KR100197443B1 - 4-beta-(aminomethylcarbonyl)-4'-o-demethyl-4-o-epi-podophyllotoxin compound, preparation thereof and antitumor agent composition containing the same - Google Patents

Abstract

The present invention is excellent in water solubility and exhibits the effect of inhibiting the growth of cancer cells, so that it can be used as an anti-drug 4β- (aminomethylcarbonyl) -4′-Ο-demethyl-4-Ο-epi- of general formula (I) The present invention relates to a grapephytotoxin compound, a method for producing the same, and an anticancer composition comprising the same.

Description

[Name of invention]

4β- (aminomethylcarbonyl) -4′-Ο-demethyl-4-Ο-epi-podophyllotoxin compound, preparation method thereof and anticancer agent composition comprising same

Detailed description of the invention

[Purpose of invention]

The present invention is a compound having excellent water solubility and antitumor activity, 4β- (aminomethylcarbonyl) -4′-Ο-demethyl-4-Ο-epi-podophyllotoxin (4β- (aminomethy lcarbonyl) -4′- Ο-demethyl-4-Ο-epi-podophyl lotoxin) compound, a preparation method thereof, and an anticancer agent composition comprising the same.

[Technical field to which the invention belongs and the prior art in that field]

Etoposide and Teniposide compounds were isolated and purified semi-synthetically from the natural product Podophyllum peltatum L. (May apple, mandrake) by Sandoz Limited in the 1960s. After its development, Bristol-Myers Squibb Co. acquired its patent right and commercialized it in the 1970s. It is currently used in combination with radiation therapy for lung cancer, malignant lymphoma, leukoma, gastric cancer, etc. It is an anticancer agent showing excellent effects [C. Keller-Julsen, M. Kuhn, A. von Wartburg, J. Med. Chem. 1971, 14 (10), 936-940; C. Keller-Julsen, M. Kuhn, J. Renz, A. von Wartburg, US Patent 3,524,844, Aug. 18, 1970]. This compound is known to have an effect on cancer cells by inhibiting DNA synthesis by inhibition of human DNA Topoisomerase II [J. C. Wang, J. Boil., Chem., 1991, 266 (11), 6659-62.

However, the etoposide and teniposide have the disadvantage of being insoluble in water and toxic, and studies to modify the 4'-Ο-demethyl-4-Ο-epi-podophyllotoxin compound to improve this have continued in recent years. It is becoming. That is, Etopophos [Mark, GS Peter, DS John, FK GB 2,207,674 A] and NK-611 [Umezawa, U.S.A.] at Bristol-Myers Squibb and Japan's Microbial Chemistry Research Foundation. H. Takeuchi, T. Kondo, S. Tanaka, W. Takita, T. Nishimura, Y. Yoshikawa, H. EP 0,196,618, Mar. 26, 1986], is entering the clinical stage for the development of anticancer drugs. However, although these etophos and NK-611 compounds have improved water solubility and increased activity, the steps for synthesizing the final compound from the starting material 4'-Ο-demethyl-4-Ο-epi-podophyllotoxin Is long and the yield is very low, especially at some stages (e.g., the combination of 4'-0-demethyl-4-0-epi-podophyllotoxin and glucoside). The price is expected to be very high.

Accordingly, studies are underway to secure compounds having a simpler structure than etophos and NK-611, and having a water solubility and activity comparable to that of etophos and NK-611. In particular, 4-anilino-4-deoxy-4'-demethyl-epi-podophyllotoxin substituted with an aniline derivative instead of glucoside (4-anilino-4-deoxy-4'-demethyl-epi-podophyl lotoxin) Series [Lee, KS; Beers, S. A .; Mori, M .; Wang, Z-Q .; Kuo, Y-H .; et al. J. Med. Chem. 1990, 33, 1364-1368] is known to have a good anti-cancer effect is being developed continuously.

Recently, 4'-demethyl-epi-podophyllotoxin and a-amino acid are composed of amide bonds (Ma, W. Li, Y. Zhongguo Yaoqu Huaxe Zazhi, 1995, 5 (3), 169-175) and compound by amine linkage of amine group of [alpha] -amino acid and 4-bromo-4-deoxy-4'-demethyl-epi-podophyllotoxin [Yan-guang, Wang; Jian-Lin, Pan; Yao-Zu, Chen; Curr, Sci. 1996, 71 (4), 312-314] have also been reported to have excellent activity.

The present inventors have studied to obtain compounds having excellent anticancer activity and low toxicity as derivatives of 4'-demethyl-epi-podophyllotoxin having a relatively simple structure, and as a result, 4'-demethyl-epi-podophyllotoxin has been studied. The derivative | guide_body by the ester bond of the hydroxyl group of and the carboxylic acid of (alpha)-amino acid was synthesize | combined, and this invention was completed.

[Technical problem to be achieved]

That is, an object of the present invention is to provide an epi-podophyllotoxin compound having excellent water solubility and excellent antitumor activity, a method for preparing the same, and an anticancer composition comprising the compound.

[Configuration and Function of Invention]

In order to achieve the above object, the present invention provides a 4β- (amino methylcarbonyl) -4′-Ο-demethyl-4-Ο-epi-podophyllotoxin compound of the general formula (I) and salts thereof.

Wherein R ₁ is hydrogen atom, hydroxy, halogen, nitro, cyano, mercapto, amino, C ₁ -C ₁₀ alkyl, C ₂ -C ₇ alkenyl, C ₁ -C ₇ alkylamino, C ₁ -C ₇ dialkylamino, C ₂ -C ₁₀ alkenoylamino, C ₁ -C ₇ alkylimino, C ₁ -C ₇ alkylthio, C ₁ -C ₇ alkylmercapto, C ₁ -C ₇ alkylsulphenyl, C ₁ -C ₇ alkylsulfinyl, C ₁ -C ₇ alkylsulfonyl, C ₁ -C ₇ alkyl sulfates, C ₁ -C ₇ alkyl sulfonamide, carboxyl, C ₁ -C ₇ alkyl, carboxy ester, C ₁ -C ₇ Alkanoyl, C ₁ -C ₇ alkylaminocarbonyl or C ₁ -C ₇ alkylcarbamoyl, wherein alkyl refers to branched, straight and cyclic alkyl, optionally alkoxy, halogen, nitro, cyano, hydroxide May be substituted with a hydroxy, C ₁ -C ₇ hydroxy alkyl, C ₁ -C ₇ alkoxyalkyl or carboxyl group; R ₁ may also form a double bond with the α-carbon of the amino acid, ie C-2, with carbon, oxygen, sulfur or nitrogen atoms; When R ₁ is a carbon atom, it may be substituted by the same or different groups, each of which is a hydrogen atom, C ₁ -C ₇ alkyl, C ₂ -C ₇ alkenyl, C ₂ -C ₇ alkenoyl, C _1- C ₇ alkoxyalkyl, C ₁ -C ₇ hydroxyalkyl, C ₁ -C ₇ aminoalkyl, C ₁ -C ₇ alkylcarboxyester, C ₁ -C ₇ alkylurea, C ₁ -C ₇ alkylcarbonate, C _1- A C ₇ alkanoyl or C ₂ -C ₁₀ carbamoyl group; When R ₁ is a nitrogen atom, a hydrogen atom, C ₁ -C ₇ alkyl, C ₂ -C ₇ alkenyl, C ₂ -C ₇ alkenoyl, C ₁ -C ₇ alkoxyalkyl, C ₁ -C ₇ hydroxyalkyl, May be substituted by a C ₁ -C ₇ aminoalkyl, C ₁ -C ₇ alkylcarboxyester, C ₁ -C ₇ alkanoyl or C ₂ -C ₁₀ carbamoyl group, which may also be substituted by aryl instead of alkyl ; R ₁ and R ₂ may be interconnected to form a heterocyclic compound and a heterocyclic aryl compound containing nitrogen, oxygen and / or sulfur atoms; R ₂ and R ₃ may be the same or different from each other, and each hydrogen atom, nitro, cyano, hydroxy, carboxy, C ₁ -C ₇ alkyl, C ₁ -C ₇ alkoxy, C ₂ -C ₇ alkenyl, C ₁ -C ₇ alkanoyl, C ₁ -C ₁₀ alkyloxycarbonyl or C ₁ -C ₁₀ alkylaminocarbonyl, wherein alkyl may be substituted by halogen, nitro, cyano, hydroxy, instead of alkyl May be substituted with heterocyclic or aryl; R ₄ represents a hydrogen atom or P (O) (OM) ₂ , where M represents a hydrogen atom or an alkali metal; The stereochemistry of the two carbons is R or S.

In addition, in the present invention, 4'-Ο-protected-4'-demethyl-epi-podophyllotoxin of the following general formula (V) and 4'-Ο-protected-4'-demethyl-epi-podophyl lotoxin Condensing the N-protected-α-amino acid of general formula (VI) with dicyclohexylcarbodiimide in a solvent in the presence or absence of 4-dimethylaminopyridine and then deprotecting the reaction. It provides a compound of the general formula (I) and a method for producing a salt thereof.

Wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined for general formula (I), and Prot ₁ and Prot ₂ represent 0- and N-protecting groups, respectively.

Furthermore, the present invention provides an anticancer composition comprising 4β- (aminomethylcarbonyl) -4′-demethyl-4-Ο-epi-podophyllotoxin of the general formula (I) or a salt thereof.

Hereinafter, the present invention will be described in detail.

The compound of the general formula (I) of the present invention is a derivative produced by the ester bond of the hydroxyl group of 4'-Ο-demethyl-epi-podophyllotoxin and the carboxylic acid of α-amino acid, Can be prepared by

As shown in the above scheme, 4'-Ο-protected 4'-demethyl-epi-podophyllotoxin (formula (V) and N-protected α-amino acid (formula (VI) is dicyclohexylcarbodii) Compound of formula (II) is prepared by condensation reaction with mid (DCC, dicyclohexylcarbodiimide) in a suitable solvent in the presence or absence of 4-dimethylaminopyridine (DMAP).

The 4'-phenolic hydroxy group of 4'-demethyl-epi-podophyllotoxin in the compound of formula (V) is protected by benzyloxycarbonyl, benzoyl, benzyl, acetyl, haloacetyl, trialkylsilyl or alkoxycarbonyl Acetyl, chloroacetyl and benzyloxycarbonyl protecting groups are preferred among these.Kolar C., Moldenhauer H., Kneisi G., J. Carbohydrate Chemistry, 1990, 9 (5), 571- 583].

It is well known to protect the amino acid of general formula (VI) with benzyloxycarbonyl, substituted benzyloxycarbonyl, benzoyl, benzyl, acetyl, haloacetyl, alkoxycarbonyl or 9-fluorenylmethoxycarbonyl, Of these, tert-butoxycarbonyl, benzyloxycarbonyl and 9-fluorenylmethoxycarbonyl protecting groups are preferred. Synthesis of Optically Active α-amino Acids, Williams, RM, Pergamon: Oxford, 1989: Amine Protecting Groups . Geiger, R .; Konig, W. In The Peptides, Vol. 3, Academic Press, New York, 1981, Chapter 1].

Reaction solvents for the condensation reaction of a compound of formula (V) with a compound of formula (VI) include benzene, toluene, dichloromethane, chloroform, ether, tetrahydrofuran, acetonitrile, alkyl esters, dimethylformamide and dimethyl sulfoxide. Any solvent that does not participate in the reaction, including seeds, is possible, but dichloromethane, chloroform, ether and tetrahydrofuran are preferred. The reaction temperature is generally possible in the range of 20 to 100 ° C, but a temperature range of 20 to 50 ° C is preferred. Under these conditions, the reaction can be completed in 1 to 24 hours.

Subsequently, the compound of general formula (II) can be manufactured by N, O-deprotection reaction, and the compound of general formula (I) can be manufactured. At this time, according to the protecting group, remove the 4'-Ο-protecting group of the compound of formula (II), remove the 4'-0-protecting group and then remove the N-protecting group, or remove the N-protecting group and then remove the 4'-Ο- protecting group. The protecting group can be removed to prepare the compound of formula (I).

Compounds having benzyloxycarbonyl and benzyl protecting groups at the 0- and N-protecting group positions in the compound of formula (II) are subjected to hydrogenation of 5 psi to 60 psi, preferably 5 to 40 psi, using Pd-C as a catalyst. Selective deprotection is possible. Deprotection solvents include all solvents that do not participate in the hydrogenation reaction, including benzene, toluene, dichloromethane, chloroform, ether, tetrahydrofuran, acetonitrile, alkyl esters, alcohols, etc. Methanol and ethanol are preferred. The reaction temperature is generally possible in the range of 20 to 100 ° C, but a temperature range of 20 to 40 ° C is preferred. Under these conditions, the reaction can be completed in 1 to 24 hours.

The benzoyl, acetyl, haloacetyl or alkoxycarbonyl protecting groups of the compound of formula (II) may optionally be removed under organic base, inorganic base or acid conditions. At this time, trifluoroacetic acid / PhSH or 10% HBr / acetic acid solution and 3M HCl are preferable as the acid, and zinc acetate is preferable as the base. As the solvent of the deprotection reaction, any solvent which does not participate in the reaction, including benzene, toluene, dichloromethane, chloroform, ether, tetrahydrofuran, alcohol or water can be used, but chloroform, dichloromethane, ethanol, methanol and water This is preferred. The reaction temperature is generally possible in the range of 0 to 100 ° C, but a temperature range of 0 to 80 ° C is preferred. Under these conditions the reaction can be completed in 1 to 24 hours. N-deprotection reactions protected with 9-fluorenylmethoxycarbonyl groups are well known in the art. Atherton and R. C. Sheppard, The Fluorenylmethoxycarbonylamino Protecting Group, in The Peptides, S. Udenfriend and J. Meienhofer, Eds., Academic Press, Orlando, FL, 1987, vol. 9, pp. 1-38] can be selectively removed by reaction with 20% piperidine at room temperature in a solvent of dichloromethane, chloroform or dimethylformamide.

The compound of general formula (I) of this invention can raise the solubility to water by forming the salt of the amine compound with an acid. Organic acids and inorganic acids may be used as the acid forming the salt. Preferred organic acids include methanesulfonic acid, oxalic acid, tartaric acid, lactic acid, acetic acid, and the like, and hydrochloric acid, sulfuric acid, phosphoric acid, and the like.

The stereochemistry of the two carbons of the compound of formula (I) is (S) or (R).

The present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited only to the following Examples.

Example 1

Step 1: Synthesis of 4-[(2S) -benzyloxycarbonylleucineyl] -4'-Ο-benzyloxycarbonyl-4'-Ο-demethyl-epi-podophyllotoxin

N-benzyloxycarbonyl-L-leucine (570 mg) and 4'- o-benzyloxycarbonyl-4'- o-demethyl- epi-podophyllotoxin (880 mg) are dissolved in 20 ml of dichloromethane. I was. DCC (443 mg) and DMAP (262 mg) were added to the solution, stirred at room temperature for 48 hours, and the resulting urea was filtered off. The filtrate was washed twice with water, dried over MgSO ₄ , dichloromethane and distilled under reduced pressure to obtain a solid compound with CHCl ₃ / n-hexane, followed by silica gel column chromatography with ethyl acetate: hexane = 1: 1 to give a pure white solid compound. Got.

Step 2: Synthesis of 4- "benzyloxycarbonyl- (2S) -leucineyl" -4'- o-demethyl-epi-podophyllotoxin (Compound No. 9)

4- [benzyloxycarbonyl-L-leucineyl] -4′-Ο-benzyloxycarbonyl-4′-Ο-demethyl-epi-podophyllotoxin (700 mg) prepared in step 1 was ethyl acetate. After dissolving in 70 ml, 5% Pd-C (70 mg) was added. Hydrogenation was performed for 30 minutes at room temperature at a hydrogen pressure of 10 psi. After removing the catalyst Pd (C) by filtration, the solvent was distilled under reduced pressure and solidified with CHCl ₃ / n-hexane to obtain compound (9).

Step 3: Synthesis of 4-"(2S) -leucineyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 10)

4-'benzyloxycarbonyl- (2S) -leucineyl'-4′-Ο-demethyl-epi-podophyllotoxin (662 mg) prepared in step 2 was dissolved in 40 ml of ethyl acetate and 10% Pd-C (220 mg) was added. Hydrogenation was performed at room temperature for 1 hour at a hydrogen pressure of 30 psi. After Pd-C was removed by filtration, the solvent was distilled under reduced pressure and solidified with CHCl ₃ / n-hexane to obtain compound (10) as a white solid.

The following compounds were prepared in the same manner as in Example 1, and mp and ¹ H NMR data of the obtained compound were as follows.

Example 2

Step 1: Synthesis of 4-[(2S) -benzyloxycarbonylisoleucineyl] -4′-Ο-benzyloxycarbonyl-4′-Ο-demethyl-epi-podophyllotoxin

Step 2: Synthesis of 4- "benzyloxycarbonyl- (2S) -isoleucineyl" -4'- o-demethyl-epi-podophyllotoxin (Compound No. 7)

Step 3: Synthesis of 4-"(2S) -isoleucineyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 8)

Example 3

Step 1: Synthesis of 4-"(2S) -benzyloxycarbonylphenylalaninyl" -4'-Ο-benzyloxycarbonyl-4'-Ο-demethyl-epi-podophyllotoxin

Step 2: Synthesis of 4- "benzyloxycarbonyl- (2S) -phenylalaninyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 19)

Step 3: Synthesis of 4-"(2S) -phenylalaninyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 21)

Example 4

Step 1: Synthesis of 4-[(2S) -benzyloxycarbonylalaninyl] -4'-Ο-benzyloxycarbonyl-4'-Ο-demethyl-epi-podophyllotoxin

Step 2: Synthesis of 4- "benzyloxycarbonyl- (2S) -alaninyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 3)

Step 3: Synthesis of 4- (L-alaninyl) -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 4)

Example 5

Step 1: Synthesis of 4-"(2S) -benzyloxycarbonylvalinyl" -4'- o-benzyloxycarbonyl-4'- o-demethyl- epi-podophyllotoxin

Step 3: Synthesis of 4-"(2S) -valinyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 6)

Example 6

Step 1: Synthesis of 4-"(2S) -benzyloxycarbonylglycinyl" -4'- o-benzyloxycarbonyl-4'- o-demethyl-epi-podophyllotoxin

Step 2: Synthesis of 4- "benzyloxycarbonylglycinyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 1)

Step 3: Synthesis of 4- (glycineyl) -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 2)

Example 7

Step 1: Synthesis of 4-"(2S) -benzyloxycarbonylphenylglycinyl" -4'- o-benzyloxycarbonyl-4'- o-demethyl-epi-podophyllotoxin

Step 2: Synthesis of 4- "benzyloxycarbonyl- (2S) -phenylglycinyl" -4'- o-demethyl-epi-podophyllotoxin (Compound No. 15)

Step 3: Synthesis of 4-"(2S) -phenylglycinyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 17)

Example 8

Step 1: Synthesis of 4-"(2R) -benzyloxycarbonylphenylglycinyl" -4'- o-benzyloxycarbonyl-4'- o-demethyl-epi-podophyllotoxin

Step 3: Synthesis of 4-"(2R) -phenylglycinyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 18)

Example 9

Step 1: Synthesis of 4-"(2S) -benzyloxycarbonylprolinyl" -4'-0-benzyloxycarbonyl-4'-0-demethyl-epi-podophyllotoxin

Step 3: Synthesis of 4-"(2S) -prolinyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 24)

Example 10

Step 1: Synthesis of 4- "9-fluorenylmethoxycarbonyl- (2S) -tryptophanyl) -4'- o-benzyloxycarbonyl-4- o-demethyl- epi-podophyllotoxin

9 ml of 9-fluorenylmethoxycarbonyl-L-tryptophan (439 mg) and 4'-Ο-benzyloxycarbonyl-4'-Ο-demethyl-epi-podophyllotoxin (500 mg) in 20 ml of dichloromethane Dissolved in. DCC (387 mg) and DMAP (229 mg) were added to this solution, and the mixture was stirred at room temperature for 5 hours. The resulting urea was removed by filtration, the filtrate was washed twice with water, dried over MgSO ₄ and dichloromethane was distilled under reduced pressure. Solidified by n-hexane: EA = 1: 1 to obtain a pure white solid compound.

Step 2: 4- (9-Fluorenylmethoxycarbonyl- (2S) -tryptophanyl) -4′-Ο-demethyl-epi-podophyllotoxin (Compound No. 25)

4- (9-Fluorenylmethoxycarbonyl-L-tryptophanyl) -4′-Ο-benzyloxycarbonyl-4′-Ο-dimethyl-epi-podophyllotoxin prepared in step 1 (500 mg) Was dissolved in ethyl acetate (20 ml) and then 10% Pd (C) (100 mg) was added. The hydrogen pressure was 30 psi, and then hydrogenated at room temperature for 7 hours to remove Pd (C) by filtration. The solvent was distilled under reduced pressure and solidified with CHCl ₃ / n-hexane to give the compound (25).

Step 3: Synthesis of 4-"(2S) -tryptophanyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 26)

4- (9-Fluorenylmethoxycarbonyl-L-tryptophanyl) -4′-Ο-demethyl-epi-podophyllotoxin (Compound No. 25) (140 mg) prepared in step 2 was added to dichloromethane 5 After dissolving in ml, piperidine (342 μl) was added to the solution and stirred at room temperature for 4 hours. Silica gel column chromatography with n-hexane: EA = 2: 1 and solidification with EA / n-hexane gave pure compound (26).

The following compounds were prepared in the same manner as in Example 10, and mp and ¹ H NMR data of the compound were as follows.

Example 11

Step 1: Synthesis of 4- "9-fluorenylmethoxycarbonyl- (2S) -methioninyl" -4'-Ο-benzyloxycarbonyl-4-Ο-demethyl-epi-podophyllotoxin

Step 2: Synthesis of 4- "9-fluorenylmethoxycarbonyl- (2S) -methioninyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 11)

Step 3: Synthesis of 4-"(2S) -methioninyl" -4'-Ο-demethyl-epi-podophyllotoxin (Compound No. 12)

Example 12

Step 1: 4-'9-Fluorenylmethoxycarbonyl- ₂ -amino- (2S) -CH ₃ S (O) CH ₂ CH ₂ CHCO] -4'-Ο-benzyloxycarbonyl-4-Ο- Synthesis of Demethyl-Epi-Podophyllotoxin

Representative compounds of the compounds of formula (I) prepared as above are shown in Table 1.

Among the compounds of the general formula (I), particularly compounds showing excellent anticancer activity are 4β- (glycinyl) -4′-Ο-demethyl-4-Ο-epi-podophyllotoxin (Compound No. 2), 4β- "(2S) -alanineyl" -4'- O-demethyl-4- O-epi-podophyllotoxin (Compound No. 4), 4β-"(2R) -phenylalaninyl"-4'- o-demethyl -4-Ο-epi-podophyllotoxin (Compound No. 22), 4β-"(2S) -prolinyl" -4'- o-demethyl-4- o-epi-podophyllotoxin (Compound No. 24) .

It is effective to use the compound of the general formula (I) of the present invention prepared as described above as an injection solution containing an aqueous solution of the compound itself or a salt with an organic acid or an inorganic acid, and can be formulated as an oral administration agent such as tablets and capsules. have. These formulations may contain, in addition to the active ingredient, diluents, lubricants and the like. Adult daily doses may be administered one to several times in the range of 1-100 mg / kg.

[Test Example 1]

[Measurement of Anticancer Activity]

Cancer cell culture

The cells used to measure anticancer activity were A-549 (non-small cell lung cancer), SK-OV-3- (adenocarcinoma, ovarian malignancy), SK-MEL-2 (malignant melanoma, metastasis to the femoral skin), XF498 (Central nervous system tumor) and HCT15 (colon adenocarcinoma), all of these cancer cells were human-derived tumor cell lines that were distributed from the National Cancer Institute of the United States (NCI) and subcultured at the Korea Research Institute of Chemical Technology. These cells were all cultured in a 37 ° C. constant temperature and 5% CO, incubator using RPMI 1640 culture supplemented with 5% fetal bovine serum as culture. Cells were passaged once every 3-4 days, 0.25% trypsin and 3 mM trans-1,2-diaminocyclohexane-N, N, N in PBS (phosphate buffered saline) solution to separate cells from adherent surfaces. A solution in which N-tetraacetic acid (CDTA) was dissolved was used.

[Activity measurement]

In 1989, the US National Cancer Institute used the sulforhodamine B assay method developed to measure the in vitro anticancer activity of drugs. In other words, the cells were passaged from the implants using trypsin-CDTA solution for the passage of passaged cells in the experiment, and the number of cells per well was 5 x 10 in a plate (96 well microplate, manufactured by Falcon). (A549, HCT15), 1 x 10 (SK-MEL-2, XF498), 2 x 10 Aliquots were made to (SK-OV-3). The aliquoted cells were incubated for 24 hours in a CO incubator and attached to the bottom, followed by removal of the culture solution with an aspirator and a solution of compound (compound Nos. 1 to 26) diluted in the culture solution at a log dose of 6 concentrations. The cells were put in 3 multiples of 100 ml each into the wells containing cells, and further incubated for 48 hours. Dimethyl sulfoxide (DMSO) was used as needed to dissolve the compound. In addition, the diluted compound solution was filtered through a 0.22 ml filter prior to addition to the cells to maintain the sterile state of the experiment.

After incubating the cells with the drug for 48 hours, the culture solution of each well was removed, and 100 ml of 10% trichloroacetic acid (TCA) was added thereto, and the cells were left at 4 ° C. for 1 hour to fix the cells to the bottom of the plate. After the fixation of the cells, the plate was washed 5-6 times with water to completely remove the remaining TCA solution and dried at room temperature so that no remaining water was left. Completely dried plates were stained with 30 ml of 1% acetic acid solution per well in 0.4% SRB solution and stained for 30 minutes, and washed again 5-6 times with 1% acetic acid solution to remove SRB that did not bind to cells. Removed. The stained cell plates are then dried at room temperature and then shaken for 10 minutes with a titer plate shaker by adding 100 ml of 10 mM unbuffered trisma base solution per well. After elution, the absorbance at 520 nm was measured using a microplate reader.

To calculate the effect of the drug on cancer cells, the number of cells (Tz) at the time of drug addition, the number of cells (C) at 48 hours incubation with the drug-free medium, and the drug at each concentration 48 The cell number (T) and the like at the time of incubation were measured and calculated by the following formula:

or

Using the data regression of the LOTUS program from the calculated values, the concentrations at which the drug inhibits the growth of cancer cells by 50%, that is, ED ₅₀ was calculated to compare the anticancer activity of each drug.

Some of the above in vitro activity measurement results were compared with etoposide (BMS, USA), an anticancer substance used worldwide.

[Test Example 2]

[Measurement of Human Gene Phase Isomerization II Enzyme Inhibition]

[Preparation of DNA Phase Isomerase II]

The cell line used in this experiment used HeLa cells purchased from ATCC. That is, HeLa cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, and then HeLa cells were collected to collect 50 mM KH containing three volumes of 1 mM EDTA, 1 mM mercaptoethanol, 0.5 mM PMSF and 10% glycol. ₂ PO ₄ (pH 7.0) buffer was added to homogenize with polytron in ice bath for 30 seconds, 2 M K- (PO4) (pH 7.0) buffer was added to make final concentration 0.3 M and then homogenized again for 30 seconds. After leaving in an ice bath for a period of time, the supernatant obtained by centrifugation at 30,000 xg for 30 minutes is partially purified by a method such as phosphocellulose chromatography.

[P ₄ unknotting analysis]

An unknotting assay is performed for IC ₅₀ determination of the synthesized Topo II inhibitor. 50 mM HEPES, pH 7.0, 50 mM KCl, 0.1 mM EDTA, 100 mM NaCl, 10 mM MgCl ₂ , 100 μg / ml fetal bovine serum albumin, 1 mM ATP, 0.4 μg P ₄ knotted DNA Enzyme was added to make the total reaction solution 20 μl and reacted at 37 ° C. for 30 minutes. The reaction was stopped by addition of SDS to a final concentration of 1%, electrophoresis on 1% agarose gel equilibrated with 50 mM tris-borate (pH 8.3), 2.5 mM EDTA solution, followed by electrophoresis of 0.5 μg / ml ethidium bromide solution. Dyeing at, taking pictures under ultraviolet light to measure the activity. Enzyme activity was measured by staining in 0.4 μg / ml ethidium bromide solution and taking pictures under ultraviolet light. Enzyme activity is 1 unit in which 50% of 0.4 μg of notched P ₄ DNA is unknotted. IC ₅₀ determined by the above method was compared and searched with the existing Topo II inhibitor etoposide.

Activity measurement results are shown in Table 2 below.

As shown in Table 2 above, all compounds of the synthesized 4β- (aminomethylcarbonyl) -4'-Ο-demethyl-4-Ο-epi-podophyllotoxin (Formula I) were tested in five cell lines. It can be seen that both have cytotoxicity at lower concentration than the comparative compound epoxide. In addition, compounds of some of these compounds (Compound Nos. 2, 4, 22 and 24) show inhibitory activity against human gene isomerase II at concentrations comparable to those of the comparative compounds. Among these compounds, 4- (glycinyl) -4′-Ο-demethyl-epi-podophyllotoxin (Compound No. 2) shows an IC value at a lower concentration than the comparative compound. Summarizing these results, it can be seen that the inhibition of DNA phase isomerase II shows a large change depending on the substituents among the derivatives of α-amino acids, and excellent activity in the case of relatively small alkyl or aryl substituents.

[Effects of the Invention]

The 4-β- (aminomethylcarbonyl) -4′-O-demethyl-4-O-epi-dopophylutoxin compound of the general formula (I) of the present invention is a novel compound, wherein the amino group of α-amino acid is an ammonium salt. In this case, the solubility in water is increased, so that it is easy to be formulated, and it has similar or superior anticancer activity to that of the comparative compound etoposide.

Claims (4)

4-β- (aminomethylcarbonyl) -4′-O-demethyl-4-O-epi-podophyllotoxin compound of the general formula (I) and salts thereof; Wherein R ₁ is hydrogen atom, hydroxy, halogen, nitro, cyano, mercapto, amino, C ₁ -C ₁₀ alkyl, C ₂ -C ₇ alkenyl, C ₁ -C ₁₀ alkoxy, C ₁ -C ₇ Alkylamino, C ₁ -C ₇ dialkylamino, C ₂ -C ₁₀ alkenoylamino, C ₁ -C ₇ alkylimino, C ₁ -C ₇ alkylthio, C ₁ -C ₇ alkylmercapto, C _1- C ₇ alkyl sulfonic phenyl, C ₁ -C ₇ alkylsulfinyl, C ₁ -C ₇ alkylsulfonyl, C ₁ -C ₇ alkyl sulfates, C ₁ -C ₇ alkyl sulfonamide, carboxyl, C ₁ -C ₇ alkyl, carboxy Esters, C ₁ -C ₇ alkanoyl, C ₁ -C ₇ alkylaminocarbonyl or C ₁ -C ₇ alkylcarbamoyl, wherein alkyl refers to branched, straight and cyclic alkyl, optionally alkoxy, halogen Can be substituted with a nitro, cyano, hydroxy, C ₁ -C ₇ hydroxyalkyl, C ₁ -C ₇ alkoxyalkyl or carboxyl group; R ₁ may also form a double bond with the α-carbon of the amino acid, ie C-2, with carbon, oxygen, sulfur or nitrogen atoms; When R ₁ is a carbon atom, it may be substituted by the same or different groups, each of which is a hydrogen atom, C ₁ -C ₇ alkyl, C ₂ -C ₇ alkenyl, C ₂ -C ₇ alkenoyl, C ₁ -C ₇ alkoxyalkyl, C ₁ -C ₇ hydroxyalkyl, C ₁ -C ₇ aminoalkyl, C ₁ -C ₇ alkylcarboxyester, C ₁ -C ₇ alkylurea, C ₁ -C ₇ alkylcarbonate, C ₁ -C ₇ alkanoyl or C ₂ -C ₁₀ carbamoyl group; When R ₁ is a nitrogen atom, a hydrogen atom, C ₁ -C ₇ alkyl, C ₂ -C ₇ alkenyl, C ₂ -C ₇ alkenoyl, C ₁ -C ₇ alkoxyalkyl, C ₁ -C ₇ hydroxyalkyl, May be substituted by a C ₁ -C ₇ aminoalkyl, C ₁ -C ₇ alkylcarboxyester, C ₁ -C ₇ alkanoyl or C ₂ -C ₁₀ carbamoyl group, which may also be substituted by aryl instead of alkyl ; R ₁ and R ₂ may be interconnected to form a heterocyclic compound and a heterocyclic aryl compound comprising nitrogen, oxygen, and / or sulfur atoms; R ₂ and R ₃ may be the same or different from each other, and each hydrogen atom, nitro, cyano, hydroxy, carboxy, C ₁ -C ₇ alkyl, C ₂ -C ₇ alkenyl, C ₁ -C ₇ alkanoyl, C ₁ -C ₁₀ alkyloxycarbonyl or C ₁ -C ₁₀ alkylaminocarbonyl, wherein alkyl may be substituted by halogen, nitro, cyano, hydroxy, and also heterocyclic or aryl instead of alkyl May be substituted with; R ₄ represents a hydrogen atom or P (O) (OM) ₂ , where M represents a hydrogen atom or an alkali metal; The stereochemistry of the two carbons is R or S. The compound of claim 1, wherein 4β- (glycinyl) -4′-O-demethyl-4-O-epi-podophyllotoxin, 4β-[(2S) -alaninyl] -4′-O-demethyl- 4-O-epi-podophyllotoxin, 4β-[(2R) -phenylalaninyl] -4'-O-demethyl-4-O-epi-podophyllotoxin or 4β-[(2S) -prolinyl]- 4'-O-demethyl-4-O-epi-podophyllotoxin. 4'-O-protected -4'-demethyl-epi-podophyllotoxin tube of the following general formula (V) The N-protected-alpha-amino acid of the following general formula (VI) is dicyclohexylcarbodiimide 4β- (aminomethylcarbonyl) -4′-O-de according to claim 1, which comprises condensation reaction in a solvent with or without 4-dimethylaminopyridine and then deprotection. Method for preparing methyl-4-O-epi-podophyllotoxin compound. Wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined in claim _1, and Prot ₁ and Prot ₂ represent 0- and N-protecting groups, respectively. An anticancer agent composition comprising the 4β- (aminomethylcarbonyl) -4′-Ο-demethyl-4-Ο-epi-podophyllotoxin compound according to claim 1.