Pyrimidopyrimidinone compounds and pharmaceutical compositions containing the same

Abstract

The present invention relates to pyrimidopyrimidinone compounds that have the effect of suppressing PD-L1 expression and pharmaceutical compositions containing the same as active ingredients. The pyrimidopyrimidinone compounds of the present invention can be effectively used in the treatment or prevention of cancer.

Description

Detailed Description of the Invention

[0001] [Technical Field] The present invention relates to pyrimidopyrimidinone compounds and pharmaceutical compositions containing the same, and more particularly to pyrimidopyrimidinone compounds that have the effect of inhibiting the expression of PD-L1 and pharmaceutical compositions containing the same as an active ingredient.

[0002] [Background technology] PD-1 (Programmed death-1, CD279) is a receptor protein on T cells that suppresses 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. Specifically, when the PD-1 receptor protein on T cells binds to its ligands, PD-L1 / 2, functional activities of T cells, including 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, and autoimmune diseases (Keir ME, Butte MJ, Freeman GJ, 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 against PD-1 and PD-L1 has been shown to restore and enhance T cell activation in many systems, including the use of monoclonal antibodies to inhibit PD-1 / PD-L1 binding in patients with cancers such as melanoma, non-small cell lung cancer, and renal cancer.

[0004] In a study targeting αvβ3 integrin to regulate PD-L1 expression in cancer cells and immune anti-cancer responses, it was found that transplantation of β3 integrin-depleted tumor cells significantly reduced primary tumor growth, a result attributed to reduced PD-L1 expression, suggesting that β3 integrin blockade can enhance the efficacy of immune anti-cancer drugs in melanoma (Proc Natl Acad Sci USA., 2019 Oct 1, 116(40), 20141-20150). The retinoblastoma protein RB, a known tumor suppressor, is known to exert its anti-cancer effects by inhibiting NF-kB activity and suppressing PD-L1 expression. Furthermore, in a melanoma model, curcumin and apigenin were shown to inhibit melanoma cell growth, and apigenin in particular significantly suppressed IFN-γ-induced PD-L1 expression. Furthermore, pretreatment of the lung cancer cell line A549 with EGCG and green tea extract (GTE) has been shown to reduce IFN-γ-induced PD-L1 expression by 40-80%. Meanwhile, in a screening study of a kinase inhibitor library that could enhance immune anti-cancer effects in melanoma, regorafenib was identified as the most potent drug among approximately 20 drugs that reduced cell surface PD-L1 expression by more than 50%. When combined with IFN-γ or immune checkpoint inhibitors (ICBs) in cells and in vivo, this drug potently promoted antitumor efficacy and strongly inhibited JAK1 / 2-STAT1 and MAPK signaling, further reducing IFN-γ-induced PD-L1 and IDO1 expression (Clin Cancer Res., 2019 Jul 15, 25(14), 4530-4541).

[0005] 3,4-Dihydropyrimido[4,5-d]pyrimidin-2-one derivatives as protein kinase inhibitors are known to inhibit the kinase activities 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, but there is no mention of the effects of these derivatives on PD-L1. Furthermore, 3-phenyl-dihydropyrimido[4,5-d]pyrimidinone derivatives have been reported to inhibit Src kinase activity with IC values ​​of 1-5 nM (IC 50 ), but there is no mention of its effect on PD-L1 (International Patent Publication No. WO2005 / 011597).

[0006] Therefore, there is a need to develop compounds that have the activity of suppressing PD-L1 expression for antitumor efficacy.

[0007] Summary of the Invention [Problem to be solved by the invention] An object of the present invention is to provide a compound represented by the following chemical formula I or a pharmaceutically acceptable salt thereof, which exhibits the effect of inhibiting the expression of PD-L1:

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

[0009] [Means for solving the problem] One embodiment of the present invention relates to a pyrimidopyrimidinone compound represented by the following chemical formula I, or a pharmaceutically acceptable salt thereof:

[0010] [ka]

[0011] In the above formula, R1 is C5~C 15 Aryl groups of C5-C15 Heteroaryl groups of C3-C 10 or a cycloalkyl group of C3 to C 10 is a heterocycloalkyl group of the formula R2 is hydrogen, a C1-C4 alkyl group, or a C1-C4 alkoxy group; R3 is hydrogen, halogen, a C1-C4 alkyl group, or a C1-C4 alkoxy group; R4 is a C1-C4 alkyl group, a C1-C4 haloalkyl group, or a C5-C 15 a substituted or unsubstituted C5-C heteroaryl group 15 or an aryl group of C5 to C 15 is a heteroaryl group of the formula:

[0012] As used herein, C5 to C 15 The aryl group includes both aromatic groups and their partially reduced derivatives. The aromatic group is a monocyclic or fused ring type having 5 to 15 carbon atoms. Representative examples of the aryl group include, but are not limited to, phenyl, naphthyl, and tetrahydronaphthyl.

[0013] As used herein, C5 to C 15 The heteroaryl group includes both heteroaromatic groups and their partially reduced derivatives. The heteroaromatic group is a monocyclic or fused ring type having 5 to 15 carbon atoms and containing one or more oxygen, sulfur, or nitrogen atoms. Representative examples of heteroaryl groups include, but are not limited to, pyridinyl, furanyl, thiophenyl, indolyl, quinolinyl, imidazolinyl, oxazolyl, dihydrooxazolyl, and thiazolyl.

[0014] As used herein, C3 to C 10The cycloalkyl group means a monocyclic or fused ring hydrocarbon having 3 to 10 carbon atoms, and examples thereof include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0015] As used herein, C3 to C 10 The heterocycloalkyl group refers to a functional group in which one or more ring carbon atoms of a monocyclic or fused ring hydrocarbon having 3 to 10 carbon atoms are substituted with oxygen, sulfur, or nitrogen, and examples thereof include, but are not limited to, thiazolidinyl, oxiranyl, and the like.

[0016] As used herein, a C1-C4 alkyl group refers to a linear or branched monovalent hydrocarbon group having 1 to 4 carbon atoms, and examples thereof include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, and t-butyl.

[0017] As used herein, the term "C1-C4 alkoxy group" refers to a linear or branched alkoxy group having 1 to 4 carbon atoms, including, but not limited to, methoxy, ethoxy, n-propanoxy, etc.

[0018] As used herein, the term "C1-C4 haloalkyl group" refers to a linear or branched hydrocarbon group having 1 to 4 carbon atoms substituted with one or more halogen atoms selected from the group consisting of fluorine, chlorine, bromine, and iodine, and includes, but is not limited to, trifluoromethyl, trichloromethyl, trifluoroethyl, and the like.

[0019] In one embodiment of the present invention, the compound of formula I is R1 is phenyl unsubstituted or substituted with halogen or hydroxy; R2 is a C1-C4 alkyl group, R3 is hydrogen, halogen, a C1-C4 alkyl group, or a C1-C4 alkoxy group; R4 is a C1-C4 alkyl group, a C1-C4 haloalkyl group, a phenyl group unsubstituted or substituted with an alkyldihydrooxazolyl group, or an alkylthiazolyl group.

[0020] As used herein, alkyldihydrooxazolyl refers to a dihydrooxazole group substituted with one or more C1-C4 alkyl groups, including, but not limited to, dimethyldihydrooxazolyl.

[0021] As used herein, alkylthiazolyl refers to a thiazolyl group substituted with one or more C1-C4 alkyl groups, including, but not limited to, t-butylthiazolyl.

[0022] In one embodiment of the present invention, the compound of formula I is R1 is phenyl, fluorophenyl or hydroxyphenyl; R2 is methyl; R3 is hydrogen, chloro, bromo, methyl or methoxy; R4 is trifluoromethylphenyl, t-butylphenyl, dimethyldihydrooxazolylphenyl or t-butylthiazolyl.

[0023] As used herein, pharmaceutically acceptable salts 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, hydroiodate, malate, malonate, and the like.

[0024] Representative compounds of the present invention are selected from the following group: 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).

[0025] The method for producing the compound of 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 typical method used in the present invention, and the unit operation procedure, reaction reagents, reaction conditions, etc. may be appropriately changed depending on the case.

[0026] [ka]

[0027] In Reaction Scheme 1, R1, R2, R3, and R4 are as defined in Chemical Formula I, and R5 is halogen or oxo (=O).

[0028] In one embodiment of the present invention, R5 is chloro, bromo, iodine or oxo (=O), in particular chloro or oxo (=O).

[0029] Reaction Scheme 1 shows a four-step process for producing a compound represented by Formula I from a compound represented by Formula II and a compound represented by Formula III as starting materials. In the first step, a compound represented by formula II is reacted with a compound represented by formula III to obtain a compound represented by formula IV.

[0030] In this case, when R5 is halogen, a compound represented by Formula IV is obtained by amination in the presence of an organic base, and when R5 is oxo (=O), a reducing agent is added to perform reductive amination to obtain a compound represented by Formula IV. The organic base may be triethylamine, N,N-diisopropylethylamine (DIPEA), or the like, and the reducing agent may be sodium borohydride (NaBH4), sodium cyanoborohydride (NaCNBH3), sodium triacetoxyborohydride (Na(CH3COO)3BH), or the like.

[0031] The reaction solvent may be acetonitrile, tetrahydrofuran, 1,4-dioxane, chloroform, dichloromethane, 1,2-dichloroethane, dimethylformamide, dimethylacetamide, methyl alcohol, ethyl alcohol, or the like, and the reaction temperature is preferably -10 to 25°C.

[0032] In the second step, triphosgene is added to the compound represented by Formula IV to form a heterocyclic urea, thereby obtaining a compound represented by Formula V. The heterocyclic urea formation reaction may be carried out in the presence of an organic base, such as triethylamine or N,N-diisopropylethylamine (DIPEA).

[0033] As the reaction solvent, acetonitrile, tetrahydrofuran, 1,4-dioxane, or the like may be used, and the reaction temperature is preferably -10 to 100°C.

[0034] In the third step, the sulfide group of the compound represented by formula V is oxidized to obtain a sulfone compound represented by formula VI having a sulfone group.

[0035] The oxidation reaction may be carried out using oxone, meta-chloroperbenzoic acid (mCPBA), or the like as an oxidizing agent.

[0036] As the reaction solvent, dichloromethane, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, 1,4-dioxane, water, or the like may be used, and the reaction temperature is preferably -10 to 100°C.

[0037] In the fourth step, the compound represented by Formula VI is subjected to a substitution reaction with an amine compound represented by Formula VII to obtain the compound represented by Formula I.

[0038] The reaction solvent may be acetonitrile, tetrahydrofuran, 1,4-dioxane, chloroform, dichloromethane, 1,2-dichloroethane, ethyl acetate, dimethylformamide, methyl alcohol, ethyl alcohol, or the like. In the substitution reaction, an organic or inorganic base such as triethylamine, N,N-diisopropylethylamine, or pyridine may be used together, or an organic acid such as trifluoroacetic acid may be used together, as necessary. The reaction temperature is 50 to 150°C, more preferably 80 to 100°C.

[0039] The compounds represented by the chemical formula II and the compounds represented by the chemical formula III may be prepared by known methods or may be commercially available products.

[0040] For example, methods for producing the compound represented by the chemical formula II and the compound represented by the chemical formula III are shown in the following reaction schemes 2 to 4. The methods described in the following reaction schemes are merely examples of methods typically used in the present invention, and the unit operation procedures, reaction reagents, reaction conditions, etc. may be appropriately changed depending on the case.

[0041] [ka]

[0042] In the reaction scheme 2, R2 is as defined in the chemical formula I, and R5 is as defined in the reaction scheme 1.

[0043] As shown in Reaction Scheme 2, the compound represented by Formula II may be prepared by reacting ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate with an amine compound represented by Formula XIII to obtain a compound represented by Formula VIII, reducing the compound represented by Formula VIII using a reducing agent such as lithium aluminum hydride (LiAlH) to obtain a compound represented by Formula IX, and then halogenating the alcohol group of the compound represented by Formula IX using thionyl chloride (SOCl) or the like, or oxidizing the alcohol group using manganese dioxide (MnO) or the like to form an aldehyde.

[0044] [ka]

[0045] In Reaction Scheme 3, R3 and R4 are as defined in Formula I, and Y is halogen or hydroxy.

[0046] In one embodiment of the present invention, Y is chloro, bromo, iodine or hydroxy, in particular chloro or hydroxy.

[0047] The compound represented by Formula III can be prepared by reacting a compound represented by Formula X with a compound represented by Formula XI in the presence of an organic base or coupling agent such as DIPEA to obtain a compound represented by Formula XII, and then reducing the nitro group of the compound represented by Formula XII to an amine group, as shown in Reaction Scheme 3. The reduction can be carried out using a combination of potassium t-butoxide and bis(pinacolato)diboron or using hydrogen gas under a palladium carbon catalyst.

[0048] [ka]

[0049] In Reaction Scheme 4, R3 and R4 are as defined in Chemical Formula I, and Y is as defined in Reaction Scheme 3.

[0050] Alternatively, the compound of Formula III can be prepared by reacting a diaminepyridine compound of Formula X' with a compound of Formula XI in the presence of an organic base or coupling agent such as DIPEA, as shown in Reaction Scheme 4.

[0051] The compound represented by the chemical formula I or a pharmaceutically acceptable salt thereof according to the present invention exhibits excellent activity in inhibiting the expression of PD-L1 (Test Example 1).

[0052] Therefore, the present invention relates to a pharmaceutical composition for inhibiting programmed death-ligand 1 (PD-L1), particularly a pharmaceutical composition for treating or preventing cancer, comprising a compound represented by formula I or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.

[0053] In one embodiment of the present invention, the compound represented by Chemical Formula I or a pharmaceutically acceptable salt thereof inhibits PD-L1 expression, thereby inducing an anti-cancer effect through the activation of immune cells, and may be useful in the treatment of colon cancer, lung cancer, breast cancer, gastric cancer, cervical cancer, bladder cancer, blood cancer, non-Hodgkin's lymphoma, etc.

[0054] The pharmaceutical composition of the present invention may be administered orally (e.g., by ingestion or inhalation) or parenterally (e.g., by injection, deposition, implantation, or suppository), and the injection may be, for example, intravenous, subcutaneous, intramuscular, or intraperitoneal. Depending on the route of administration, the pharmaceutical composition of the present invention may be formulated as tablets, capsules, granules, fine subtilase, powder, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, sprays, and the like. The various forms of the pharmaceutical composition of the present invention described above may be prepared by known techniques using pharmaceutically acceptable carriers commonly used for each dosage form. Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrating agents, lubricants, preservatives, antioxidants, isotonic agents, buffers, coating agents, sweeteners, solubilizers, bases, dispersing agents, wetting agents, suspending agents, stabilizers, coloring agents, and the like.

[0055] The pharmaceutical composition according to the present invention contains the compound of the present invention or a pharmaceutically acceptable salt thereof in an amount ranging from about 0.01 to 95% by weight, depending on the form of the drug.

[0056] The specific dosage of the pharmaceutical composition of the present invention may vary depending on the species, body weight, sex, severity of the disease, and the doctor's judgment of the mammal, including humans, being treated. Preferably, for oral administration, 0.01 to 50 mg of the active ingredient is administered per kg of body weight per day, and for 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 doctor's judgment, etc.

[0057] [Effects of the Invention] The compounds according to the present invention have the effect of suppressing the expression of PD-L1, and therefore can be effectively used in pharmaceutical compositions for treating or preventing cancer.

[0058] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the change in tumor volume over time after administration of a compound of the present invention to mice in which cancer was induced with B16F10 cells.

[0059] [FIG. 2] A graph showing the weight of tumors excised from mice in which cancer was induced with B16F10 cells and then administered with a compound of the example.

[0060] [Figure 3] Western blot analysis of the degree of PD-L1 expression suppression in tumors excised from mice after administration of a compound of the present invention to mice with cancer induced using B16F10 cells.

[0061] Figure 4 shows the results of Western blot analysis of the inhibitory effect of PD-L1 expression after treatment of PD-L1-overexpressing cells with compounds of the examples.

[0062] [Mode for Carrying Out the Invention] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and that the scope of the present invention is not limited to these examples.

[0063] Preparation Example 1: Preparation of Compound Represented by Formula II Preparation Example 1-1: 5-(chloromethyl)-N-methyl-2-(methylthio)pyrimidin-4-amine (II-1)

[0064] [ka]

[0065] 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 40% methylamine solution (3.54 g, 45.55 mmol) were added. The mixture was stirred at 20–25°C for 16 hours. Upon completion of the reaction, the precipitated salt was filtered off, and the solvent was removed under reduced pressure. The concentrated residue was then 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%).

[0066] 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).

[0067] The obtained ethyl 4-(methylamino)-2-(methylthio)pyrimidine-5-carboxylate 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 the mixture was stirred at 20–25 °C for 2 h. Upon completion of the reaction, 5 mL of water, 15 mL of 15% sodium hydroxide solution, and 15 mL of water were added dropwise, successively, to the reaction mixture to terminate the reaction. The mixture was then stirred for 1 h. The resulting white precipitate was filtered, and the filtrate was extracted twice with ethyl acetate. The combined organic layer was extracted with brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give (4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methanol (IX-1, 6.00 g, 84%).

[0068] 1H 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).

[0069] 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. 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.

[0070] Preparation Example 1-2: 4-(methylamino)-2-(methylthio)pyrimidine-5-carbaldehyde (II-2)

[0071] [ka]

[0072] 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 stirring at 20-25°C for 20 hours. Upon 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.

[0073] 1 H NMR (400MHz, CDCl3): δ 9.70 (s, 1H), 8.56(bs, 1H), 8.30(s,1H), 3.12 (d, 3H), 2.57(s, 3H) ES-MS m / z: 184.18 [M+H] + .

[0074] Preparation Example 2: Preparation of Compound Represented by Formula III Production Example 2-1: N-(5-amino-6-methylpyridin-3-yl)-3-(trifluoromethyl)benzamide (III-1)

[0075] [ka]

[0076] 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, which was then stirred at 20–25 °C for 1 h. Upon completion of the reaction, the mixture was extracted sequentially with dichloromethane, distilled water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography to give N-(6-methyl-5-nitropyridin-3-yl)-3-(trifluoromethyl)benzamide XII-1 (1.76 g, 83%).

[0077] The 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, and 0.32 g of 10% palladium carbon was added. The mixture was stirred under a hydrogen atmosphere for 3 hours. 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.

[0078] 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).

[0079] Preparation Example 2-2: N-(5-amino-6-methylpyridin-3-yl)-3-(t-butyl)benzamide (III-2)

[0080] [ka]

[0081] 3-(t-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 catalytic 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 give 3-(t-butyl)benzoyl chloride XI-2 (0.67 g, Quant.) without further purification.

[0082] 6-Methyl-5-nitropyridin-3-amine X-1 (0.90 g, 5.88 mmol) was dissolved in 50 mL of dichloromethane. The 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, followed by stirring at 20–25 °C for 1 h. Upon completion of the reaction, the mixture was extracted sequentially with dichloromethane, distilled water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography to give 3-(t-butyl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide XII-2 (1.39 g, 76%).

[0083] ES-MS m / z: 314.25 [M+H] + .

[0084] The 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, and 0.32 g of 10% palladium carbon was added and stirred under a hydrogen atmosphere for 3 hours. 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%).

[0085] 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) ES-MS m / z: 239.31 [M+H] + .

[0086] Preparation Example 2-3: N-(5-amino-6-methoxypyridin-3-yl)-3-(trifluoromethyl)benzamide (III-3)

[0087] [ka]

[0088] 2-Methoxy-3,5-dinitropyridine (0.55 g, 2.76 mmol) was dissolved in 20 mL of ethyl alcohol, followed by the addition of 6N hydrochloric acid (4.00 mL) and iron (1.30 g) and heating to reflux for 1 hour. Upon completion of the reaction, the mixture was cooled to 20-25°C, neutralized with 5M sodium hydroxide solution, and then 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%).

[0089] 1H NMR (400MHz, CDCl3): δ 3.23 (s, 3H), 3.90 (br, 4H), 6.46 (s, 1H), 6.69 (s, 1H) ES-MS m / z: 140.11 [M+H] + .

[0090] The 2-methoxypyridine-3,5-diamine X'-1 (0.21 g, 1.51 mmol) obtained above was dissolved in 30 mL of acetone and cooled to 0 °C. 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. Upon completion of the reaction, the solvent was concentrated and the mixture was extracted sequentially with 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 yield 0.17 g (45%) of the title compound.

[0091] 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) ES-MS m / z: 312.10 [M+H] + .

[0092] Preparation Example 2-4: N-(5-amino-6-methylpyridin-3-yl)-4-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)benzamide (III-4)

[0093] [ka]

[0094] 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. 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 h. Upon completion of the reaction, the mixture was extracted sequentially with dichloromethane, distilled water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography to give 4-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide XII-6 (1.99 g, 86%).

[0095] ES-MS m / z: 355.25 [M+H] + .

[0096] The 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, and 0.30 g of 10% palladium carbon was added. The mixture was stirred under a hydrogen atmosphere for 5 hours. Then, the same procedure as in Preparation 2-1 was repeated to obtain 1.64 g (92%) of the title compound.

[0097] ES-MS m / z: 325.25 [M+H] + .

[0098] Preparation Example 2-5: N-(5-amino-6-methylpyridin-3-yl)-3-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)benzamide (III-5)

[0099] [ka]

[0100] 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. 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 h. Upon completion of the reaction, the mixture was extracted sequentially with dichloromethane, distilled water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography to give 3-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)-N-(6-methyl-5-nitropyridin-3-yl)benzamide XII-7 (1.39 g, 60%).

[0101] The obtained 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) was dissolved in 30 mL of methyl alcohol, and 0.30 g of 10% palladium carbon was added. The mixture was stirred under a hydrogen atmosphere for 3 hours. Then, the same procedure as in Preparation 2-1 was carried out to obtain 1.17 g (92%) of the title compound.

[0102] ES-MS m / z: 325.16 [M+H] + .

[0103] Preparation Example 2-6: N-(5-amino-6-methoxypyridin-3-yl)-3-(t-butyl)benzamide (III-6)

[0104] [ka]

[0105] 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-(t-butyl)benzoyl chloride XI-2 (0.50 g, 2.54 mmol) were added and stirred for 2 h. Upon completion of the reaction, the solvent was concentrated and the mixture was extracted sequentially with ethyl acetate, distilled water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield 0.34 g (45%) of the title compound.

[0106] ES-MS m / z: 300.28 [M+H] + .

[0107] Preparation Example 2-7: N-(5-amino-6-methoxypyridin-3-yl)-3-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)benzamide (III-7)

[0108] [ka]

[0109] 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. 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 h. Upon completion of the reaction, the solvent was concentrated and the mixture was extracted sequentially with ethyl acetate, distilled water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield 0.34 g (43%) of the title compound.

[0110] ES-MS m / z: 341.36 [M+H] + .

[0111] Preparation Example 2-8: N-(5-amino-6-chloropyridin-3-yl)-3-(trifluoromethyl)benzamide trifluoroacetate (III-8)

[0112] [ka]

[0113] t-Butyl (5-amino-2-chloropyridin-3-yl)carbamate (1.20 g, 4.92 mmol) was dissolved in 60 mL of dichloromethane and 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 h. Upon completion of the reaction, the mixture was extracted sequentially with 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 yield 1.59 g (78%) of t-butyl (2-chloro-5-(3-(trifluoromethyl)benzamido)pyridin-3-ylcarbamate.

[0114] 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).

[0115] 1.50 g (3.61 mmol) of the t-butyl (2-chloro-5-(3-(trifluoromethyl)benzamido)pyridin-3-ylcarbamate obtained above was dissolved in dichloromethane:trifluoroacetic acid (1:1) and stirred at 20-25°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 trifluoroacetate salt of the title compound.

[0116] 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).

[0117] Preparation Example 2-9: N-(5-amino-6-chloropyridin-3-yl)-3-(t-butyl)benzamide trifluoroacetate (III-9)

[0118] [ka]

[0119] t-Butyl (5-amino-2-chloropyridin-3-yl)carbamate (0.84 g, 3.45 mmol) was dissolved in 50 mL of dichloromethane and 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 for 5 hours at 20-25 ° C. Next, t-butyl (5-(3-(t-butyl)benzamido)-2-chloropyridin-3-yl)carbamate (0.90 g, 65%) was obtained in the same manner as in Preparation Example 2-14.

[0120] The obtained t-butyl (5-(3-(t-butyl)benzamido)-2-chloropyridin-3-yl)carbamate (0.88 g, 2.18 mmol) was dissolved in dichloromethane:trifluoroacetic acid (1:1), and then 0.88 g of the trifluoroacetate salt of the title compound was quantitatively obtained by the same method as in Preparation Example 2-14.

[0121] 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).

[0122] Preparation Example 2-10: N-(5-amino-6-chloropyridin-3-yl)-2-(t-butyl)thiazole-4-carboxamide trifluoroacetate (III-10)

[0123] [ka]

[0124] t-Butyl (5-amino-2-chloropyridin-3-yl)carbamate (0.84 g, 3.45 mmol) was dissolved in 50 mL of dichloromethane and 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 the mixture was stirred for 5 hours at 20-25 °C. t-Butyl (5-(2-(t-butyl)thiazole-4-carboxamido)-2-chloropyridin-3-yl)carbamate (0.94 g, 67%) was obtained in the same manner as in Preparation Example 2-14.

[0125] The obtained t-butyl (5-(2-(t-butyl)thiazole-4-carboxamido)-2-chloropyridin-3-yl)carbamate (0.92 g, 2.24 mmol) was dissolved in dichloromethane:trifluoroacetic acid (1:1), and then treated in the same manner as in Preparation Example 2-14 to quantitatively obtain 0.92 g of the trifluoroacetate salt of the title compound.

[0126] Preparation Example 3: Preparation of Compound Represented by Formula IV 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)

[0127] [ka]

[0128] 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 was dissolved in 30 mL of acetonitrile and slowly added dropwise to the reaction solution, followed by stirring at 20-25°C for 1 hour. The reaction solvent was concentrated under reduced pressure, and the mixture was extracted sequentially 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.

[0129] 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) ES-MS m / z: 463.03 [M+H] + .

[0130] Preparation 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)

[0131] [ka]

[0132] 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.

[0133] 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) ES-MS m / z: 451.14 [M+H] + .

[0134] Preparation 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)

[0135] [ka]

[0136] Compound III-2 (2.08 g, 7.33 mmol) prepared in Preparation Example 2-2, instead of compound III-1, 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 and stirred for 15 minutes. Sodium cyanoborohydride (2.30 g, 36.68 mmol) was added to the reaction solution, which was then heated to 45°C and stirred for 18 hours. After the reaction was completed, the mixture was cooled to 20-25°C, concentrated under reduced pressure, and extracted with ethyl acetate and 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.

[0137] ES-MS m / z: 451.14 [M+H] + .

[0138] 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)

[0139] [ka]

[0140] 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.

[0141] 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) ES-MS m / z: 527.97 [M+H] + .

[0142] Preparation 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)

[0143] [ka]

[0144] 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.

[0145] ES-MS m / z: 492.54 [M+H] + .

[0146] Preparation 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)

[0147] [ka]

[0148] 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.

[0149] ES-MS m / z: 492.54 [M+H] + .

[0150] Preparation 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)

[0151] [ka]

[0152] 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.

[0153] ES-MS m / z: 467.61 [M+H] + .

[0154] Preparation 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)

[0155] [ka]

[0156] 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.

[0157] ES-MS m / z: 508.55 [M+H] + .

[0158] 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)

[0159] [ka]

[0160] 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.

[0161] Preparation 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)

[0162] [ka]

[0163] 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.

[0164] Preparation 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)

[0165] [ka]

[0166] 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.

[0167] 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).

[0168] Preparation Example 4: Preparation of Compound Represented by Formula V 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)

[0169] [ka]

[0170] Compound IV-1 (0.70 g, 1.51 mmol) prepared in Preparation 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 stirred at 100°C for 3 hours. Upon completion of the reaction, 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. The residue was purified by column chromatography to yield 0.37 g (50%) of the title compound.

[0171] 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) ES-MS m / z: 489.50 [M+H] + .

[0172] Preparation 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)

[0173] [ka]

[0174] 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.

[0175] 1H 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) ES-MS m / z: 477.60 [M+H] + .

[0176] 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)

[0177] [ka]

[0178] 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.

[0179] 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) ES-MS m / z: 505.09 [M+H] + .

[0180] Preparation Example 4-4: 4-(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-4)

[0181] [ka]

[0182] 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.

[0183] ES-MS m / z: 518.09 [M+H] + .

[0184] Preparation 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)

[0185] [ka]

[0186] 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.

[0187] ES-MS m / z: 518.09 [M+H] + .

[0188] Preparation 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)

[0189] [ka]

[0190] 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.

[0191] ES-MS m / z: 493.62 [M+H] + .

[0192] Preparation 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)

[0193] [ka]

[0194] 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.

[0195] ES-MS m / z: 534.65 [M+H] + .

[0196] 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)

[0197] [ka]

[0198] 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.

[0199] Preparation 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)

[0200] [ka]

[0201] 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.

[0202] Preparation 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)

[0203] [ka]

[0204] 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.

[0205] Preparation Example 5: Preparation of Compound Represented by Formula VI 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)

[0206] [ka]

[0207] Compound V-1 (0.37 g, 0.76 mmol) prepared in Preparation Example 4-1 was dissolved in tetrahydrofuran:distilled water (2:1, 30 mL), and the oxidizing agent oxone (4.66 g, 7.57 mmol) was added. The mixture was stirred at 65°C for 5 hours. After completion of the reaction, the reaction solvent was concentrated under reduced pressure and washed sequentially 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 0.17 g (44%) of the title compound.

[0208] 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) ES-MS m / z: 521.60 [M+H] + .

[0209] Preparation 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)

[0210] [ka]

[0211] 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.

[0212] 1 H 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) ES-MS m / z: 509.72 [M+H] + .

[0213] 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)

[0214] [ka]

[0215] The title compound (90 mg, 97%) was obtained in the same manner as in Preparation Example 5-1, except that Compound V-3 (0.09 g, 0.17 mmol) prepared in Preparation Example 4-3 was used instead of Compound V-1. 1 H NMR (400MHz, 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) ES-MS m / z: 537.01 [M+H] + .

[0216] Preparation 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)

[0217] [ka]

[0218] 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.

[0219] ES-MS m / z: 550.25 [M+H] + .

[0220] Preparation 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)

[0221] [ka]

[0222] 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.

[0223] ES-MS m / z: 550.21 [M+H] + .

[0224] Preparation 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)

[0225] [ka]

[0226] 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.

[0227] 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) ES-MS m / z: 525.61 [M+H] + .

[0228] Preparation 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)

[0229] [ka]

[0230] 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.

[0231] ES-MS m / z: 566.50 [M+H] + .

[0232] 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)

[0233] [ka]

[0234] 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.

[0235] Preparation 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)

[0236] [ka]

[0237] 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.

[0238] Preparation 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)

[0239] [ka]

[0240] 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.

[0241] Example: Preparation of Compounds of Formula I 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)

[0242] [ka]

[0243] Compound VI-1 (0.17 g, 0.33 mmol) prepared in Preparation 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 extracted sequentially with ethyl acetate, water, and brine. The organic layer was dried over anhydrous sodium cyanide and concentrated under reduced pressure. The concentrated residue was purified by column chromatography to obtain 87 mg (50%) of the title compound.

[0244] 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) ES-MS m / z: 534.62 [M+H] + .

[0245] 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)

[0246] [ka]

[0247] The same procedure as in Example 1 was repeated except that compound VI-2 (0.21 g, 0.41 mmol) prepared in Preparation Example 5-2 was used instead of compound VI-1 to obtain 86 mg (40%) of the title compound.

[0248] 1 H 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) ES-MS m / z: 522.72 [M+H] + .

[0249] 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)

[0250] [ka]

[0251] The same procedure as in Example 1 was repeated except that compound VI-3 (90 mg, 0.17 mmol) prepared in Preparation Example 5-3 was used instead of compound VI-1 to obtain 25 mg (27%) of the title compound.

[0252] 1 H NMR (400MHz, 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) ES-MS m / z: 550.07 [M+H] + .

[0253] 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)

[0254] [ka]

[0255] The same procedure as in Example 1 was repeated except that compound VI-4 (0.20 g, 0.36 mmol) prepared in Preparation Example 5-4 was used instead of compound VI-1 to obtain 0.10 g (55%) of the title compound.

[0256] 1H NMR (400MHz, 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) ES-MS m / z: 563.72 [M+H] + .

[0257] 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)

[0258] [ka]

[0259] The same procedure as in Example 1 was repeated except that compound VI-5 (0.23 g, 0.42 mmol) prepared in Preparation Example 5-5 was used instead of compound VI-1 to obtain 0.14 g (58%) of the title compound.

[0260] 1 H NMR (400MHz, 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) ES-MS m / z: 563.62 [M+H] + .

[0261] 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)

[0262] [ka]

[0263] The same procedure as in Example 1 was repeated except that compound VI-6 (0.17 g, 0.32 mmol) prepared in Preparation Example 5-6 was used instead of compound VI-1 to obtain 83 mg (47%) of the title compound.

[0264] 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) ES-MS m / z: 538.64 [M+H] + .

[0265] 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)

[0266] [ka]

[0267] The same procedure as in Example 1 was repeated except that compound VI-7 (0.14 g, 0.25 mmol) prepared in Preparation Example 5-7 was used instead of compound VI-1 to obtain 65 mg (44%) of the title compound.

[0268] 1 H NMR (400MHz, 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) ES-MS m / z: 579.65 [M+H] + .

[0269] 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)

[0270] [ka]

[0271] The same procedure as in Example 1 was repeated except that compound VI-8 (0.20 g, 0.37 mmol) prepared in Preparation Example 5-8 was used instead of compound VI-1 to obtain 92 mg (46%) of the title compound.

[0272] 1H NMR (400MHz, 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) ES-MS m / z: 553.97 [M+H] + .

[0273] 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)

[0274] [ka]

[0275] The same procedure as in Example 1 was repeated except that compound VI-9 (0.21 g, 0.40 mmol) prepared in Preparation Example 5-9 was used instead of compound VI-1 to obtain 98.3 mg (46%) of the title compound.

[0276] 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) ES-MS m / z: 542.14 [M+H] + .

[0277] 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)

[0278] [ka]

[0279] The same procedure as in Example 1 was repeated except that compound VI-10 (0.18 g, 0.33 mmol) prepared in Preparation Example 5-10 was used instead of compound VI-1 to obtain 0.12 g (64%) of the title compound.

[0280] 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) ES-MS m / z: 549.16 [M+H] + .

[0281] 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)

[0282] [ka]

[0283] 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, and 4-aminophenol (1.00 g, 9.24 mmol) and trifluoroacetic acid (0.71 mL, 9.24 mmol) were added, followed by stirring for 7 hours at 85° C. 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.

[0284] 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) ES-MS m / z: 569.16 [M+H] + .

[0285] Test Example 1: Analysis of PD-L1 expression inhibitory activity The PD-L1 promoter gene fragment was inserted into the pGL4.14-[luc2 / Hygro] vector (Promega, Madison, WI) to generate a promoter-reporter construct for measuring gene transcription activity as follows.

[0286] Primers containing KpnI and XhoI restriction enzyme sites were constructed for PD-L1, followed by polymerase chain reaction (PCR). The promoter DNA was isolated by agarose gel electrophoresis. The pGL4.14-[luc2 / Hygro] vector, which contains firefly luciferase as a reporter gene, was treated with the same restriction enzymes and then subjected to agarose gel electrophoresis and gel extraction. The purified PD-L1 promoter gene and pGL4.14-[luc2 / Hygro] vector were reacted with In-fusion DNA ligase (Takara) at 50°C for 1 hour and then transformed into E. coli to prepare a promoter-reporter construct containing the PCSK9 promoter, designated pGL4.14-PD-L1.

[0287] To measure the transcriptional activity of the PD-L1 gene promoter, A549 cells were transiently transfected with the PD-L1 promoter-reporter construct as follows, and the luciferase activity of the cell extracts was measured.

[0288] 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 counted immediately before adding DNA and plated at 2.5 × 10 cells per well in a 12-well plate. 5A 1ml volume of the cell line was prepared. The DNA-Lipofectamine complex was carefully mixed with A549 cells and incubated in a 37°C incubator for 6 hours. Transfected cells were then screened by changing the medium, and a luciferase assay was performed to identify A549 cells transfected with PD-L1. This cell line was designated A549 pGL4.14-PD-L1 and further experiments were carried out. IFNγ (50ng / ml) was used as a PD-L1 inducer.

[0289] PD-L1 expression conditions were established in Beas-2B cells, a human normal lung epithelial cell line, using EGF and IFNγ, which are known to induce PD-L1. At the RNA level, treatment with EGF at concentrations of 25, 50, or 100 ng / ml did not result in PD-L1 activation, and only IFNγ increased PD-L1. Furthermore, PD-L1 mRNA levels increased starting 2 hours after treatment with 50 ng / ml IFNγ, with the most significant increase occurring 4 hours after treatment with 50 ng / ml IFNγ.

[0290] Similarly, PD-L1 protein levels were examined in Beas-2B cells. Treatment with IFNγ did not significantly change the expression level up to 8 hours after treatment, but PD-L1 expression increased from 8 hours onward. Therefore, the protein was lysed 20 hours after treatment with 50 ng / ml IFNγ.

[0291] 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, and the PD-L1 expression inhibition rate was evaluated using a luminometer. For comparison, a similar experiment was also performed using GS9973 (Entospletinib), which is known to have PD-L1 expression inhibitory activity.

[0292] The PD-L1 expression inhibition rate was calculated using the following mathematical formula 1. [Mathematical formula 1] PD-L1 expression suppression rate (%) = 100-[(A / B) x 100] In the above formula, A is the value when the drug is treated, and B is the value when the drug is not treated. The results are shown in Table 1 below.

[0293] [Table 1]

[0294] From Table 1, it can be seen that the pyrimidopyrimidinone compounds according to the present invention have superior PD-L1 expression inhibitory activity compared to GS9973 (Entospletinib), and that such PD-L1 expression inhibitory activity is concentration-dependent.

[0295] Test Example 2: In vivo efficacy evaluation of PD-L1 expression inhibitors 1 × 10 melanoma B16F10 cells per 6-week-old male C57BL / 6 mouse 6 The disease model was created by subcutaneously injecting the cells at 100 μl into the back of the neck of a mouse. The day after the creation of the disease model was designated as day 1 of administration, and the cells were orally administered once daily for 14 days as shown in Table 2 below. The tumors were excised on day 15 after the start of the study. The change in tumor volume and tumor size were measured.

[0296] The results are shown in Figures 1 and 2.

[0297] [Table 2]

[0298] 1 and 2, it can be seen that compounds I-2, I-3, I-8 and I-9 have tumor growth inhibitory potency of 22 to 59% compared to the control group.

[0299] Test Example 3: Western blot analysis Test Example 3-1: After the in vivo tumor suppression test using compound I-8 in Test Example 2, the inhibitory effect of PD-L1 expression in tumor cells isolated from mice was confirmed by Western blot analysis. For comparison, the inhibitory effect of PD-L1 expression in tumor cells isolated from control mice was also confirmed by Western blot analysis.

[0300] Proteins were analyzed using an electron chemiluminescence (ECL) kit (elpis biotec, EBP-1073) on ChemiDoc. TM The results were confirmed using an imaging system (Bio-Rad), and are shown in Figure 3.

[0301] From Figure 3, it can be seen that the expression of PD-L1 was reduced when compound I-8 was administered compared to the control group.

[0302] Test Example 3-2: The inhibitory effect of the compounds of the examples on PD-L1 overexpressing cells was confirmed by Western blot analysis.

[0303] To examine changes in PD-L1 protein levels, 6 × 10 NCI-H358 (NSCLC) cell lines were used. 5 After 24 hours, NCI-H358 (NSCLC) cells were treated with IFN-γ (20 ng / ml) to overexpress PD-L1, and then treated with 1 μM or 10 μM of the compounds. After 24 hours, the proteins were analyzed using an electron chemiluminescence (ECL) kit (Elpis Biotec, EBP-1073) using ChemiDoc TM The results were confirmed using an imaging system (Bio-Rad). The results of Western blot analysis are shown in Figure 4.

[0304] From Figure 4, it can be seen that the compounds of the examples exhibit a concentration-dependent inhibitory effect on PD-L1 expression compared to the control group that was not treated with the drug. [Brief explanation of the drawings]

[0305] [Figure 1]1 is a graph showing the change in tumor volume over time after administration of a compound of an example to mice in which cancer was induced with B16F10 cells. [Figure 2] 1 is a graph showing the weight of tumors excised from mice in which cancer was induced with B16F10 cells and then the compounds of the examples were administered to the mice. [Figure 3] The results show the results of Western blotting to measure the degree of suppression of PD-L1 expression in tumors excised from mice after administration of a compound of the example to mice in which cancer was induced using B16F10 cells. [Figure 4] Figure 1 shows the results of Western blot analysis of the inhibitory effect of PD-L1 expression after treatment of PD-L1 overexpressing cells with compounds of the examples.

Claims

1. A pyrimidopyrimidinone compound represented by the following chemical formula I or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 In the above formula, R １ is phenyl unsubstituted or substituted with halogen or hydroxy, R ２ is C １ ~C ４ is an alkyl group of the formula R ３ is hydrogen, halogen, C １ ~C ４ or an alkyl group of C １ ~C ４ is an alkoxy group of the formula R ４ is C １ ~C ４ alkyl group of C １ ~C ４ phenyl substituted or unsubstituted with a haloalkyl group of the formula (I), alkyldihydrooxazolyl, or alkylthiazolyl.

2. R １ is phenyl, fluorophenyl or hydroxyphenyl, R ２ is methyl, R ３ is hydrogen, chloro, bromo, methyl or methoxy, R ４ The pyrimidopyrimidinone compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein is trifluoromethylphenyl, t-butylphenyl, dimethyldihydrooxazolylphenyl, or t-butylthiazolyl.

3. 2. The pyrimidopyrimidinone compound according to claim 1, which is 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).

4. A pharmaceutical composition for inhibiting programmed death-ligand 1 (PD-L1), comprising the pyrimidopyrimidinone compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, which is an anticancer agent.

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

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