Figure 3 shows the results of Western blot analysis to determine the degree of inhibition of PD-L1 expression in tumors extracted from mice after administration of the example compound to mice induced with B16F10 cells.

[0106] Figure 4 shows the results of measuring the PD-L1 expression inhibition efficacy by Western blot after treatment of PD-L1 overexpressing cells with the example compound.

[0107] Hereinafter, the present invention will be described in more detail by way of examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0108]

[0109] Manufacturing Example 1: Manufacturing of a compound of chemical formula II

[0110] Manufacturing Example 1-1: 5-(chloromethyl)-N-methyl-2-(methylthio)pyrimidin-4-amine (II-1)

[0111]

[0112] Ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate (10.00 g, 42.98 mmol) was dissolved in 100 mL of tetrahydrofuran, and triethylamine (7.37 mL, 52.86 mmol) and a 40% solution of methylamine (3.54 g, 45.55 mmol) were added, followed by stirring at 20–25°C for 16 h. After the reaction was complete, the precipitated salt was filtered, and the solvent was removed under reduced pressure. 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 obtain ethyl 4-(methylamino)-2-(methylthio)pyrimidine-5-carboxylate (VIII-1, 8.82 g, 90%).

[0113] 1 H NMR (400MHz, CDCl3): δ 1.37 (t, 3H), 2.55 (s, 3H), 3.09 (d, 3H), 4.33 (q, 2H), 8.18 (bs, 1H), 8.61 (s, 1H)

[0114] Ethyl 4-(methylamino)-2-(methylthio)pyrimidine-5-carboxylate VIII-1 (8.80 g, 38.72 mmol) obtained above 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 to 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 sequentially and slowly added dropwise to the reaction mixture, and the reaction was terminated and stirred for 1 hour. The formed white precipitate was filtered, and the filtrate was extracted twice with ethyl acetate, and the combined organic layers were extracted with brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain (4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methanol (IX-1, 6.00 g, 84%).

[0115] 1 H NMR (400MHz, CDCl3): δ 2.53 (s, 3H), 3.05 (d, 3H), 4.40 (s, 2H), 5.22 (m, 1H), 6.95 (br,1H), 7.96 (s, 1H)

[0116] 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, cooled to 0°C, thionyl chloride (5.59 mL, 77.19 mmol) was slowly added dropwise, and stirred at 70°C for 4 hours. The resulting white solid was filtered to obtain 7.20 g (100%) of the title compound.

[0117]

[0118] Manufacturing Example 1-2: 4-(methylamino)-2-(methylthio)pyrimidine-5-carbaldehyde (II-2)

[0119]

[0120] (4-(Methylamino)-2-(methylthio)pyrimidin-5-yl)methanol IX-1 (0.74 g, 4.00 mmol) was added 15 mL of dichloromethane, manganese dioxide (3.47 g, 39.95 mmol) was added, and the mixture was stirred at 20 to 25°C for 20 hours. After the reaction was completed, 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.

[0121] 1 H NMR (400 MHz, CDCl3): δ 9.70 (s, 1H), 8.56 (bs, 1H), 8.30 (s, 1H), 3.12 (d, 3H), 2.57 (s, 3H)

[0122] ES-MS m / z: 184.18 [M+H] +

[0123]

[0124] Manufacturing Example 2: Manufacturing of a compound of chemical formula III

[0125] Manufacturing Example 2-1: N-(5-amino-6-methylpyridin-3-yl)-3-(trifluoromethyl)benzamide (III-1)

[0126]

[0127] 6-Methyl-5-nitropyridin-3-amine X-1 (1.00 g, 6.53 mmol) was dissolved in 50 mL of dichloromethane and 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 the mixture was stirred at 20–25°C for 1 hour. After the reaction was completed, dichloromethane, distilled water, and brine were sequentially extracted. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated residue was purified by column chromatography to obtain N-(6-methyl-5-nitropyridin-3-yl)-3-(trifluoromethyl)benzamide XII-1 (1.76 g, 83%).

[0128] N-(6-methyl-5-nitropyridin-3-yl)-3-(trifluoromethyl)benzamide XII-1 (1.16 g, 3.57 mmol) obtained above was dissolved in 50 mL of methyl alcohol, 0.32 g of 10% palladium carbon was added, and the mixture was stirred for 3 hours under a hydrogen atmosphere. Upon completion of the reaction, the reaction mixture was filtered through celite, washed with methanol, and concentrated under reduced pressure to obtain 1.04 g (99%) of the title compound.

[0129] 1 H NMR (400MHz, CDCl3): δ 2.55 (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)

[0130]

[0131] Manufacturing Example 2-2: N-(5-amino-6-methylpyridin-3-yl)-3-(t-butyl)benzamide (III-2)

[0132]

[0133] 3-(t-butyl)benzoic acid (0.6 g, 3.37 mmol) was suspended in 20 mL of dichloromethane, and oxalyl chloride (0.58 mL, 6.73 mmol) and 1 to 2 drops of a catalytic amount of dimethylformamide were added. The mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solvent was concentrated under reduced pressure to obtain 3-(t-butyl)benzoyl chloride XI-2 (0.67 g, Quant) without purification.

[0134] 6-Methyl-5-nitropyridin-3-amine X-1 (0.90 g, 5.88 mmol) was dissolved in 50 mL of dichloromethane, and 3-(t-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 at 0°C, and the mixture was stirred at 20 to 25°C for 1 hour. After the reaction was completed, dichloromethane, distilled water, and brine were sequentially extracted. 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-(t-butyl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide XII-2 (1.39 g, 76%).

[0135] ES-MS m / z: 314.25 [M+H] +

[0136] 3-(t-butyl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide XII-2 (1.39 g, 4.44 mmol) obtained above was dissolved in 40 mL of methyl alcohol, 0.32 g of 10% palladium carbon was added, and the mixture was stirred for 3 hours under a hydrogen atmosphere. Upon completion of the reaction, the reaction mixture was filtered through celite, washed with methanol, and concentrated under reduced pressure to obtain the title compound III-2 (1.18 g, 94%).

[0137] 1 H NMR (400MHz, CDCl3): δ 1.34(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)

[0138] ES-MS m / z: 239.31 [M+H] +

[0139]

[0140] Manufacturing Example 2-3: N-(5-amino-6-methoxypyridin-3-yl)-3-(trifluoromethyl)benzamide (III-3)

[0141]

[0142] 2-Methoxy-3,5-dinitropyridine (0.55 g, 2.76 mmol) was dissolved in 20 mL of ethyl alcohol, 6N hydrochloric acid (4.00 mL) and iron (1.30 g) were added, and the mixture was heated and refluxed for 1 hour. After the reaction was completed, it was cooled to 20-25 °C, neutralized with 5 M sodium hydroxide solution, and the reaction mixture was filtered through Celite. 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 obtain 2-methoxypyridine-3,5-diamine X'-1 (0.21 g, 55%).

[0143] 1 H NMR (400MHz, CDCl3): δ 3.23 (s, 3H), 3.90 (br, 4H), 6.46 (s, 1H), 6.69 (s, 1H)

[0144] ES-MS m / z: 140.11 [M+H] +

[0145] 2-Methoxypyridine-3,5-diamine X'-1 (0.21 g, 1.51 mmol) obtained above was dissolved in 30 mL of acetone, cooled to 0 ℃, 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 concentrated, and sequential extraction was performed using ethyl acetate, distilled water, and brine. 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.

[0146] 1 H NMR (400MHz, CDCl3): δ 3.23 (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)

[0147] ES-MS m / z: 312.10 [M+H] +

[0148]

[0149] Manufacturing Example 2-4: N-(5-amino-6-methylpyridin-3-yl)-4-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)benzamide (III-4)

[0150]

[0151] 6-Methyl-5-nitropyridin-3-amine X-1 (1.00 g, 6.52 mmol) was dissolved in 50 mL of dichloromethane, cooled to 0 °C, N,N-diisopropylethylamine (3.41 mL, 19.59 mmol) and 4-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)benzoyl chloride XI-3 (1.55 g, 6.52 mmol) were added, and stirred for 2 hours. After the reaction was completed, extraction was performed sequentially using dichloromethane, distilled 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 4-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide XII-6 (1.99 g, 86%).

[0152] ES-MS m / z: 355.25 [M+H] +

[0153] 4-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide XII-6 (1.95 g, 5.50 mmol) obtained above was dissolved in 50 mL of methyl alcohol, 0.30 g of 10% palladium carbon was added, and the mixture was stirred for 5 hours under a hydrogen atmosphere. Thereafter, 1.64 g (92%) of the title compound was obtained in the same manner as in Manufacturing Example 2-1.

[0154] ES-MS m / z: 325.25 [M+H] +

[0155]

[0156] Manufacturing Example 2-5: N-(5-amino-6-methylpyridin-3-yl)-3-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)benzamide (III-5)

[0157]

[0158] 6-Methyl-5-nitropyridin-3-amine X-1 (1.00 g, 6.52 mmol) was dissolved in 60 mL of tetrahydrofuran, cooled to 0 °C, N,N-diisopropylethylamine (3.41 mL, 19.59 mmol) and 3-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)benzoyl chloride XI-4 (1.55 g, 6.52 mmol) were added, and stirred for 2 hours. After the reaction was completed, extraction was performed sequentially using dichloromethane, distilled 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 3-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide XII-7 (1.39 g, 60%).

[0159] 3-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide XII-7 (1.39 g, 3.92 mmol) obtained above was dissolved in 30 mL of methyl alcohol, 0.30 g of 10% palladium carbon was added, and the mixture was stirred for 3 hours under a hydrogen atmosphere. Thereafter, 1.17 g (92%) of the title compound was obtained in the same manner as in Manufacturing Example 2-1.

[0160] ES-MS m / z: 325.16 [M+H] +

[0161]

[0162] Manufacturing Example 2-6: N-(5-amino-6-methoxypyridin-3-yl)-3-(t-butyl)benzamide (III-6)

[0163]

[0164] 2-Methoxypyridine-3,5-diamine X'-1 (0.44 g, 3.18 mmol) was dissolved in 40 mL of acetone, cooled to 0 ℃, triethylamine (0.48 g, 4.77 mmol) and 3-(t-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 concentrated, and sequential extraction was performed with ethyl acetate, distilled water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 0.34 g (45%) of the title compound.

[0165] ES-MS m / z: 300.28 [M+H] +

[0166]

[0167] Manufacturing Example 2-7: N-(5-amino-6-methoxypyridin-3-yl)-3-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)benzamide (III-7)

[0168]

[0169] 2-Methoxypyridine-3,5-diamine X'-1 (0.40 g, 2.87 mmol) was dissolved in 40 mL of acetone, cooled to 0 ℃, triethylamine (0.44 g, 4.31 mmol) and 3-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)benzoyl chloride XI-4 (0.55 g, 2.30 mmol) were added, and stirred for 2 hours. After the reaction was completed, the solvent was concentrated, and sequential extraction was performed with ethyl acetate, distilled water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 0.34 g (43%) of the title compound.

[0170] ES-MS m / z: 341.36 [M+H] +

[0171]

[0172] Manufacturing Example 2-8: N-(5-amino-6-chloropyridin-3-yl)-3-(trifluoromethyl)benzamide trifluoroacetate (III-8)

[0173]

[0174] t-Butyl (5-amino-2-chloropyridin-3-yl)carbamate (1.20 g, 4.92 mmol) was dissolved in 60 mL of dichloromethane, cooled to 0 °C, N,N-diisopropylethylamine (2.51 mL, 14.77 mmol) and 3-(trifluoromethyl)benzoyl chloride XI-1 (1.13 g, 5.42 mmol) were added, and stirred at 20-25 °C for 4 hours. After the reaction was completed, extraction was performed sequentially using dichloromethane, distilled water, and saturated sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 1.59 g (78%) of t-butyl (2-chloro-5-(3-(trifluoromethyl)benzamido)pyridin-3-ylcarbamate.

[0175] 1 H NMR (400MHz, CDCl3): δ 1.53 (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).

[0176] 1.50 g (3.61 mmol) of t-butyl (2-chloro-5-(3-(trifluoromethyl)benzamido)pyridin-3-ylcarbamate obtained above was dissolved in dichloromethane:trifluoroacetic acid (1:1) and stirred at 20 to 25°C for 5 hours. After completion of the reaction, the reaction solvent was concentrated under reduced pressure to obtain 1.10 g (71%) of the title compound trifluoroacetate.

[0177] 1H NMR (400MHz, DMSO-d6): δ 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).

[0178]

[0179] Manufacturing Example 2-9: N-(5-amino-6-chloropyridin-3-yl)-3-(t-butyl)benzamide trifluoroacetate (III-9)

[0180]

[0181] t-Butyl (5-amino-2-chloropyridin-3-yl)carbamate (0.84 g, 3.45 mmol) was dissolved in 50 mL of dichloromethane, cooled to 0 °C, N,N-diisopropylethylamine (1.76 mL, 10.34 mmol) and 3-(t-butyl)benzoyl chloride XI-2 (0.68 g, 3.45 mmol) were added, and stirred at 20 to 25 °C for 5 hours. Thereafter, t-butyl (5-(3-(t-butyl)benzamido)-2-chloropyridin-3-yl)carbamate (0.90 g, 65%) was obtained using the same method as in Preparation Example 2-14.

[0182] The t-butyl (5-(3-(t-butyl)benzamido)-2-chloropyridin-3-yl)carbamate (0.88 g, 2.18 mmol) obtained above was dissolved in dichloromethane:trifluoroacetic acid (1:1), and 0.88 g of the title compound trifluoroacetate was quantitatively obtained using the same method as in Manufacturing Example 2-14.

[0183] 1 H NMR (400MHz, DMSO-d6): δ 1.38 (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).

[0184]

[0185] Manufacturing Example 2-10: N-(5-amino-6-chloropyridin-3-yl)-2-(t-butyl)thiazole-4-carboxamide trifluoroacetate (III-10)

[0186]

[0187] t-Butyl (5-amino-2-chloropyridin-3-yl)carbamate (0.84 g, 3.45 mmol) was dissolved in 50 mL of dichloromethane, cooled to 0 °C, N,N-diisopropylethylamine (1.76 mL, 10.34 mmol) and 2-(t-butyl)thiazole-4-carbonyl chloride XI-5 (0.70 g, 3.45 mmol) were added, and stirred at 20 to 25 °C for 5 hours. t-Butyl (5-(2-(t-butyl)thiazole-4-carboxamido)-2-chloropyridin-3-yl)carbamate (0.94 g, 67%) was obtained using the same method as in Preparation Example 2-14.

[0188] The t-butyl (5-(2-(t-butyl)thiazole-4-carboxamido)-2-chloropyridin-3-yl)carbamate (0.92 g, 2.24 mmol) obtained above was dissolved in dichloromethane:trifluoroacetic acid (1:1), and 0.92 g of the title compound trifluoroacetate was quantitatively obtained using the same method as in Manufacturing Example 2-14.

[0189]

[0190] Manufacturing Example 3: Manufacturing of a compound of chemical formula IV

[0191] Manufacturing Example 3-1: N-(6-methyl-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)-3-(trifluoromethyl)benzamide (IV-1)

[0192]

[0193] Compound III-1 (0.60 g, 2.03 mmol) prepared in Preparation Example 2-1 was dissolved in 50 mL of acetonitrile, and N,N-diisopropylethylamine (1.06 mL, 6.10 mmol) was added at 0°C. Compound II-1 (0.40 g, 1.63 mmol) prepared in Preparation Example 1-1, dissolved in 30 mL of acetonitrile, was slowly added dropwise to the reaction solution, and the mixture was stirred at 20 to 25°C for 1 hour. The reaction solvent was concentrated under reduced pressure, and sequentially 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 0.70 g (70%) of the title compound.

[0194] 1 H NMR (400MHz, CDCl3): δ 2.55 (s, 3H), 3.05 (d, 3H), 4.16 (s, 2H), 5.57 (m, 1H), 6.88 (dd, 1H), 7.29 (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)

[0195] ES-MS m / z: 463.03 [M+H] +

[0196]

[0197] Manufacturing Example 3-2: 3-(t-butyl)-N-(6-methyl-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-2)

[0198]

[0199] Instead of compound III-1, compound III-2 (0.90 g, 3.18 mmol) prepared in Preparation Example 2-2 and compound II-1 (0.76 g, 3.16 mmol) prepared in Preparation Example 1-1 were used to obtain 0.80 g (56%) of the title compound in the same manner as in Preparation Example 3-1.

[0200] 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)

[0201] ES-MS m / z: 451.14 [M+H] +

[0202]

[0203] Manufacturing Example 3-3: 3-(t-butyl)-N-(6-methyl-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-2)

[0204]

[0205] 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 methyl alcohol, and acetic acid (0.88 mL, 15.28 mmol) and compound II-2 (1.12 g, 6.11 mmol) prepared in Preparation Example 1-2 were added, followed by stirring for 15 minutes. Sodium cyanoborohydride (2.30 g, 36.68 mmol) was added to the reaction solution, and 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 concentrated under reduced pressure, and extracted with ethyl acetate and a saturated sodium bicarbonate solution. 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.

[0206] ES-MS m / z: 451.14 [M+H] +

[0207]

[0208] Manufacturing Example 3-4: N-(4-methoxy-3-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)-3-(trifluoromethyl)benzamide (IV-3)

[0209]

[0210] Instead of compound III-1, compound III-3 (0.17 g, 0.69 mmol) prepared in Preparation Example 2-3 was used, and 0.10 g (30%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0211] 1 H NMR (400MHz, CDCl3): δ 2.55 (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)

[0212] ES-MS m / z: 527.97 [M+H] +

[0213]

[0214] Manufacturing Example 3-5: 4-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)-N-(6-methyl-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-4)

[0215]

[0216] Instead of compound III-1, compound III-4 (1.00 g, 3.08 mmol) prepared in Preparation Example 2-4 was used, and 0.45 g (30%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0217] ES-MS m / z: 492.54 [M+H] +

[0218]

[0219] Manufacturing Example 3-6: 3-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)-N-(6-methyl-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-5)

[0220]

[0221] Instead of compound III-1, compound III-5 (1.00 g, 3.08 mmol) prepared in Preparation Example 2-5 was used, and 0.40 g (30%) of the title compound was obtained in the same manner as in Preparation Example 3-1.

[0222] ES-MS m / z: 492.54 [M+H] +

[0223]

[0224] Manufacturing Example 3-7: 3-(t-butyl)-N-(6-methoxy-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-6)

[0225]

[0226] Instead of compound III-1, compound III-6 (0.34 g, 1.14 mmol) prepared in Preparation Example 2-6 was used in the same manner as Preparation Example 3-1 to obtain 0.18 g (51%) of the title compound.

[0227] ES-MS m / z: 467.61 [M+H] +

[0228]

[0229] Manufacturing Example 3-8: 3-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)-N-(6-methoxy-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-7)

[0230]

[0231] Instead of compound III-1, compound III-7 (0.34 g, 1.00 mmol) prepared in Preparation Example 2-7 was used in the same manner as Preparation Example 3-1 to obtain 0.15 g (45%) of the title compound.

[0232] ES-MS m / z: 508.55 [M+H] +

[0233]

[0234] Manufacturing Example 3-9: N-(6-chloro-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)-3-(trifluoromethyl)benzamide (IV-8)

[0235]

[0236] Instead of compound III-1, compound III-8 (1.15 g 1.99 mmol) prepared in Preparation Example 2-8 was used to obtain 0.26 g (41%) of the title compound in the same manner as in Preparation Example 3-1.

[0237]

[0238] Manufacturing Example 3-10: 3-(t-butyl)-N-(6-chloro-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)benzamide (IV-9)

[0239]

[0240] Instead of compound III-1, compound III-9 (1.17 g 2.91 mmol) prepared in Preparation Example 2-9 was used in the same manner as Preparation Example 3-1 to obtain 0.28 g (41%) of the title compound.

[0241]

[0242] Manufacturing Example 3-11: 2-(t-butyl)-N-(6-chloro-5-(((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methyl)amino)pyridin-3-yl)thiazole-4-carboxamide (IV-10)

[0243]

[0244] Instead of compound III-1, compound III-10 (0.67 g 3.33 mmol) prepared in Preparation Example 2-10 was used in the same manner as Preparation Example 3-1 to obtain 0.32 g (41%) of the title compound.

[0245] 1 H NMR (400MHz, CDCl3): δ 9.24 (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).

[0246]

[0247] Manufacturing Example 4: Manufacturing of a compound of chemical formula V

[0248] Manufacturing 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)

[0249]

[0250] Compound Ⅳ-1 (0.70 g, 1.51 mmol) prepared in Manufacturing Example 3-1 was dissolved in 60 mL of 1,4-dioxane, and then N,N-diisopropylethylamine (0.78 mL, 4.54 mmol) and triphosgene (0.63 g, 2.12 mmol) were added at 0°C, and the mixture was 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.

[0251] 1 H NMR (400MHz, CDCl3): δ 2.55 (s, 3H), 2.63 (s, 3H), 3.65 (d, 3H), 5.16 (s, 2H), 6.88 (dd, 1H), 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)

[0252] ES-MS m / z: 489.50 [M+H] +

[0253]

[0254] Manufacturing Example 4-2: 3-(t-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)

[0255]

[0256] Instead of compound IV-1, compound IV-2 (0.80 g, 1.78 mmol) prepared in Manufacturing Example 3-2 was used in the same manner as in Manufacturing Example 4-1, to obtain 0.50 g (60%) of the title compound.

[0257] 1 H NMR (400MHz, CDCl3): δ 1.34 (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)

[0258] ES-MS m / z: 477.60 [M+H] +

[0259]

[0260] Manufacturing 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)

[0261]

[0262] Instead of compound IV-1, compound IV-3 (0.10 g, 0.21 mmol) prepared in Manufacturing Example 3-4 was used, and 0.10 g (95%) of the title compound was obtained in the same manner as in Manufacturing Example 4-1.

[0263] 1 H NMR (400MHz, CDCl3): δ 2.55 (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)

[0264] ES-MS m / z: 505.09 [M+H] +

[0265]

[0266] Manufacturing Example 4-4: 4-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)-N-(6-methyl-5-(1-methyll-7-(methylthio)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)pyridin-3-yl)benzamide (V-4)

[0267]

[0268] Instead of compound IV-1, compound IV-4 (0.40 g, 0.81 mmol) prepared in Manufacturing Example 3-5 was used in the same manner as in Manufacturing Example 4-1, to obtain 0.32 g (76%) of the title compound.

[0269] ES-MS m / z: 518.09 [M+H] +

[0270]

[0271] Manufacturing Example 4-5: 3-(4,4-dimethyl-4,5-dihydrooxazol-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)

[0272]

[0273] Instead of compound IV-1, compound IV-5 (0.40 g, 0.81 mmol) prepared in Preparation Example 3-6 was used in the same manner as in Preparation Example 4-1, to obtain 0.35 g (83%) of the title compound.

[0274] ES-MS m / z: 518.09 [M+H] +

[0275]

[0276] Manufacturing Example 4-6: 3-(t-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)

[0277]

[0278] Instead of compound IV-1, compound IV-6 (0.18 g, 0.39 mmol) prepared in Manufacturing Example 3-7 was used in the same manner as in Manufacturing Example 4-1, to obtain 0.17 g (90%) of the title compound.

[0279] ES-MS m / z: 493.62 [M+H] +

[0280]

[0281] Manufacturing Example 4-7: 3-(4,4-dimethyl-4,5-dihydrooxazol-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)

[0282]

[0283] Instead of compound IV-1, compound IV-7 (0.15 g, 0.30 mmol) prepared in Preparation Example 3-8 was used in the same manner as in Preparation Example 4-1, to obtain 0.15 g (93%) of the title compound.

[0284] ES-MS m / z: 534.65 [M+H] +

[0285]

[0286] Manufacturing 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)

[0287]

[0288] Instead of compound Ⅳ-1, compound Ⅳ-8 (0.27 g, 0.56 mmol) prepared in Manufacturing Example 3-9 was used in the same manner as in Manufacturing Example 4-1, to obtain 0.27 g (96%) of the title compound.

[0289]

[0290] Manufacturing Example 4-9: 3-(t-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)

[0291]

[0292] Instead of compound IV-1, compound IV-9 (0.28 g, 0.59 mmol) prepared in Manufacturing Example 3-10 was used in the same manner as in Manufacturing Example 4-1, to obtain 0.23 g (78%) of the title compound.

[0293]

[0294] Manufacturing Example 4-10: 2-(t-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)

[0295]

[0296] Instead of compound Ⅳ-1, compound Ⅳ-10 (0.31 g, 0.65 mmol) prepared in Manufacturing Example 3-11 was used in the same manner as in Manufacturing Example 4-1, to obtain 0.25 g (77%) of the title compound.

[0297]

[0298] Manufacturing Example 5: Manufacturing of a compound of chemical formula VI

[0299] Manufacturing 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)

[0300]

[0301] Compound V-1 (0.37 g, 0.76 mmol) prepared in Manufacturing Example 4-1 was dissolved in tetrahydrofuran:distilled water (2:1, 30 mL), and oxone (4.66 g, 7.57 mmol), an oxidizing agent, was added, followed by stirring at 65°C for 5 hours. After completion of the reaction, the reaction solvent was concentrated under reduced pressure, washed sequentially with ethyl acetate, water, and brine, 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.

[0302] 1 H NMR (400MHz, CDCl3): δ 2.70 (s, 3H), 3.38 (s, 3H), 3.40 (s, 3H), 4.42 (d, 2H), 6.76 (m, 1H), 7.36 (m, 1H), 7.48 - 7.99 (m, 4H), 8.25 (m, 1H), 9.12(s, 1H)

[0303] ES-MS m / z: 521.60 [M+H] +

[0304]

[0305] Manufacturing Example 5-2: 3-(t-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)

[0306]

[0307] Instead of compound V-1, compound V-2 (0.51 g, 1.07 mmol) prepared in Manufacturing Example 4-2 was used in the same manner as in Manufacturing Example 5-1, to obtain 0.22 g (42%) of the title compound.

[0308] 1H NMR (400MHz, CDCl3): δ 1.34 (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)

[0309] ES-MS m / z: 509.72 [M+H] +

[0310]

[0311] Manufacturing 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)

[0312]

[0313] Instead of compound V-1, compound V-3 (0.09 g, 0.17 mmol) prepared in Manufacturing Example 4-3 was used, and 90 mg (97%) of the title compound was obtained in the same manner as in Manufacturing Example 5-1.

[0314] 1 H NMR (400 MHz, CDCl3): δ 3.10 (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)

[0315] ES-MS m / z: 537.01 [M+H] +

[0316]

[0317] Manufacturing Example 5-4: 4-(4,4-dimethyl-4,5-dihydrooxazol-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)

[0318]

[0319] Instead of compound V-1, compound V-4 (0.30 g, 0.58 mmol) prepared in Manufacturing Example 4-4 was used, and 0.24 g (76%) of the title compound was obtained in the same manner as in Manufacturing Example 5-1.

[0320] ES-MS m / z: 550.25 [M+H] +

[0321]

[0322] Manufacturing Example 5-5: 3-(4,4-dimethyl-4,5-dihydrooxazol-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)

[0323]

[0324] Instead of compound V-1, compound V-5 (0.35 g, 0.58 mmol) prepared in Manufacturing Example 4-5 was used, and 0.24 g (76%) of the title compound was obtained in the same manner as in Manufacturing Example 5-1.

[0325] ES-MS m / z: 550.21 [M+H] +

[0326]

[0327] Manufacturing Example 5-6: 3-(t-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)

[0328]

[0329] Instead of compound V-1, compound V-6 (0.17 g, 0.35 mmol) prepared in Manufacturing Example 4-6 was used in the same manner as in Manufacturing Example 5-1, to obtain 0.17 g (91%) of the title compound.

[0330] 1 H NMR (400MHz, CDCl3): δ 1.36 (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)

[0331] ES-MS m / z: 525.61 [M+H] +

[0332]

[0333] Manufacturing Example 5-7: 3-(4,4-dimethyl-4,5-dihydrooxazol-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)

[0334]

[0335] Instead of compound V-1, compound V-7 (0.15 g, 0.28 mmol) prepared in Manufacturing Example 4-7 was used in the same manner as in Manufacturing Example 5-1, to obtain 0.14 g (89%) of the title compound.

[0336] ES-MS m / z: 566.50 [M+H] +

[0337]

[0338] Manufacturing 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)

[0339]

[0340] Instead of compound V-1, compound V-8 (0.26 g, 0.51 mmol) prepared in Manufacturing Example 4-8 was used, and 0.21 g (77%) of the title compound was obtained in the same manner as in Manufacturing Example 5-1.

[0341]

[0342] Manufacturing Example 5-9: 3-(t-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)

[0343]

[0344] Instead of compound V-1, compound V-9 (0.23 g, 0.46 mmol) prepared in Manufacturing Example 4-9 was used, and 0.21 g (86%) of the title compound was obtained in the same manner as in Manufacturing Example 5-1.

[0345]

[0346] Manufacturing Example 5-10: 2-(t-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)

[0347]

[0348] Instead of compound V-1, compound V-10 (0.26 g, 0.52 mmol) prepared in Manufacturing Example 4-10 was used in the same manner as in Manufacturing Example 5-1, to obtain 0.19 g (67%) of the title compound.

[0349]

[0350] Example: Preparation of a compound of formula I

[0351] 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)

[0352]

[0353] Compound VI-1 (0.17 g, 0.33 mmol) prepared in Manufacturing Example 5-1 was dissolved in 10 ml of 1,4-dioxane, added to 4 ml of aniline, and stirred at 100°C for 3 hours. After completion of the reaction, the mixture was sequentially extracted with ethyl acetate, water, and brine, 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 87 mg (50%) of the title compound.

[0354] 1 H NMR (400MHz, DMSO-d6): δ 2.55 (s, 3H), 3.09 (s, 3H), 4.42 (d, 2H), 6.46-7.54 (m, 6H), 7.75 (m, 2H), 8.01 (m, 1H), 8.17 (m, 1H), 9.60(s, 1H), 10.36(s, 1H)

[0355] ES-MS m / z: 534.62 [M+H] +

[0356]

[0357] Example 2: 3-(t-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)

[0358]

[0359] 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.

[0360] 1H NMR (400MHz, DMSO-d6): δ 1.34 (s, 9H), 2.55 (s, 3H), 3.11 (s, 3H), 4.42 (d, 2H), 6.46-7.54 (m, 9H), 7.75 (m, 1H), 7.98 (m, 1H), 8.25 (m, 1H), 9.53(s, 1H), 10.35(s, 1H)

[0361] ES-MS m / z: 522.72 [M+H] +

[0362]

[0363] 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)

[0364]

[0365] 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.

[0366] 1 H NMR (400 MHz, DMSO-d6): 3.35 (s, 3H), δ 3.90 (s, 3H), 4.63 (s, 2H), 6.95 (t, 1H), 7.29 (t, 1H), 7.70 (d, 2H), 7.79 (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)

[0367] ES-MS m / z: 550.07 [M+H] +

[0368]

[0369] Example 4: 4-(4,4-dimethyl-4,5-dihydrooxazol-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)

[0370]

[0371] Instead of compound VI-1, compound VI-4 (0.20 g, 0.36 mmol) prepared in Preparation Example 5-4 was used to obtain 0.10 g (55%) of the title compound in the same manner as in Example 1.

[0372] 1 H NMR (400 MHz, DMSO-d6): δ 1.31 (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)

[0373] ES-MS m / z: 563.72 [M+H] +

[0374]

[0375] Example 5: 3-(4,4-dimethyl-4,5-dihydrooxazol-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)

[0376]

[0377] Instead of compound VI-1, compound VI-5 (0.23 g, 0.42 mmol) prepared in Preparation Example 5-5 was used in the same manner as in Example 1 to obtain 0.14 g (58%) of the title compound.

[0378] 1 H NMR (400 MHz, DMSO-d6): δ 1.41 (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)

[0379] ES-MS m / z: 563.62 [M+H] +

[0380]

[0381] Example 6: 3-(t-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)

[0382]

[0383] 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.

[0384] 1 H NMR (400MHz, DMSO-d6): δ 1.34 (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, 1H), 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)

[0385] ES-MS m / z: 538.64 [M+H] +

[0386]

[0387] Example 7: 3-(4,4-dimethyl-4,5-dihydrooxazol-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)

[0388]

[0389] 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 was obtained in the same manner as in Example 1.

[0390] 1 H NMR (400 MHz, DMSO-d6): δ 1.34 (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)

[0391] ES-MS m / z: 579.65 [M+H] +

[0392]

[0393] 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)

[0394]

[0395] Instead of compound Ⅵ-1, compound Ⅵ-8 (0.20 g, 0.37 mmol) prepared in Preparation Example 5-8 was used to obtain 92 mg (46%) of the title compound in the same manner as in Example 1.

[0396] 1 H NMR (400 MHz, DMSO-d6): δ 3.33 (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)

[0397] ES-MS m / z: 553.97 [M+H] +

[0398]

[0399] Example 9: 3-(t-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)

[0400]

[0401] Instead of compound Ⅵ-1, compound Ⅵ-9 (0.21 g, 0.40 mmol) prepared in Preparation Example 5-9 was used in the same manner as in Example 1 to obtain 98.3 mg (46%) of the title compound.

[0402] 1 H NMR (400MHz, DMSO-d6): δ 1.31 (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)

[0403] ES-MS m / z: 542.14 [M+H] +

[0404]

[0405] Example 10: 2-(t-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)

[0406]

[0407] 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.

[0408] 1 H NMR (400MHz, DMSO-d6): δ 1.47 (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)

[0409] ES-MS m / z: 549.16 [M+H] +

[0410]

[0411] 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)

[0412]

[0413] Instead of compound Ⅵ-1, compound Ⅵ-8 (1.00 g, 1.85 mmol) prepared in Preparation Example 5-8 was dissolved in 20 mL of 1,4-dioxane, 4-aminophenol (1.00 g, 9.24 mmol) and trifluoroacetic acid (0.71 mL, 9.24 mmol) were added, and the mixture was stirred at 85°C for 7 hours. After completion of the reaction, 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.

[0414] 1 H NMR (400MHz, DMSO-d6): δ 3.33 (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)

[0415] ES-MS m / z: 569.16 [M+H] +

[0416]

[0417] Test Example 1: Analysis of PD-L1 expression inhibition activity

[0418] The PD-L1 promoter gene region was inserted into the pGL4.14-[luc2 / Hygro] vector (promega, Madison, WI) as follows to prepare a promoter-reporter construct for measuring gene transcription activity.

[0419] After constructing primers including KpnI and XhoI restriction enzyme sites for PD-L1, polymerase chain reaction (PCR) was performed, and promoter region DNA was isolated by agarose gel electrophoresis. The pGL4.14-[luc2 / Hygro] vector containing firefly luciferase as a reporter gene was treated with the same restriction enzymes, and then isolated by agarose gel electrophoresis and gel extraction. The purely isolated PD-L1 promoter gene region and the pGL4.14-[luc2 / Hygro] vector were ligated with In-fusion DNA ligase (Takara) at 50°C for 1 hour, and then transformed into E. coli to produce a promoter-reporter construct with the PCSK9 promoter inserted, which was named pGL4.14-PD-L1.

[0420] To measure the transcriptional activity of the PD-L1 gene promoter, the PD-L1 promoter-reporter construct was transiently transfected into A549 cells as follows, and then the luciferase activity of the cell extract was measured.

[0421] 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 for the process of forming complexes between DNA and Lipofectamine 2000. A549 cells were seeded at 2.5 × 10 per well in 12-well plates by checking the number of cells just before adding DNA. 5A dog (1 ml) was prepared. The DNA-lipofectamine complex was carefully mixed with A549 cells and incubated at 37°C for 6 hours. Afterwards, the medium was changed to identify transfected cells. A549 cells transfected with PD-L1 were identified through a luciferase test. This cell line was named the A549 pGL4.14-PD-L1 cell line and experiments were conducted. IFNγ (50 ng / ml) was used as a PD-L1 inducer.

[0422] We established the expression conditions of PD-L1 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, PD-L1 activation was not observed when EGF was treated at concentrations of 25, 50, and 100 ng / ml, and only IFNγ was confirmed to increase PD-L1. In addition, the mRNA level of PD-L1 increased after 2 hours of treatment with 50 ng / ml of IFNγ, and the mRNA level was confirmed to increase most significantly after 4 hours of treatment with 50 ng / ml of IFNγ.

[0423] Similarly, when the protein level of PD-L1 was confirmed in Beas-2B cells, no significant change was observed before 8 hours when treated with IFNγ, but an increase in the expression level of PD-L1 was confirmed after 8 hours. Therefore, the protein was lysed and used 20 hours after treatment with 50 ng / ml of IFNγ.

[0424] The A549 pGL4.14-PD-L1 cell line was treated with the pyrimidopyrimidinone derivatives of the examples at concentrations of 0.1 μM and 1.0 μM, respectively, and the PD-L1 expression inhibition rate was evaluated using a luminometer. For comparison, the same experiment was performed with GS9973 (Entospletinib), which is known to have PD-L1 expression inhibitory activity.

[0425] The PD-L1 expression inhibition rate was calculated using the following mathematical formula 1.

[0426] [Mathematical Formula 1]

[0427] PD-L1 expression inhibition rate (%) = 100-[(A / B) Х 100]

[0428] In the above formula, A is the value when drug is treated, and B is the value when drug is not treated.

[0429]

[0430] The results are shown in Table 1 below.

[0431] PD-L1 expression inhibition rate in A549 (%) 0.1 μM 1.0 μMGS9973 (Entospletinib) 10.347.1I-112.887.1I-232.672.5I-329.392.5I-420.182.5I-519.481.9I-613.994.1I-713.185.3I-860.497.5I-948.897.2I-1067.098.0I-1165.098.0

[0432] Through the above Table 1, it can be confirmed that the pyrimido pyrimidinone compound according to the present invention has superior PD-L1 expression inhibition activity compared to GS9973 (Entospletinib), and that this PD-L1 expression inhibition activity appears in a concentration-dependent manner.

[0433]

[0434] Test Example 2: In vivo efficacy evaluation of PD-L1 expression inhibitors

[0435] Melanoma B16F10 cells were injected into 1 × 10 6-week-old male C57BL / 6 mice. 6 A disease model was created by subcutaneously injecting 100 μl of cells into the dorsal aspect of mice. The following day after the creation of the disease model was designated Day 1 of administration, and oral administration was performed once daily for 14 days as shown in Table 2 below. Tumors were excised on the 15th day of the test. Changes in tumor volume and tumor size were measured.

[0436] The results are shown in Figures 1 and 2.

[0437]

[0438] Number of animals by group and sex Animal number Dosage (mg / kg) G1 Normal control M81 ~ 8-G2I-8M89 ~ 1610 G3I-9M817 ~ 2410 G4I-2M825 ~ 3220 G5I-3M833 ~ 4020

[0439] Through Figures 1 and 2, it can be confirmed that compounds I-2, I-3, I-8, and I-9 have tumor growth inhibition ability of 22 to 59% compared to the control group.

[0440]

[0441] Test Example 3: Western Blot Analysis

[0442] Test Example 3-1:

[0443] After an in vivo tumor inhibition test using Compound I-8 of Test Example 2, the PD-L1 expression inhibition efficacy in tumor cells extracted from mice was confirmed through Western blot analysis. For comparison, the PD-L1 expression inhibition efficacy in tumor cells extracted from control mice was also confirmed through Western blot analysis.

[0444] Proteins were quantified using a chemiluminescence (ECL) kit (elpis biotec, EBP-1073) using ChemiDoc TM The results were confirmed using an imaging system (Bio-rad). The results are shown in Figure 3.

[0445] Through Figure 3, it can be confirmed that the expression of PD-L1 is reduced compared to the control group when compound I-8 is administered.

[0446]

[0447] Test Example 3-2:

[0448] The inhibitory effect of PD-L1 expression on PD-L1 overexpressing cells was confirmed through Western blot analysis after treatment with the example compound.

[0449] To observe changes in PD-L1 protein levels, 6×10 NCI-H358 (NSCLC) cell lines were cultured 5 cells / ml. After 24 hours, the NCI-H358 (NSCLC) cell line was treated with IFN-γ (20 ng / ml) to overexpress PD-L1, and then 24 hours later, the compound was additionally treated at 1 μM and 10 μM, respectively. After 24 hours, the protein was measured using a chemiluminescence (ECL) kit (elpis biotec, EBP-1073) using ChemiDoc TM It was confirmed using an imaging system (Bio-rad). The results of Western blot analysis are shown in Figure 4.

[0450] Through Figure 4, it can be confirmed that the example compounds exhibit a concentration-dependent PD-L1 expression inhibitory effect compared to the control group that was not treated with the drug.

Claims

1. A pyrimido pyrimidinone compound of the following chemical formula I or a pharmaceutically acceptable salt thereof: [Chemical Formula I] In the above formula, R 1 Silver C 5 -C 15 Aryl group of C 5 -C 15 Heteroaryl group of C 3 -C 10 Cycloalkyl group of or C 3 -C 10 is a heterocycloalkyl group, R 2 is hydrogen, C 1 -C 4 Alkyl group of or C 1 -C 4 is an alkoxy group, R 3 is hydrogen, halogen, C 1 -C 4 Alkyl group of or C 1 -C 4 is an alkoxy group, R 4 is C 1 -C 4 Alkyl group of C 1 -C 4 Haloalkyl group or C 5 -C 15 C substituted or unsubstituted with a heteroaryl group 5 -C 15 Aryl group of or C 5 -C 15 is a heteroaryl group.

2. In paragraph 1, R 1 is phenyl, which is optionally substituted with halogen or hydroxyl, R 2 is C 1 -C 4 is an alkyl group, R 3 is hydrogen, halogen, C 1 -C 4 Alkyl group of or C 1 -C 4 is an alkoxy group, R 4 is C 1 -C 4 Alkyl group of C 1 -C 4 A pyrimido pyrimidinone compound, or a pharmaceutically acceptable salt thereof, which is a phenyl substituted or unsubstituted with a haloalkyl group, an alkyldihydrooxazolyl, or an alkylthiazolyl.

3. In paragraph 1, R 1 is phenyl, fluorophenyl or hydroxyphenyl, R 2 is methyl, R 3 is hydrogen, chloro, bromo, methyl or methoxy, R 4 A pyrimido pyrimidinone compound or a pharmaceutically acceptable salt thereof, wherein the pyrimido pyrimidinone compound is trifluoromethylphenyl, t-butylphenyl, dimethyldihydrooxazolylphenyl or t-butylthiazolyl.

4. In paragraph 1, a pyrimido pyrimidinone compound selected from the following compounds or a pharmaceutically acceptable salt thereof: 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-(t-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-dihydrooxazol-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-dihydrooxazol-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-(t-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-dihydrooxazol-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-(t-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-(t-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).

5. A pharmaceutical composition for inhibiting programmed death-Ligand 1 (PD-L1), comprising a pyrimido pyrimidinone compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition which is an anticancer agent in paragraph 5.

7. A pharmaceutical composition for the treatment or prevention of colon cancer, lung cancer, breast cancer, stomach cancer, cervical cancer, bladder cancer, blood cancer or non-Hodgkin's lymphoma in clause 6.