TETRACYCLIC COMPOUND USED AS CDC7 INHIBITOR

Abstract

A tetracyclic compound as a Cdc7 inhibitor. Specifically disclosed is a compound represented by formula (I), an isomer thereof, or a pharmaceutically acceptable salt thereof.

##STR00001##

Claims

1. A compound of formula (I), an isomer thereof or a pharmaceutically acceptable salt thereof: ##STR00032## wherein, the carbon atom with “*” can be a chiral carbon atom present in a form of a single (R) or (S) enantiomer or in a form enriched in one enantiomer; X is selected from the group consisting of O, NH and NCH.sub.3; L is selected from the group consisting of —CH.sub.2—CH.sub.2—CH.sub.2—, —CH.sub.2—O—CH.sub.2—, —CH.sub.2—S—CH.sub.2—, —CH.sub.2—NH—CH.sub.2—, —NH—CH.sub.2—CH.sub.2—, —S—CH.sub.2—CH.sub.2— and —O—CH.sub.2—CH.sub.2—; R.sub.1 is selected from the group consisting of H, halogen, CN, C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, phenyl, and 5-6 membered heteroaryl, wherein the C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, phenyl, and 5-6 membered heteroaryl are each independently optionally substituted with 1, 2 or 3 R.sub.a, the 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms or heteroatom groups each independently selected from the group consisting of O, S, N and NH; R.sub.2 is selected from R.sub.b, R.sub.3 is selected from NH.sub.2, and R.sub.4 is selected from H; alternatively, R.sub.2 is selected from R.sub.c, and R.sub.3 and R.sub.4 are joined to form a ring A optionally substituted with 1, 2 or 3 R.sub.e, wherein the ring A is selected from the group consisting of C.sub.6-14 aryl, 5-14 membered heteroaryl, 5-12 membered heterocycloalkenyl and 4-14 membered heterocycloalkyl each independently containing 1, 2 or 3 heteroatoms or heteroatom groups independently selected from the group consisting of O, S, N and NR.sub.d; R.sub.a is each independently selected from the group consisting of F, Cl, Br, I, OH, CN, NH.sub.2, CH.sub.3 and ##STR00033## R.sub.b is selected from the group consisting of H and C.sub.1-6 alkyl, the C.sub.1-6 alkyl being optionally substituted with 1, 2 or 3 R; R.sub.c is selected from the group consisting of H, F, Cl, Br, I and C.sub.1-3 alkyl; R.sub.d is selected from the group consisting of H and C.sub.1-4 alkyl; R is selected from the group consisting of —OCH.sub.3, —OCH.sub.2CH.sub.3, —O—CH(CH.sub.3).sub.2, cyclopropyl, cyclopentyl, phenyl, pyrazolyl, pyridinyl, NH.sub.2, —NHCH.sub.3 and —N(CH.sub.3).sub.2; R.sub.e is selected from the group consisting of F, Cl, Br, I, OH, CN, COOH, NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2, CH.sub.3, CH.sub.2CH.sub.3, CF.sub.3, —OCH.sub.3, —OCH.sub.2CH.sub.3, —O—CH(CH.sub.3).sub.2, —C(═O)OCH.sub.3, —C(═O)CH.sub.3 and —C(═O)CH.sub.2CH.sub.3.

2-18. (canceled)

19. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.1 is selected from the group consisting of H, F, Cl, Br, I, CN, CH.sub.3, CH.sub.2CH.sub.3, cyclopropyl, phenyl and pyridinyl, wherein the CH.sub.3, CH.sub.2CH.sub.3, cyclopropyl, phenyl and pyridinyl are each independently optionally substituted with 1, 2 or 3 R.sub.a.

20. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 19, wherein R.sub.1 is selected from the group consisting of H, F, Cl, Br, I, CN, CH.sub.3, CH.sub.2CH.sub.3, CF.sub.3, cyclopropyl, phenyl and pyridinyl.

21. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 20, wherein R.sub.1 is selected from H.

22. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.b is selected from the group consisting of H, methyl, ethyl, isopropyl, propyl, butyl and isobutyl.

23. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.e is selected from the group consisting of H, methyl, ethyl and F.

24. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.d is selected from the group consisting of H, methyl, ethyl, propyl, isopropyl and n-butyl.

25. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 1, wherein the ring A is selected from 5-9 membered heterocycloalkyl containing 1 heteroatom or heteroatom group selected from the group consisting of N and NR.sub.d.

26. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 25, wherein the ring A is selected from the group consisting of ##STR00034##

27. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 26, wherein the structural unit ##STR00035## is selected from the group consisting of ##STR00036##

28. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 27, wherein the structural unit ##STR00037## is selected from the group consisting of ##STR00038##

29. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 1, wherein the structural unit ##STR00039## is selected from ##STR00040##

30. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 1, wherein L is selected from —CH.sub.2—CH.sub.2—CH.sub.2—.

31. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from: ##STR00041## wherein the carbon atom with “*” can be a chiral carbon atom present in a form of a single (R) or (S) enantiomer or in a form enriched in one enantiomer; R.sub.1 is as defined in claim 1; the ring A is as defined in claim 1.

32. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 1, selected from the group consisting of: ##STR00042##

33. The compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 32, selected from the group consisting of: ##STR00043## ##STR00044##

34. A pharmaceutical composition comprising a therapeutically effective amount of the compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable carrier.

35. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the compound, the isomer thereof or the pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition thereof.

36. The method according to claim 35, wherein the cancer is selected from colorectal cancer or pancreatic cancer.

Description

DETAILED DESCRIPTION

[0080] The present invention is described in detail below by way of examples. However, this is by no means disadvantageously limiting the scope of the present invention. Although the present invention has been described in detail herein and specific examples have also been disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific examples without departing from the spirit and scope of the present invention.

Example 1. Compounds 1-1 and 1-2

[0081] ##STR00028##

[0082] Preparation of Compound 1B:

[0083] Compound 1A (20 g, 158.54 mmol) was stirred in N,N-dimethylformamide dimethyl acetal (53.82 g, 451.66 mmol) at 100° C. for 3 h. The reaction system was concentrated to dryness under reduced pressure to give compound 1B without purification, which was directly used in the next step. LCMS (ESI) m/z: 182 (M+1).

[0084] Preparation of Compound 1C:

[0085] Hydrazine hydrate (6.96 g, 139.05 mmol) was added to a solution of 1B (28 g, 154.5 mmol) in methanol (80 mL) at 0° C. The reaction system was heated to 90° C. and stirred for 2 h. The reaction system was concentrated to dryness under reduced pressure. The residue was stirred in 150 mL of a mixture (ethyl acetate/petroleum ether=1/9, v/v) for 10 min, and the resulting mixture was filtered to give solid compound 1C. LCMS (ESI) m/z: 151 (M+1).

[0086] Preparation of Compound 1D:

[0087] Compound 1C (3.0 g, 19.98 mmol), 3,4-dihydro-2H-pyran (2.52 g, 29.96 mmol) and trifluoroacetic acid (228 mg, 2.0 μmol) were stirred in tetrahydrofuran (50 mL) at 50° C. for 5 h. The reaction system was cooled to room temperature. Water (20 mL) was added, followed by ethyl acetate (50 mL×2) for extraction. The organic phase was washed with saturated brine (10 mL×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated by rotary evaporation. The residue was purified by column chromatography (1000 mesh silica gel, petroleum ether/ethyl acetate=100/1 to 3/1) to give compound 1D. LCMS (ESI) m/z: 235 (M+1). .sup.1H NMR (deuterated methanol, 400 MHz) δppm 8.21 (s, 1H), 5.33-5.42 (m, 1H), 4.02-4.11 (m, 1H), 3.70-3.77 (m, 1H), 2.95-3.02 (m, 2H), 2.68-2.74 (m, 2H), 1.94-2.05 (m, 6H), 1.57-1.82 (m, 4H).

[0088] Preparation of Compound 1E:

[0089] To a solution of compound 1D (3.5 g, 14.94 mmol) in tetrahydrofuran (50 mL) was added sodium hydride (1.19 g, 29.88 mmol, 60%) at 20° C. under nitrogen atmosphere. The reaction system was stirred at 20° C. for 0.5 h. Ethyl formate (1.66 g, 22.41 mmol) was then added, and the reaction was continued for another 2 h. The reaction system was quenched with saturated ammonium chloride solution (20 mL) and extracted with ethyl acetate (80 mL×2). The organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated by rotary evaporation to give compound 1E. LCMS (ESI) m/z: 263 (M+1).

[0090] Preparation of Compound 1F:

[0091] To a solution of compound 1E (3.9 g, 14.87 mmol) in ethanol (15 mL) was added hydroxylamine hydrochloride (1.08 g, 14.87 mmol) at 25° C. The reaction system was stirred at 90° C. for 2 h and filtered to give compound 1F. LCMS (ESI) m/z: 176 (M+1). .sup.1H NMR (deuterated methanol, 400 MHz) δppm 8.39-8.47 (m, 1H), 8.25-8.32 (m, 1H), 3.12-3.23 (m, 2H), 2.85-2.94 (m, 2H), 2.06-2.17 (m, 2H).

[0092] Preparation of Compound 1G:

[0093] To a solution of compound 1F (2.5 g, 14.27 mmol) in ethanol (20 mL) was added sodium methoxide (1.54 g, 28.54 mmol) at 20° C. under nitrogen atmosphere. The reaction system was stirred at 75° C. for 2 h. The reaction system was cooled to room temperature, quenched with saturated ammonium chloride solution (5 mL) and extracted with ethyl acetate (20 mL×2). The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated by rotary evaporation to give compound 1G. LCMS (ESI) m/z: 176 (M+1).

[0094] Preparation of Compound 1H:

[0095] Compound 1G (1.7 g, 9.7 mmol), 3,4-dihydro-2H-pyran (1.06 g, 12.62 mmol) and trifluoroacetic acid (111 mg, 970.39 μmol) were stirred in tetrahydrofuran (50 mL) at 60° C. for 3 h. The reaction system was cooled to room temperature and concentrated by rotary evaporation. The residue was purified by column chromatography (1000 mesh silica gel, petroleum ether/ethyl acetate=10/1 to 1/1) to give compound 1H. LCMS (ESI) m/z: 260 (M+1).

[0096] Preparation of Compound 1J:

[0097] Compound 1H (0.5 g, 1.93 mmol), compound 11 (399 mg, 2.89 mmol) and potassium carbonate (799 mg, 5.78 mmol) were stirred in N,N-dimethylformamide (10 mL) at 100° C. for 3 h. The reaction system was cooled to room temperature and concentrated by rotary evaporation. The residue was purified by column chromatography (1000 mesh silica gel, petroleum ether/ethyl acetate=10/1 to 1/1) to give compound 1J. LCMS (ESI) m/z: 317 (M+1).

[0098] Preparation of Compound 1K

[0099] Sodium (29 mg, 1.25 mmol) was added to ethanol (9 mL) at 20° C. After sodium completely disappeared, compound 1J (0.2 g, 623.47 μmol) was added. The reaction system was then refluxed for 5 h. The reaction system was concentrated to dryness under reduced pressure. The residue was purified by reversed-phase flash chromatography (Agela Technologies, C18, 20-35 μm, 0.1% aqueous formic acid solution/acetonitrile) to give compound 1K. LCMS (ESI) m/z: 317 (M+1). .sup.1H NMR (deuterated methanol, 400 MHz) δ ppm 7.97 (s, 1H), 5.27-5.16 (m, 1H), 3.96-3.89 (m, 1H), 3.66-3.58 (m, 1H), 2.89-2.82 (m, 2H), 2.56-2.49 (m, 2H), 2.06-1.92 (m, 4H), 1.72-1.44 (m, 4H).

[0100] Preparation of Compound 1M:

[0101] To a solution of compound 1K (181 mg, 1.04 mmol) and compound 1L (110 mg, 347.71 μmol) in dichloromethane (10 mL) was added diisopropylethylamine (180 mg, 1.39 mmol) at 20° C. under nitrogen atmosphere. The reaction system was stirred at 20° C. for 5 h. The reaction system was concentrated to dryness to give crude compound 1M, which was directly used in the next step. LCMS (ESI) m/z: 454 (M+1).

[0102] Preparation of Compound 1N:

[0103] Compound 1M (150.7 mg, 347.71 μmol) was dissolved in a solvent mixture of methanol (10 mL) and water (10 mL) at 20° C., and sodium hydroxide (139.09 mg, 3.48 mmol) was then added. The reaction system was warmed to 70° C. and stirred for 30 min. Methanol was evaporated. Water (10 mL) was added, followed by ethyl acetate (50 mL×2) for extraction. The organic phase was washed with saturated brine (10 mL×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated by rotary evaporation to give crude compound 1N, which was directly used in the next step. LCMS (ESI) m/z: 436 (M+1).

[0104] Preparation of Examples 1-1 and 1-2:

[0105] To a solution of compound 1N (0.075 g, 172.21 μmol) in dichloromethane (3 mL) was dropwise added trifluoroacetic acid (2 mL, 27.01 mmol) at 25° C. The reaction system was stirred at room temperature for 1 h. The reaction system was concentrated. The residue was purified by reversed-phase flash chromatography (Agilent, C18 reversed-phase column, 20-35 μm, 0.1% formic acid aqueous solution/acetonitrile). The resulting product was separated by SFC (Daicel CHIRALCEL OJ-H (250 mm×30 mm, 5 m); mobile phase: carbon dioxide as phase A, methanol containing 0.1% ammonium hydroxide as phase B; elution gradient: 30%-30% phase B, 3.95 min) to give two fractions, which were re-purified by preparative high performance liquid chromatography (column: Phenomenex Synergi C18 150×25×10 μm; mobile phase: 0.05% diluted hydrochloric acid as phase A, acetonitrile as phase B; elution gradient: 11%-31% phase B, 11 min) to give compound 1-1 and compound 1-2.

[0106] Determined using the SFC analytical method below, the retention times of compound 1-1 and compound 1-2 were 2.089 min and 1.919 min, respectively.

[0107] SFC analytical method:

[0108] Column: Chiralcel OD-3 50×4.6 mm I.D., 3 μm; mobile phase: carbon dioxide as phase A, ethanol containing 0.05% diethylamine as phase B; gradient elution: 5%-40% phase B; flow rate: 3 mL/min; wavelength: 220 nm; column temperature: 35° C.; back pressure: 100 Bar.

[0109] Compound 1-1: LCMS (ESI) m/z: 352 (M+1). .sup.1H NMR (400 MHz, deuterated methanol) δppm 8.50-7.90 (m, 1H), 4.06-3.95 (m, 1H), 3.43 (br s, 4H), 3.18-3.09 (m, 1H), 3.04-2.86 (m, 2H), 2.58-2.47 (m, 1H), 2.38-2.28 (m, 1H), 2.20-1.80 (m, 8H).

[0110] Compound 1-2: LCMS (ESI) m/z: 352 (M+1). .sup.1H NMR (400 MHz, deuterated methanol) δppm 8.53-8.00 (m, 1H), 4.21-3.78 (m, 1H), 3.72-3.37 (m, 3H), 3.18-2.77 (m, 4H), 2.61-2.41 (m, 1H), 2.35-2.23 (m, 1H), 2.17-1.85 (m, 8H).

Example 2. Compounds 2-1 and 2-2

[0111] ##STR00029##

[0112] Preparation of Compound 2B:

[0113] To a solution of compound 1K (500 mg, 1.57 mmol) and compound 2A (1.07 g, 4.0 μmol) in dichloromethane (10 mL) was added diisopropylethylamine (608 mg, 4.71 mmol) at 20° C. under nitrogen atmosphere. The reaction system was stirred at 20° C. for 5 h. The reaction system was concentrated to dryness. The residue was purified by reversed-phase flash chromatography (Agilent, C18 reversed-phase column, 20-35 μm, 0.1% formic acid aqueous solution/acetonitrile) to give compound 2B. LCMS (ESI) m/z: 530 (M+1).

[0114] Preparation of Compound 2C:

[0115] To compound 2B (0.4 g, 755.31 μmol) was added dropwise a 35% solution of hydrobromic acid in acetic acid (5 mL) at room temperature. The reaction system was stirred at 20° C. for 1 h. The reaction system was concentrated to give crude compound 2C, which was directly used in the next step. LCMS (ESI) m/z: 330 (M+1).

[0116] Preparation of Compound 2-1:

[0117] Compound 2C (238 mg, 722.63 μmol) was dissolved in methanol (10 mL) at room temperature, and sodium hydroxide (289 mg, 7.23 mmol) was added. The reaction system was warmed to 70° C. and stirred for 30 min. Methanol was evaporated. The residue was purified by preparative high performance liquid chromatography (column: Phenomenex Synergi C18 150×25×10 μm; mobile phase: 0.05% diluted hydrochloric acid as phase A, acetonitrile as phase B; elution gradient: 6%-26% phase B, 9 min) to give compound 2-1. LCMS (ESI) m/z: 312 (M+1). .sup.1H NMR (400 MHz, deuterated methanol) 6 ppm 8.24-8.17 (m, 1H), 4.82-4.75 (m, 1H), 3.71-3.61 (m, 1H), 3.55-3.46 (m, 1H), 3.20-3.13 (m, 2H), 3.11-3.03 (m, 2H), 2.70-2.55 (m, 1H), 2.24-2.17 (m, 2H), 2.17-2.17 (m, 1H), 2.17-2.10 (m, 2H).

[0118] Preparation of Compound 2E:

[0119] To a solution of compound 1K (300 mg, 941.05 μmol) and compound 2D (0.504 g, 1.88 mmol) in dichloromethane (10 mL) was added diisopropylethylamine (365 mg, 2.82 mmol) at 20° C. under nitrogen atmosphere. The reaction system was stirred at 20° C. for 5 h. The reaction system was concentrated to dryness to give crude compound 2E. LCMS (ESI) m/z: 548 (M+1).

[0120] Preparation of Compound 2F:

[0121] To compound 2E (0.5 g, 913.07 μmol) was added dropwise a 30% solution of hydrobromic acid in acetic acid (5 mL) at room temperature. The reaction system was stirred at 20° C. for 1 h. The reaction system was concentrated to give crude compound 2F, which was directly used in the next step. LCMS (ESI) m/z: 330 (M+1).

[0122] Preparation of Compound 2-2:

[0123] The compound was prepared as described for example compound 2-1. LCMS (ESI) m/z: 312 (M+1). .sup.1H NMR (400 MHz, deuterated methanol) 6 ppm 8.25-8.15 (m, 1H), 4.83-4.75 (m, 1H), 3.71-3.61 (m, 1H), 3.54-3.46 (m, 1H), 3.21-3.13 (m, 2H), 3.11-3.03 (m, 2H), 2.69-2.57 (m, 1H), 2.24-2.12 (m, 5H).

[0124] Determined using the SFC analytical method below, the retention times of compound 1-1 and compound 1-2 were 0.991 min and 1.298 min, respectively.

[0125] SFC analytical method:

[0126] Column: Chiralpak AD-3 50×4.6 mm I.D., 3 μm; mobile phase: carbon dioxide as phase A, ethanol containing 0.05% diethylamine as phase B; gradient elution: isocratic elution with 40% phase B; flow rate: 3 mL/min; wavelength: 220 nm; column temperature: 35° C.; back pressure: 100 Bar.

Example 3. Compounds 3-1 and 3-2

[0127] ##STR00030##

[0128] Preparation of Compound 3-1:

[0129] Compound 2-1(50.00 mg, 160.14 μmol), 37% aqueous formaldehyde solution (22 μL, 800.69 μmol) and sodium cyanoborohydride (30 mg, 480.41 μmol) were stirred in methanol (5 mL) at 20° C. for 1 h. The reaction system was concentrated. The residue was purified by preparative high performance liquid chromatography (column: Phenomenex Synergi C18 150×25×10 μm; mobile phase: 0.05% aqueous hydrochloric acid solution as phase A, acetonitrile as phase B; elution gradient: 6%-26% acetonitrile, time: 12 min) to give compound 3-1. LCMS (ESI)m/z: 326 (M+1). .sup.1H NMR (400 MHz, deuterated methanol) 6 ppm 8.30-8.19 (m, 1H), 4.68-4.57 (m, 1H), 4.03-3.90 (m, 1H), 3.51-3.36 (m, 1H), 3.22-3.15 (m, 2H), 3.13 (s, 3H), 3.11-3.05 (m, 2H), 2.85-2.73 (m, 1H), 2.40-2.27 (m, 1H), 2.26-2.12 (m, 4H).

[0130] Preparation of Compound 3-2:

[0131] The compound was prepared as described for compound 3-1. LCMS (ESI)m/z: 326 (M+1). .sup.1H NMR (400 MHz, deuterated methanol) 6 ppm 8.30 (s, 1H), 4.67-4.56 (m, 1H), 4.03-3.90 (m, 1H), 3.46-3.39 (m, 1H), 3.23-3.17 (m, 2H), 3.12 (s, 3H), 3.12-3.07 (m, 2H), 2.87-2.73 (m, 1H), 2.43-2.28 (m, 1H), 2.27-2.14 (m, 4H).

[0132] Determined using the SFC analytical method below, the retention times of compound 1-1 and compound 1-2 were 1.190 min and 1.116 min, respectively.

[0133] SFC analytical method:

[0134] Column: Chiralcel OJ-3 50×4.6 mm I.D., 3 μm; mobile phase: carbon dioxide as phase A, methanol containing 0.05% diethylamine as phase B; gradient elution: 5%-40% phase B; flow rate: 3 mL/min; wavelength: 220 nm; column temperature: 35° C.; back pressure: 100 Bar.

Example 4. Compound 4

[0135] ##STR00031##

[0136] Preparation of Compound 4B:

[0137] To a solution of compound 4A (0.375 g, 2.62 mmol) and diisopropylamine (1.02 g, 7.86 mmol) in dichloromethane (5 mL) was added 1,1-carbonyldiimidazole (0.425 g, 2.62 mmol) at 20° C. under nitrogen atmosphere. The reaction system was concentrated to dryness under reduced pressure to give compound 4B.

[0138] Preparation of Compound 4C:

[0139] To a solution of compound 1K (300 mg, 869.83 μmol) and compound 4B (500 mg, 2590 μmol) in N,N-dimethylformamide (10 mL) was added diisopropylethylamine (450 mg, 3480 μmol) at 20° C. The reaction system was stirred at 100° C. for 12 h and then at 120° C. for 36 h. The reaction system was concentrated to dryness to give crude compound 4C. LCMS (ESI) m/z: 424 (M+1).

[0140] Preparation of Compound 4:

[0141] To a solution of compound 4C (0.357 g, 842.96 μmol) in dichloromethane (3 mL) was dropwise added trifluoroacetic acid (2 mL, 27.01 mmol) at room temperature. The reaction system was stirred at 20° C. for 2 h. The reaction system was concentrated. The residue was purified by preparative high performance liquid chromatography (column: Phenomenex Synergi C18 150×25×10 μm; mobile phase: 0.05% diluted hydrochloric acid as phase A, acetonitrile as phase B; elution gradient: 0%-25% acetonitrile, time: 10 min) to give compound 4. LCMS (ESI) m/z: 340 (M+1). .sup.1H NMR (400 MHz, deuterated methanol) 6 ppm 8.05-8.01 (m, 1H), 4.23-4.16 (m, 1H), 3.74-3.66 (m, 1H), 3.16-3.11 (m, 2H), 3.07-3.03 (m, 2H), 2.93 (s, 3H), 2.37-2.29 (m, 1H), 2.18-2.09 (m, 2H), 2.06 (m, 5H), 1.81-1.71 (m, 1H).

Experimental Example 1. Detection of Inhibitory Effect of Compounds Against Activity of Cdc7/DBF4 Kinase

[0142] Materials:

[0143] Cdc7/DBF4 kinase detection kit purchased from Promega; and

[0144] Nivo multi-marker analyzer (PerkinElmer).

[0145] Method:

[0146] An enzyme, a substrate, adenosine triphosphate and an inhibitor were diluted with the kinase buffer in the kit.

[0147] A test compound was serially 5-fold diluted to an 8th concentration, i.e., from 10 μM to 0.13 nM, with the DMSO concentration being 5%, and the duplicate well experiment was set up. To a microplate were added 1 μL of inhibitors of various concentration gradients, 2 μL of CDC7/DBF4 kinase (6.25 ng), 2 μL of a mixture of substrate and ATP (10 μM adenosine triphosphate, 0.2 μg/μL substrate), and the final concentration gradient of the compound was diluted from 2 μM to 0.025 nM. The reaction system was left reacting at 25° C. for 60 min. After the reaction was completed, 5 μL of ADP-Glo reagent was added to each well, and the reaction was continued at 25° C. for 40 min. After the reaction was completed, 10 μL of the kinase detection reagent was added to each well. After 30 min of reaction at 25° C., the chemiluminescence was read using a multi-marker analyzer, with an integration time of 0.5 s.

[0148] Data Analysis:

[0149] The original data were converted to inhibition rate using the equation (Sample−Min)/(Max−Min)×100%, and the IC.sub.50 value was then curve fitted using four parameters (obtained from the “log(inhibitor) vs. response-Variableslope” model in GraphPadPrism). Table 1 provides the inhibitory activity of the compounds disclosed herein against Cdc7/DBF4 kinase.

[0150] Results: see Table 1.

TABLE-US-00001 TABLE 1 Inhibitory activity of the compounds disclosed herein against Cdc7/DBF4 kinase Compound Cdc7/DBF4 IC.sub.50 Compound Cdc7/DBF4 IC.sub.50 number (nmol) number (nmol) Compound 1-1 1.49 Compound 2-2 1.41 Compound 1-2 2.8 Compound 3-1 0.6 Compound 2-1 0.95 Compound 3-2 1.97

[0151] Conclusion: The compounds disclosed herein exhibit good inhibitory activity against Cdc7/DBF4 kinase.

Experimental Example 2. Detection of Inhibitory Effect of Compounds Against Activity of Colo205 Cells

[0152] Materials:

[0153] 1640 medium; fetal bovine serum; penicillin/streptomycin antibiotics purchased from Wisent;

[0154] CellTiter-Glo (chemiluminescence detection reagent for cell viability) reagent purchased from Promega;

[0155] COLO205 cell line purchased from Wuhan Procell Life Science&Technology Co., Ltd; and

[0156] Nivo multi-marker analyzer (PerkinElmer).

[0157] Method:

[0158] COLO205 cells were plated on to white 96-well plates by adding 80 μL of cell suspension (containing 3000 COLO205 cells) to each well. The cell plate was incubated in a CO.sub.2 incubator overnight.

[0159] A test compound was serially 3-fold diluted to an 8th concentration, i.e., from 2 mM to 920 nM, and the duplicate well experiment was set up. 78 μL of medium was added to an intermediate plate, 2 μL of the serially diluted compound was transferred to corresponding wells of the intermediate plate, and after mixing, the mixture was transferred to the cell plate at 20 μL per well. The concentration of the compound transferred to the cell plate ranged from 10 μM to 4.57 nM. The cell plate was incubated in a CO.sub.2 incubator for 3 days. Another cell plate was read for signal values on the day of compound addition, and these values were used as the maximum values (the Max value in the equation below) in data analysis. The chemiluminescence detection reagent for cell viability was added to this cell plate at 25 μL per well and the luminescence signals were stabilized by incubation at room temperature for 10 min. Readings were taken using a multi-marker analyzer.

[0160] The chemiluminescence detection reagent for cell viability was added to the cell plate at 25 μL per well and the luminescence signals were stabilized by incubation at room temperature for 10 min. Readings were taken using a multi-marker analyzer.

[0161] Data Analysis:

[0162] The original data were converted to inhibition rate using the equation (Sample−Min)/(Max−Min)×100%, and the IC.sub.50 value was then curve fitted using four parameters (obtained from the “log(inhibitor) vs. response-Variableslope” model in GraphPadPrism). Table 2 provides the inhibitory activity of the compounds disclosed herein against COLO205 cell proliferation.

[0163] Results: see Table 2.

TABLE-US-00002 TABLE 2 Inhibitory activity of the compounds disclosed herein against COLO205 cell proliferation Compound number Colo205 IC.sub.50 (nmol) Compound 1-2 51.72

[0164] Conclusion: The compounds disclosed herein exhibit good inhibitory activity against COLO205 cells.