KR20010097480A - CDK inhibitors having a new structure - Google Patents

Abstract

The present invention relates to novel compounds of formula (I), pharmaceutically acceptable salts, hydrates, solvates and isomers thereof, useful as inhibitors of Cyclin Dependent Kinase (hereinafter referred to as "CDK"). The present invention also relates to an anticancer composition comprising the compound of formula 1 as an active ingredient together with a pharmaceutically acceptable carrier.

In the above formula

X represents hydrogen, halogen or C ₁ -C ₄ -alkyl,

Y represents nitro or substituted or unsubstituted amino,

Z represents hydrogen or C ₁ -C ₄ -alkyl.

Description

CDV inhibitors having a new structure

The present invention relates to novel compounds of formula (I), pharmaceutically acceptable salts, hydrates, solvates and isomers thereof, useful as inhibitors of Cyclin Dependent Kinase (hereinafter referred to as "CDK").

[Formula 1]

Wherein X represents hydrogen, halogen or C ₁ -C ₄ -alkyl,

Y represents nitro or substituted or unsubstituted amino,

Z represents hydrogen or C ₁ -C ₄ -alkyl.

The present invention also relates to an anticancer composition comprising the compound of formula 1 as an active ingredient together with a pharmaceutically acceptable carrier.

Full-scale research at the molecular level of cell division began to develop in the late 1980s through the study of frog egg division, characterization of various cell growths and radioactive mutations in yeast, and Rb, a tumor suppressor. In the 1990s, it was discovered that small cell cycle regulators regulate cell division processes such as cell growth, differentiation, development, aging, and apoptosis through their regulatory functions. It helped to understand the pathology more accurately. A representative example is cancer. In the process of transforming normal cells into cancer cells, cell growth regulation is often lost. In other words, many abnormal cell cycle regulators have been found in cancer cells, and some of them show a deep correlation between invasion / metastasis, which is the most problematic problem in cancer pathology. In particular, inducing overexpression or knock-out of cell growth regulators using a transgenic animal results in cell cycle deregulation of cancer from these cancers. It can be seen that is a direct cause of the cause.

Cell growth is under positive and negative control, as with all other biological controls. The major pathway of cell cycle regulation to date is due to the activity of cyclin-dependent kinases, and studies of many cancer cells and carcinogenesis mechanisms have shown that the positive or negative regulation of kinase activity leads to the occurrence of cancer. . That is, cancer may occur when balanced control is not achieved or timely control is not made.

Representative mammalian cyclin dependent kinases include cyclin dependent kinase 4 (CDK4), which is active in the mid-G1 phase of the cell cycle, CDK2, which is active in the mid-G1 and S phases, and CDC2, which is active in the G2-M phase ( CDK1). It is known that CDK4 and CDK2 are regulated by G1-S cell cycle checkpoint and CDC2 is regulated by G2-M checkpoint. Various cancer cells show abnormalities in the regulation of CDK4, CDK2, and CDC2 (CDK1), and it was confirmed that abnormalities of these enzymes, which are artificially induced in transgenic animals, actually cause cancer. Thus, these representative cyclin dependent kinases CDK4, CDK2, CDC2 (CDK1) are in very good positions as targets for anticancer agents.

The results reported so far regarding the association between these CDKs and cancer disease are described in more detail below.

The association between abnormal regulation of CDK4 activity and cancer development has been observed in several cancer tissues. Deletion of the p16 and p15 genes or overexpression of cyclin D1 has been identified in several cancers, suggesting that a malignant phenotype of cancer may occur if CDK4 activity is not regulated. In addition, p16 knockout mice have been reported to have a higher incidence of cancer than p53 knockout mice, indicating that the loss of p16 function in CDK4 regulation is the cause of cancer. These results suggest that p16 may play a role downstream in NIH 3T3 cells overexpressing Ras or src. Conversely, when the p16 or p21 were expressed in cells transformed with ras, it was observed that the modified phenotype is changed to the normal phenotype. These experimental evidences seem to prove that deregulation of CDK4 activity is a clear cause of cancer, and furthermore, it is likely to play a role in maintaining the phenotype of cancer cells. Therefore, the inhibitor of CDK4 is highly likely to show anticancer effect.

On the other hand, in the case of CDK2, overexpression of cyclin E is observed in some breast cancers, which is closely associated with metastasis of breast cancer, which inhibits cell death and reduces anchorage independent growth in serum conditions where cyclin E overexpression is low. Hyperproliferation and neoplasia of mammary epithelial cells was observed in transgenic animals overexpressing CDK2 using the MMTV promoter. It strongly suggests that it may be associated with, indicating that the inhibitor of CDK2 can act as an anticancer agent.

In addition, CDC2 (CDK1), CDK3, CDK5, CDK6, and CDK7 have been found to play an important role in each stage of cell division, and they are divided into cyclin dependent kinase (CDKs) families. In addition, in the case of cyclin, cyclins A, B, C, D2, D3, D4, F and G, in addition to cyclin D1 or cyclin E mentioned above, belong to the same family.

Based on these accumulated findings, the development of these inhibitors has recently begun in recognition that inhibitors that effectively inhibit these cyclin dependent kinases will be useful as anticancer agents.

Compounds effective as CDKs inhibitors developed so far include the flavopyridols of Formula 2 (see European Patent Nos. 0,241,003 (1987) and 0,366,061 (1990)).

In addition, CDKs inhibitors of formula (3) having a purine structure have recently been reported (WO 97/16447), and structurally completely different compounds of formula (4) have been reported to be effective CDK inhibitors (see WO 98). / 33798).

However, the CDK inhibitors developed to date have not yet exhibited sufficiently satisfactory effects. Thus, the inventors of the present invention have conducted intensive studies on inhibitors of these CDKs enzymes, particularly flavone-based compounds. It was confirmed that the compound of 1 effectively inhibits the above-described CDKs enzymes and completed the present invention.

Accordingly, an object of the present invention is to provide a novel compound that inhibits CDKs activity and an anticancer composition containing the compound as an active ingredient. CDKs include CDK2, CDK4 and CDC2 (CDK1), CDK3, CDK5, CDK6, CDK7, and the like, and cyclin cyclin D1 and cyclin E and cyclin A, B, C, D2, D3, D4, F, G It includes everything.

Hereinafter, the present invention will be described in detail.

The present invention relates to novel compounds of formula (1), pharmaceutically acceptable salts, hydrates, solvates and isomers thereof, which exhibit anticancer effects by inhibiting CDKs activity:

[Formula 1]

In the above formula,

X represents hydrogen, halogen or C ₁ -C ₄ -alkyl,

Y represents nitro or substituted or unsubstituted amino,

Z represents hydrogen or C ₁ -C ₄ -alkyl.

The compound of formula 1 according to the present invention may have an asymmetric carbon center and may include a double bond, depending on the type of substituent, so that the compound may exist as individual enantiomers, diastereomers or geometric isomers. It may also be present as a mixture thereof. Accordingly, such isomers or mixtures thereof are also within the scope of the present invention.

The compounds according to the invention can also form pharmaceutically acceptable salts. Such pharmaceutically acceptable salts include acids that form non-toxic acid addition salts containing pharmaceutically acceptable anions, such as inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, formic acid, Organic carbon acids such as citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, etc., sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid Acid addition salts formed by and the like are included.

Among the compounds represented by Chemical Formula 1, which exhibit CDKs inhibitory activity, a preferred compound is X represents hydrogen or chloro, Y represents nitro or substituted or unsubstituted amino, wherein the substituent of amino is p-nitrobenzoyl, (parent Pololin-4-yl) carbonyl, benzyl, p-toluenesulfonyl, 2,4-difluorophenylcarbamoyl and (4-methylpiperazin-1-yl) carbonyl, and Z is A compound representing hydrogen or methyl.

Representative compounds among the compounds of the formula (1) include the following compounds.

7-amino-2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one;

7-amino-6-chloro-2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one;

N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-nitrobenzeneamide;

7-amino-2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one;

N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-morpholinecarboxamide;

7- (benzylamino) -2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one;

N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-methylbenzenesulfonamide;

N- (2,4-difluorophenyl) -N '-[2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] urea;

N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-methyl-1-piperazinecarboxamide;

2- (3,4-dihydroxyphenyl) -3-hydroxy-8-methyl-7-nitro-4H-chromen-4-one;

7-amino-2- (3,4-dihydroxyphenyl) -3-hydroxy-8-methyl-4H-chromen-4-one; And

N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-8-methyl-4-oxo-4H-chromen-7-yl] -4-methyl-1-piperazinecarboxamide .

On the other hand, the compound of formula 1 according to the present invention can be prepared according to the method as described below. However, the reaction conditions such as the preparation method of the compound according to the present invention, for example, the reaction solvent, the base, and the amount of the reactant are not limited to those described below, but are described in the present specification or known in the art. Various combinations of the disclosed methods can be readily prepared by any combination, and such combinations are conventional techniques that are generalized to those skilled in the art.

Compound of Formula 1 according to the present invention was synthesized through the following synthetic route, it is shown in Schemes 1 to 7 by schematizing it.

As shown in Scheme 1, compound [7] obtained through compound [5] and compound [6] was mixed with 3 equivalents of piperonal and 3 equivalents of sodium hydroxide in an 80% ethanol aqueous solution as a solvent at room temperature. After reacting for 15 hours, compound [8] is prepared. Compound [8] was reacted with an excess of 10% aqueous sodium hydroxide solution and excess hydrogen peroxide in a methanol solvent at room temperature for 3 hours or more to prepare compound [9].

As shown in Scheme 2, compound [9] was reacted with at least 3 equivalents of borontribromide in methylene chloride solvent at room temperature for 2 hours to prepare the compound of Example 2. Meanwhile, compound [10] can be prepared by reducing compound [9] with palladium (Pd) under hydrogen pressure in a methanol solvent, and the obtained compound [10] in a methylene chloride solvent with at least 3 equivalents of borontribromide at room temperature. The compound of Example 1 was prepared by reacting for 2 hours.

As shown in Scheme 3, compound [10] is reacted with 3 equivalents of triethylamine and 3 equivalents of 4-nitrobenzoyl chloride in a methylene chloride solvent to prepare compound [11]. Compound thus obtained [11] ] Is reacted with at least 3 equivalents of borontribromide in a methylene chloride solvent at room temperature for 2 hours to prepare the compound of Example 3.

As shown in Scheme 4, compound [10] was obtained by reacting compound [10] in the same manner as in Scheme 3, except that 4-nitrobenzenesulfonyl chloride was used instead of 4-nitrobenzoyl chloride. To prepare the compound of Example 4. Also, except that morpholine-4-carbonyl chloride is used instead of 4-nitrobenzoyl chloride, the reaction is carried out in the same manner as in Scheme 3 to obtain Compound [13], to prepare the compound of Example 5.

As shown in Scheme 5, compound [10] is reacted with 1 equivalent of benzyl bromide in dimethylformamide solvent to prepare compound [14], and the obtained compound [14] is present in triethylamine base in methylene chloride solvent. Under reaction with paratoluenesulfonylchloride to prepare compound [15]. Subsequently, the obtained compound [15] was reacted with at least 3 equivalents of boron tribromide in a methylene chloride solvent at room temperature for 2 hours to prepare the compound of Example 7.

As shown in Scheme 6, compound [14] is reacted with triphosgene in methylene chloride solvent, followed by reaction with 2,4-difluoroaniline to prepare compound [16], and then obtained compound [16]. Was reacted with at least 3 equivalents of borontribromide in a methylene chloride solvent at room temperature for 2 hours to prepare the compound of Example 8.

As shown in Scheme 7, compound [18] was prepared by reacting compound [17] with 3 equivalents of piperonal and 3 equivalents of sodium hydroxide in an 80% aqueous ethanol solution as a solvent at room temperature for 15 hours. . Compound [19] was prepared by reacting the obtained compound [18] with an excess of 10% aqueous sodium hydroxide solution and excess hydrogen peroxide at room temperature for 3 hours. Reaction of the obtained compound [19] with triphosgene in a methylene chloride solvent followed by reaction with N-methylpiperazine affords compound [20], which is combined with at least 3 equivalents of borontribromide in methylene chloride solvent at room temperature. The compound of Example 12 was prepared by reacting for 2 hours.

As mentioned above, the above-described preparation method is only one example, and may be replaced by any other method as long as it shows the same result, and compounds not specifically mentioned may be easily prepared using the preparation method. . Also, which method is chosen is a matter at the discretion of the skilled person. For specific reaction conditions, reference may be made to the following Preparation Examples and Examples.

After the reaction is completed, the product can be separated and purified by conventional workup methods such as chromatography, recrystallization and the like.

The compound of formula 1 according to the present invention can be usefully used as an anticancer agent because of its excellent inhibitory activity against CDKs. Accordingly, the present invention provides another anticancer composition comprising a compound of Formula 1, a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof as an active ingredient together with a pharmaceutically acceptable carrier. The purpose.

The total daily dose to be administered to the host in a single dose or in separate doses when administering a compound of the present invention for clinical purposes is preferably in the range of 1 to 50 mg per kilogram of body weight, but the specific dose level for a particular patient is determined by the specific compound to be used, Weight, sex, health status, diet, time of administration, method of administration, rate of excretion, drug mixing and the severity of the disease may vary.

The compounds of the present invention can be administered in injectable and oral formulations as desired.

Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, can be prepared using suitable dispersing, wetting or suspending agents according to the known art. Solvents that can be used include water, Ringer's solution and isotonic NaCl solution, and sterile fixed oils are conventionally employed as a solvent or suspending medium. Any non-irritating fixed oil may be used for this purpose, including mono- and diglycerides, and fatty acids such as oleic acid may be used in the preparation of injectables.

Solid dosage forms for oral administration may be capsules, tablets, pills, powders and granules, and capsules and tablets are particularly useful. Tablets and pills are preferably prepared with enteric agents. Solid dosage forms are prepared by mixing the active compound of formula 1 according to the present invention with a carrier selected from one or more inert diluents such as sucrose, lactose, starch and the like, lubricants such as magnesium stearate, disintegrants and binders.

In the case where the compound of the present invention is to be clinically administered to obtain a desired anticancer effect, the active compound of Formula 1 may be administered simultaneously with at least one component selected from known anticancer agents. Anticancer agents that can be administered in combination with the compounds of the present invention in this manner include 5-fluorouracil, cisplatin, doxorubicin, taxol, gemcitabine, and the like.

However, the compound-containing preparation according to the present invention for the purpose of anticancer effect is not limited to those described above, and any agent useful for the treatment and prevention of cancer may be included.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, these Examples and Experimental Examples are only for better understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Preparation Example 1 Synthesis of 3-amino-4-chlorophenol

4-chloro-3-nitrophenol (13.0 g, 74.9 mmol) and tin chloride (32 g) were dissolved in ethanol (200 mL) and stirred at room temperature for 20 hours. After completion of the reaction, the solvent was concentrated, ethyl acetate (300 mL) was added to dissolve and washed with water (100 mL). The solvent was concentrated to give 9.7 g (90% yield) of the title compound.

¹ H NMR (CD ₃ OD, ppm); δ 6.94 (1H, d), 6.28 (1H, d), 6.09 (1H, dd)

FAB MS (m / e) = 144 [M ⁺ +1]

Preparation Example 2 Synthesis of 3- (acetylamino) -4-chlorophenyl Acetate

The compound (7.8 g, 54.3 mmol) obtained in Preparation Example 1 was dissolved in ethyl acetate (150 mL), acetic anhydride (15 mL) and triethylamine (15 mL) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction was washed with water (100mlx2) and the solvent was concentrated to give 8.9g (72% yield) of the title compound.

¹ H NMR (CDCl ₃ , ppm); δ 8.21 (1H, s), 7.64 (1H, s), 7.34 (1H, d), 6.80 (1H, d), 2.30 (3H, s), 2.27 (3H, s)

FAB MS (m / e) = 228 [M ⁺ +1]

Preparation Example 3 Synthesis of N- (4-acetyl-2-chloro-5-hydroxyphenyl) acetamide

Compound (8.80 g, 38.7 mmol) and trichloroaluminum (12.3 g) obtained in Preparation Example 2 were mixed and heated at 140 ° C. for 2 hours. After the reaction, methanol (70 mL) was added to dissolve and concentrated. It was dissolved in ethyl acetate (200 mL) again, washed with water (100 mL × 2), concentrated and washed with methylene chloride (20 mL) to give 5.62 g (yield 63.9%) of the title compound.

¹ H NMR (CDCl ₃ , ppm); δ 8.15 (1H, s), 7.70 (1H, s), 2.56 (3H, s), 2.26 (3H, s)

FAB MS (m / e) = 228 [M ⁺ +1]

Preparation Example 4 of N- {4-[(E) -3- (1,3-benzodioxol-5-yl) -2-propenyl] -2-chloro-5-hydroxyphenyl} acetamide synthesis

Compound (3.0 g, 13.2 mmol), piperonal (2.5 g) and sodium hydroxide (1.5 g) obtained in Preparation Example 3 were mixed in an 80% aqueous ethanol solution (50 mL) and stirred at room temperature for 15 hours. After completion of the reaction, water (100 mL) was added and stirred for about 10 minutes. The white solid obtained at this time was filtered, washed with water and diethyl ether and dried to yield 3.8 g (yield 80.0%) of the title compound.

¹ H NMR (DMSO-d ₆ , ppm); δ 9.49 (1H, s), 8.44 (1H, s), 7.90 (1H, s), 7.80- 7.71 (3H, m), 7.38 (2H, d), 7.01 (2H, d), 6.13 (2H, s ), 2.19 (3H, s)

FAB MS (m / e) = 360 [M ⁺ +1]

Preparation Example 5 Synthesis of 7-amino-2- (1,3-benzodioxol-5-yl) -6-chloro-3-hydroxy-4H-chromen-4-one

The compound (3.5 g, 9.72 mmol) obtained in Preparation Example 4 was dissolved in methanol (50 mL), 30% hydrogen peroxide solution (3 mL) and 10% aqueous sodium hydroxide solution (3 mL) were added, followed by stirring for 4 hours. After completion of the reaction, water (100 mL) was added and stirred for about 10 minutes. The white solid obtained at this time was filtered and washed with water and diethyl ether to obtain 3.2 g (yield 99.3%) of the title compound.

¹ H NMR (DMSO-d ₆ , ppm); δ 9.21 (1H, s), 7.83 (1H, s), 7.71 (1H, d), 7.67 (1H, s), 7.09 (1H, d), 6.85 (1H, s), 6.48 (2H, s), 6.11 (2H, s)

FAB MS (m / e) = 332 [M ⁺ +1]

Preparation Example 6 Synthesis of 7-amino-2- (1,3-benzodioxol-5-yl) -3-hydroxy-4H-chromen-4-one

The compound (3.0 g, 9.04 mmol) obtained in Preparation Example 5 was dissolved in methanol (50 mL), Pd / C (100 mg) was added, and the mixture was stirred under hydrogen pressure for 3 hours. Upon completion of the reaction, the solid was filtered off, concentrated and washed with methylene chloride (20 mL) to give 2.3 g (yield 85.5%) of the title compound.

¹ H NMR (DMSO-d ₆ , ppm); δ 8.51 (1H, d), 8.48 (1H, d), 8.42 (1H, s), 7.68 (1H, d), 7.42 (1H, d), 7.33 (1H, s), 6.74 (2H, s)

FAB MS (m / e) = 298 [M ⁺ +1]

Preparation Example 7 of 2- (1,3-benzodioxol-5-yl) -7-[(4-nitrobenzoyl) amino] -4-oxo-4H-chromen-3-yl 4-nitrobenzoate synthesis

The compound (100 mg, 0.34 mmol) obtained in Preparation Example 6 was dissolved in methylene chloride (30 mL), triethylamine (0.3 mL) and 4-nitrobenzoyl chloride (0.3 mL) were added, followed by stirring for about 3 hours. After completion of the reaction, the reaction was washed with water (30 mL × 2) and the solvent was concentrated. The residue was separated by column chromatography (ethyl acetate / n-hexane = 1/2, v / v) to give 70 mg (yield 35.0%) of the title compound.

¹ H NMR (CDCl ₃ , ppm); δ 8.40-8.32 (4H, m), 8.20 (1H, d), 7.92-7.88 (4H, m), 7.50-7.40 (2H, m), 7.38-7.25 (2H, m), 7.01 (1H, d) , 6.03 (2H, s)

FAB MS (m / e) = 596 [M ⁺ +1]

Preparation Example 8 2- (1,3-benzodioxol-5-yl) -7-{[(4-nitrophenyl) sulfonyl] amino} -4-oxo-4H-chromen-3-yl 4- Synthesis of Nitrobenzenesulfonate

80 mg of the title compound was obtained in the same manner as in Preparation Example 7, except that 120 mg (0.404 mmol) of the compound obtained in Preparation Example 6, and 0.3 ml of 4-nitrosulfonyl chloride instead of 4-nitrobenzoyl chloride were used. (Yield 26.6%) was obtained.

¹ H NMR (CDCl ₃ , ppm); δ 8.20 (1H, d), 8.17 (2H, d), 8.06 (2H, d), 7.95 (1H, d), 7.80 (2H, d), 7.66 (1H, d), 7.13 (1H, d), 6.88 (1H, s), 6.60 (1H, d), 6.54 (1H, d), 6.40 (1H, s), 5.81 (2H, s)

FAB MS (m / e) = 668 [M ⁺ +1]

Preparation Example 9 2- (1,3-benzodioxol-5-yl) -7-[(morpholin-4-yl) carbonylamino] -4-oxo-4H-chromen-3-yl (parent Synthesis of Polin-4-yl) carboxylate

Same as in Preparation Example 7, except that 110 mg (0.37 mmol) of the compound obtained in Preparation Example 6 was used and 0.32 ml of (morpholin-4-yl) carbonyl chloride was used instead of 4-nitrobenzoylchloride. This resulted in 90 mg (46.5% yield) of the title compound.

¹ H NMR (CDCl ₃ , ppm); δ 7.90 (1H, d), 7.81 (1H, s), 7.46 (1H, d), 7.34 (1H, s), 7.10 (1H, d), 6.91 (1H, d), 6.06 (2H, s), 3.80-3.66 (8H, m), 3.42-3.26 (8H, m)

FAB MS (m / e) = 524 [M ⁺ +1]

Preparation Example 10 Synthesis of 2- (1,3-benzodioxol-5-yl) -7- (benzylamino) -3- (benzyloxy) -4H-chromen-4-one

The compound (140 mg, 0.47 mmol) obtained in Preparation Example 6 was dissolved in dimethylformamide (30 mL), potassium carbonate (200 mg, 1.45 mmol) and benzyl bromide (0.2 mL, 1.68 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Stirred. After completion of the reaction, the resultant was concentrated, dissolved in ethyl acetate (40 mL), washed with water (30 mL × 2), and concentrated to give 130 mg (yield 57.9%) of the title compound.

¹ H NMR (CDCl ₃ , ppm); δ 8.10 (1H, d), 7.53 (1H, d), 7.47 (1H, s), 7.42-7.30 (10H, m), 6.85 (1H, d), 6.63 (1H, d), 6.52 (1H, s ), 6.03 (2H, s), 5.12 (2H, s), 4.67 (2H, s)

FAB MS (m / e) = 478 [M ⁺ +1]

Preparation Example 11 Synthesis of 7-amino-2- (1,3-benzodioxol-5-yl) -3- (benzyloxy) -4H-chromen-4-one

The compound (240 mg, 0.81 mmol) obtained in Preparation Example 6 was dissolved in dimethylformamide (30 mL), potassium carbonate (130 mg, 0.94 mmol) and benzyl bromide (0.1 mL, 0.84 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Stirred. After completion of the reaction, the resultant was concentrated, dissolved in ethyl acetate (40 mL), washed with water (30 mL × 2), and concentrated to give 220 mg (yield 70.1%) of the title compound.

¹ H NMR (CDCl ₃ , ppm); δ 8.04 (1H, d), 7.58 (1H, d), 7.43-7.30 (5H, m), 6.85 (1H, d), 6.69 (1H, d), 6.59 (1H, s), 6.05 (2H, s ), 5.10 (2H, s)

FAB MS (m / e) = 388 [M ⁺ +1]

Preparation Example 12 N- [2- (1,3-benzodioxol-5-yl) -3- (benzyloxy) -4-oxo-4H-chromen-7-yl] -4-methylbenzenesulfonamide Synthesis of

The compound (70 mg, 0.18 mmol) obtained in Preparation Example 11 was dissolved in methylene chloride (20 mL), paratoluenesulfonyl chloride (80 mg) and triethylamine (0.3 mL) were added thereto, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the mixture was washed with water (20 mL × 2), concentrated and separated by column chromatography (ethyl acetate / hexane = 3/1, v / v) to give 50 mg (yield 51.2%) of the title compound.

¹ H NMR (CDCl ₃ , ppm); δ 8.10 (1H, d), 7.76 (2H, d), 7.59 (1H, s), 7.49 (1H, s), 7.35-7.25 (8H, m), 7.16 (1H, s), 6.97 (1H, s ), 6.88 (1H, d), 6.06 (2H, s), 5.08 (2H, s), 2.38 (3H, s)

FAB MS (m / e) = 542 [M ⁺ +1]

Preparation Example 13: N- [2- (1,3-benzodioxol-5-yl) -3- (benzyloxy) -4-oxo-4H-chromen-7-yl] -N '-(2, Synthesis of 4-difluorophenyl) urea

The compound (75 mg, 0.19 mmol) obtained in Preparation Example 11 was dissolved in methylene chloride (30 mL), triphosphogen (150 mg) and triethylamine (0.3 mL) were added thereto, and the mixture was stirred at room temperature for 2 hours. Thereafter, 2,4-difluoroaniline (170 mg) was added and the mixture was further stirred for 2 hours. After completion of the reaction, the mixture was washed with water (20 mL × 2) and concentrated. The residue was separated by column chromatography (ethyl acetate / hexane = 2/1, v / v) to give 57 mg (yield 55.3%) of the title compound.

¹ H NMR (CD ₃ OD, ppm); δ 8.11 (1H, d), 7.83 (2H, d), 7.39 (1H, d), 7.32 (1H, d), 7.22 (1H, s), 7.03-6.98 (5H, m), 6.90 (1H, d ), 6.63-6.57 (2H, m), 5.78 (2H, s), 4.76 (2H, s)

FAB MS (m / e) = 543 [M ⁺ +1]

Preparation Example 14 N- [2- (1,3-benzodioxol-5-yl) -3- (benzyloxy) -4-oxo-4H-chromen-7-yl] -4-methyl-1- Synthesis of Piperazine Carboxamide

The compound (65 mg, 0.17 mmol) obtained in Preparation Example 11 was dissolved in methylene chloride (25 mL), followed by addition of triphosgene (140 mg) and triethylamine (0.2 mL), followed by stirring at room temperature for 2 hours. Thereafter, N-methylpiperazine (150 mg) was added and the mixture was further stirred for 2 hours. After completion of the reaction, the mixture was washed with water (25 mL × 2) and concentrated. The residue was separated by column chromatography (ethyl acetate) to give 47 mg (53.8%) of the title compound.

¹ H NMR (CDCl ₃ , ppm); δ 8.12 (1H, d), 8.00 (1H, s), 7.61 (1H, d), 7.52 (1H, s), 7.36-7.25 (5H, m), 7.04 (1H, d), 6.85 (1H, d ), 6.15 (1H, s), 6.03 (2H, s), 5.10 (2H, s), 3.57-3.55 (4H, m), 2.48-2.46 (4H, m), 2.33 (3H, s)

FAB MS (m / e) = 514 [M ⁺ +1]

Preparation Example 15 Synthesis of 1- (2-hydroxy-3-methyl-4-nitrophenyl) -1-ethanone

2-Methyl-3-nitrophenol (1.59 g, 10.4 mmol), trichloroaluminum (1.38 g) and acetylchloride (1.3 mL) were mixed in nitrobenzene (13 mL) and heated at 120 ° C. for 2 hours. After the reaction, the mixture was diluted with ethyl acetate and the solid was filtered and then extracted using 1N aqueous sodium hydroxide solution (100 mL). The mixture was neutralized with 1N aqueous hydrochloric acid solution, extracted with ethyl acetate (100 mL), and concentrated to give 1.02 g (yield 50.2%) of the title compound.

¹ H NMR (CDCl ₃ , ppm); δ 7.72 (1H, d), 7.29 (1H, d), 2.71 (3H, s), 2.36 (3H, s)

FAB MS (m / e) = 196 [M ⁺ +1]

Preparation Example 16 (E) -3- (1,3-benzodioxol-5-yl) -1- (2-hydroxy-3-methyl-4-nitrophenyl) -2-propen-1-one Synthesis of

Compound (0.9g, 4.61mmol), piperonal (1.2g) and sodium hydroxide (300mg) obtained in Preparation Example 15 were mixed in an 80% aqueous ethanol solution (20ml) and stirred at room temperature for 15 hours. After completion of the reaction, water (20 ml) was added and the mixture was stirred for about 10 minutes. The white solid obtained at this time was filtered, washed with water and diethyl ether and dried to obtain 0.8 g (yield 53.0%) of the title compound.

¹ H NMR (DMSO-d ₆ , ppm); δ 7.72 (1H, d), 7.54 (1H, d), 7.48 (1H, d), 7.32 (1H, s), 7.29 (1H, d), 7.12 (1H, d), 6.92 (1H, s), 6.13 (1H, s), 2.71 (3H, s), 2.36 (3H, s)

FAB MS (m / e) = 328 [M ⁺ +1]

Preparation Example 17 Synthesis of 2- (1,3-benzodioxol-5-yl) -3-hydroxy-8-methyl-7-nitro-4H-chromen-4-one

The compound (0.7 g, 2.14 mmol) obtained in Preparation Example 16 was dissolved in methanol (30 mL), 30% hydrogen peroxide solution (1 mL) and 10% aqueous sodium hydroxide solution (1 mL) were added, followed by stirring for 4 hours. After the reaction was completed, water (70 mL) was added thereto, followed by stirring for about 10 minutes. The white solid obtained at this time was filtered and washed with water and diethyl ether to give 520 mg (yield 71.2%) of the title compound.

¹ H NMR (DMSO-d ₆ , ppm); δ 8.11 (1H, d), 7.90 (1H, d), 7.78 (1H, s), 7.66 (1H, d), 6.93 (1H, d), 6.10 (2H, s), 2.69 (3H, s)

FAB MS (m / e) = 342 [M ⁺ +1]

Preparation Example 18 Synthesis of 7-amino-2- (1,3-benzodioxol-5-yl) -3-hydroxy-8-methyl-4H-chromen-4-one

The compound (200 mg, 0.59 mmol) obtained in Preparation Example 17 was dissolved in methanol (30 mL), Pd / C (10 mg) was added thereto, and the mixture was stirred under hydrogen pressure for 2 hours. The solid formed after the reaction was filtered, concentrated and washed with methylene chloride (10 mL) to give 160 mg (86.8% yield) of the title compound.

¹ H NMR (DMSO-d ₆ , ppm); δ 7.75 (1H, d), 7.68 (1H, s), 7.64 (1H, d), 7.11 (1H, d), 6.75 (1H, d), 6.11 (2H, s), 6.06 (2H, s), 2.26 (3H, s)

FAB MS (m / e) = 312 [M ⁺ +1]

Preparation Example 19 2- (1,3-benzodioxol-5-yl) -8-methyl-7-{[(4-methyl-1-piperazinyl) carbonyl] amino} -4-oxo-4H Synthesis of -chromen-3-yl 4-methyl-1-piperazincarboxylate

The compound (100 mg, 0.32 mmol) obtained in Preparation Example 18 was dissolved in methylene chloride (30 mL), triphosphene (200 mg) and triethylamine (0.2 mL) were added, and the mixture was stirred at room temperature for 2 hours. Thereafter, N-methylpiperazine (0.2 ml) was added to the reaction solution, followed by further stirring for 1 hour. After completion of the reaction, the mixture was washed with water (20 mL × 2) and separated by column chromatography (ethyl acetate) to obtain 80 mg (yield 44.3%) of the title compound.

¹ H NMR (CDCl ₃ , ppm); δ 8.04 (1H, d), 7.80 (1H, d), 7.74 (1H, s), 7.43 (1H, d), 6.98 (1H, d), 6.06 (2H, s), 3.58-3.54 (8H, m ), 2.60-2.56 (2H, s), 2.48 (3H, s), 2.35 (3H, s), 2.32 (3H, s)

FAB MS (m / e) = 564 [M ⁺ +1]

Example 1: Synthesis of 7-amino-2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one

The compound (25 mg, 0.084 mol) obtained in Preparation Example 6 was dissolved in methylene chloride (5 ml) and reacted with an excess of boron tribromide (BBr ₃ , 0.1 ml) at room temperature for 2 hours. The reaction solution was concentrated and the resulting solid was washed with methylene chloride and 10% methanol / methylene chloride, filtered and dried to give 15 mg (62.6%) of the title compound.

¹ H NMR (DMSO-d ₆ , ppm); δ 8.49 (1H, d), 8.45 (1H, d), 8.41 (1H, s), 7.65 (1H, d), 7.42 (1H, d), 7.33 (1H, s)

FAB MS (m / e) = 286 [M ⁺ +1]

Example 2: Synthesis of 7-amino-6-chloro-2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one

15 mg (yield 61.7%) of the title compound were obtained in the same manner as in Example 1, except that 25 mg (0.076 mmol) of the compound obtained in Preparation Example 5 was used instead of the compound obtained in Preparation Example 6.

¹ H NMR (DMSO-d ₆ , ppm); δ 7.82 (1H, s), 7.63 (1H, s), 6.87 (1H, d), 6.80 (1H, s), 6.62 (1H, d), 6.44 (2H, s)

FAB MS (m / e) = 320 [M ⁺ +1]

Example 3: Synthesis of N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-nitrobenzeneamide

15 mg (yield 69.0%) of the title compound were obtained in the same manner as in Example 1, except that 30 mg (0.050 mmol) of the compound obtained in Preparation Example 7 was used instead of the compound obtained in Preparation Example 6.

¹ H NMR (DMSO-d ₆ , ppm); δ 8.41-8.39 (3H, m), 8.17 (2H, d), 8.15-8.13 (2H, m), 7.89 (1H, d), 7.62 (1H, d), 6.98 (1H, d)

FAB MS (m / e) = 435 [M ⁺ +1]

Example 4: Synthesis of 7-amino-2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one

11 mg (yield 57.8%) of the title compound were obtained in the same manner as in Example 1, except that 35 mg (0.052 mmol) of the compound obtained in Preparation Example 8 was used instead of the compound obtained in Preparation Example 6.

¹ H NMR (DMSO-d ₆ , ppm); δ 7.79 (1H, d), 7.76 (1H, d), 7.62 (1H, dd), 7.01- 6.94 (2H, m)

FAB MS (m / e) = 364 [M ⁺ +1]

Example 5: Synthesis of N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-morpholinecarboxamide

Except for using the compound 34mg (0.065mmol) obtained in Preparation Example 9 instead of the compound obtained in Preparation Example 6 was carried out in the same manner as in Example 1 to obtain 13mg (yield 50.2%) of the title compound.

¹ H NMR (DMSO-d ₆ , ppm); δ 7.88 (1H, d), 7.78 (1H, s), 7.42 (1H, d), 7.30 (1H, s), 7.05 (1H, d), 6.91 (1H, d), 3.65-3.55 (4H, m ), 3.30-3.23 (4H, m)

FAB MS (m / e) = 399 [M ⁺ +1]

Example 6: Synthesis of 7- (benzylamino) -2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one

19 mg (yield 65.7%) of the title compound were obtained in the same manner as in Example 1 except for using 37 mg (0.077 mmol) of the compound obtained in Preparation Example 10 instead of the compound obtained in Preparation Example 6.

¹ H NMR (DMSO-d ₆ , ppm); δ 7.74 (1H, d), 7.61 (1H, s), 7.46-7.34 (5H, m), 7.28 (1H, d), 6.85 (1H, d), 6.81 (1H, d), 6.49 (1H, s ), 4.41 (3H, s)

FAB MS (m / e) = 376 [M ⁺ +1]

Example 7: Synthesis of N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-methylbenzenesulfonamide

12 mg (yield 31.4%) of the title compound were obtained in the same manner as in Example 1, except that 47 mg (0.087 mmol) of the compound obtained in Preparation Example 12 was used instead of the compound obtained in Preparation Example 6.

¹ H NMR (DMSO-d ₆ , ppm); δ 7.96 (1H, d), 7.80-7.73 (4H, m), 7.37-7.34 (3H, m), 7.13 (1H, d), 6.96 (1H, s), 2.35 (3H, s)

FAB MS (m / e) = 440 [M ⁺ +1]

Example 8: N- (2,4-difluorophenyl) -N '-[2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7- Synthesis of urea

12 mg (yield 30.3%) of the title compound were obtained in the same manner as in Example 1, except that 49 mg (0.090 mmol) of the compound obtained in Preparation Example 13 was used instead of the compound obtained in Preparation Example 6.

¹ H NMR (DMSO-d ₆ , ppm); δ 9.60 (1H, s), 8.71 (1H, s), 8.10 (2H, d), 8.01 (1H, d), 7.71 (1H, s), 7.59 (1H, d), 7.35 (1H, t), 7.21 (1H, d), 7.10 (1H, t), 6.89 (1H, d)

FAB MS (m / e) = 441 [M ⁺ +1]

Example 9: N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-methyl-1-piperazinecarboxamide Synthesis of

12 mg (yield 37.4%) of the title compound were obtained in the same manner as in Example 1, except that 40 mg (0.078 mmol) of the compound obtained in Preparation Example 14 was used instead of the compound obtained in Preparation Example 6.

¹ H NMR (DMSO-d ₆ , ppm); δ 8.05-8.00 (2H, m), 7.69 (1H, s), 7.56 (1H, d), 7.43 (1H, d), 6.89 (1H, d), 4.32-4.28 (4H, m), 3.53-3.41 (4H, m), 2.84 (3H, s)

FAB MS (m / e) = 412 [M ⁺ +1]

Example 10 Synthesis of 2- (3,4-Dihydroxyphenyl) -3-hydroxy-8-methyl-7-nitro-4H-chromen-4-one

80 mg (yield 55.3%) of the title compound were obtained in the same manner as in Example 1 except for using 150 mg (0.439 mmol) of the compound obtained in Preparation Example 17 instead of the compound obtained in Preparation Example 6.

¹ H NMR (DMSO-d ₆ , ppm); δ 8.10 (1H, d), 7.92 (1H, d), 7.78 (1H, s), 7.62 (1H, d), 6.90 (1H, d), 2.69 (3H, s)

FAB MS (m / e) = 330 [M ⁺ +1]

Example 11: Synthesis of 7-amino-2- (3,4-dihydroxyphenyl) -3-hydroxy-8-methyl-4H-chromen-4-one

The compound (32 mg, 0.097 mmol) obtained in Example 10 was dissolved in methanol (20 mL), Pd / C (8 mg) was added, and the mixture was stirred under hydrogen pressure for 2 hours. After completion of the reaction, the solid was filtered and concentrated to give 21 mg (yield 72.3%) of the title compound.

¹ H NMR (CD ₃ OD, ppm); δ 7.80 (1H, s), 7.76 (1H, d), 7.73 (1H, d), 6.90 (1H, d), 6.78 (1H, d), 2.33 (3H, s)

FAB MS (m / e) = 300 [M ⁺ +1]

Example 12 N- [2- (3,4-Dihydroxyphenyl) -3-hydroxy-8-methyl-4-oxo-4H-chromen-7-yl] -4-methyl-1-pipe Synthesis of Razine Carboxamide

12 mg (yield 34.0%) of the title compound was obtained in the same manner as in Example 1, except that 47 mg (0.083 mmol) of the compound obtained in Preparation Example 19 was used instead of the compound obtained in Preparation Example 6.

¹ H NMR (CD ₃ OD, ppm); δ 7.93 (1H, d), 7.51 (1H, d), 7.39-7.37 (2H, m), 7.02 (1H, d), 3.61-3.58 (4H, m), 3.54-3.51 (4H, m), 2.37 (3H, s)

FAB MS (m / e) = 426 [M ⁺ +1]

Experimental Example 1

Inhibitory Activity of CDK2 and CDK4

Experiments on CDK2 inhibitory activity can be found in the Kitagawa method [Kitagawa, M. et al., Oncogene 9 ; 2549, 1994], and CDK4 inhibitory activity was measured by Carlson method (Carlson, BA et al., Cancer Research 56 ; 2473, 1996].

Active CDK2 / cyclin A is a combination of histidine-labeled human CDK2 protein and cyclin A protein, purified from insect cells infected simultaneously with baculovirus expressing His-CDK2 gene and baculovirus expressing cyclin A gene. A unit activity of 14 nmole / min / mg and a Km of ATP of 22 µM were used. Active CDK4 / cyclin D1 is a combination of human CDK4 protein and cyclin D1 protein linked to GST (glutathione-S-transferase), expressed and purified in insect cells, with a unit activity of 57 nmole / min / mg and a Km for ATP of 940 μM. Was used. As the substrate of the enzyme, amino acids 780 to 928 at the C-terminus of the human RB protein were labeled with GST protein at the N-terminus thereof, expressed in bacteria, and purified.

CDK2 / cyclin A and CDK4 / cyclin D1 enzyme activity measurements were performed as follows. About 100 ng of enzyme was added for 30 min at 30 ° C. in 100 μl of 20 mM Tris (pH 8.0), 100 mM NaCl, 10 mM MgCl ₂ buffer, including 20 μg of GST-RB protein, 100 μM ATP, 5 μCi p32-γ-ATP Reacted. Then EDTA solution was added to the concentration was 20mM to terminate the enzyme reaction. 30 μl of 50% glutathione beads (purchased from Pharmacia) were then added to attach the GST-RB to the beads, which were then washed three times with 20 mM Tris (pH 8.0), 100 mM NaCl, 10 mM EDTA solution and scintillation count. counting) was measured. In order to analyze the inhibitory ability of the compound, an appropriate concentration of inhibitor was added to the enzyme reaction solution and enzyme activity was measured according to the above-described method.

Inhibitory activity against CDK2 and CDK4 of the compound of formula 1 according to the present invention measured according to this method is shown as an IC50 value (see Table 1 below).

Experimental Example 2

Acute Toxicity Test

To investigate the acute oral toxicity of the compounds obtained in Examples 1 and 5, solutions containing the compounds in different concentrations were administered orally at a dose of 10 ml per kg to male mice of the ICR strain. After oral administration, mortality and 7 days of symptoms were observed, and the median lethal dose (LD50, mg / kg) was calculated according to the Litchfield-Wilcoxon method and the results are shown in Table 2 below.

Test compound LD50 (mg / kg) Example 1 > 3,000 Example 5 > 3,000

Claims (4)

Compounds of Formula 1, pharmaceutically acceptable salts, hydrates, solvates and isomers thereof: [Formula 1] In the above formula X represents hydrogen, halogen or C ₁ -C ₄ -alkyl, Y represents nitro or substituted or unsubstituted amino, Z represents hydrogen or C ₁ -C ₄ -alkyl. The compound of claim 1, wherein X represents hydrogen or chloro, Y represents nitro or substituted or unsubstituted amino, wherein the substituent of amino is p-nitrobenzoyl, (morpholin-4-yl) carbonyl , Benzyl, p-toluenesulfonyl, 2,4-difluorophenylcarbamoyl and (4-methylpiperazin-1-yl) carbonyl and Z represents hydrogen or methyl. The method of claim 1, 7-amino-2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one; 7-amino-6-chloro-2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one; N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-nitrobenzeneamide; 7-amino-2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one; N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-morpholinecarboxamide; 7- (benzylamino) -2- (3,4-dihydroxyphenyl) -3-hydroxy-4H-chromen-4-one; N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-methylbenzenesulfonamide; N- (2,4-difluorophenyl) -N '-[2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] urea; N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-4-oxo-4H-chromen-7-yl] -4-methyl-1-piperazinecarboxamide; 2- (3,4-dihydroxyphenyl) -3-hydroxy-8-methyl-7-nitro-4H-chromen-4-one; 7-amino-2- (3,4-dihydroxyphenyl) -3-hydroxy-8-methyl-4H-chromen-4-one; And N- [2- (3,4-dihydroxyphenyl) -3-hydroxy-8-methyl-4-oxo-4H-chromen-7-yl] -4-methyl-1-piperazinecarboxamide Compound selected from among. An anticancer composition comprising a compound of formula (I), a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof as defined in claim 1 as an active ingredient with a pharmaceutically acceptable carrier.