JPWO2006080463A1 - Cancer therapeutic agent and recurrence preventive agent using vitamin K hydroquinone derivative - Google Patents

Abstract

Provided is an excellent cancer therapeutic agent, cancer recurrence preventive agent or action enhancer containing at least one of carboxylic acid esters of vitamin K hydroquinone represented by the following general formula (I) or a salt thereof. Wherein R1 and R2 are each a hydrogen atom, or an amino acid, N-acylamino acid, N-alkylamino acid, N, N-dialkylamino acid, pyridinecarboxylic acid and their hydrohalides, alkylsulfonates or sugars. Represents a carboxylic acid residue having a nitrogen substituent selected from the residues of acid salts, or a dicarboxylic acid residue selected from residues of dicarboxylic acids and alkali metal salts thereof, and at least one of R 1 and R 2 represents a nitrogen substituent. (R3 represents a hydrogen atom or a group represented by the general formula (II) or the general formula (III), and n represents an integer of 1 to 14).

Description

Related applications

  This application claims the priority of Japanese Patent Application No. 2005-22301 filed on Jan. 28, 2005, and is incorporated herein.

  The present invention relates to a drug applied to cancer diseases, particularly a cancer therapeutic agent using a vitamin K hydroquinone derivative, a cancer preventive agent, and an auxiliary agent for enhancing the action of a quinone anticancer agent.

Natural vitamin K is phylloquinone (vitamin K ₁ ) and menaquinone-4 (vitamin K _{2 (20)} ), but these natural vitamins K are Prothrombin and other γ-carboxyglutamic acid residues (Gla). It is essential for biosynthesis of vitamin K-dependent proteins and is used as a hemostatic agent and an osteoporosis therapeutic agent. In the biosynthesis of vitamin K-dependent proteins, vitamin K is converted to vitamin K hydroquinone, a two-electron reductant, and an enzyme that converts glutamic acid residues (Glu) to γ-carboxyglutamic acid residues (Gla) (γ-glutamyl carboxylase) It is known to act as a cofactor for When vitamin K is deficient or when the vitamin K cycle is inhibited by a coumarin-based drug such as warfarin, glazing is incomplete and deγ-carboxylated vitamin K-dependent protein is produced.

On the other hand, phylloquinone (vitamin K ₁ ) and menaquinone-4 (vitamin K _{2 (20)} ), which are natural vitamin K, are known to have an antitumor effect on cancer cells (Non-patent Documents 1 and 2). In particular, menaquinone-4 (vitamin K _{2 (20)} ) releases abnormal prothrombin (DCP, Des-γ-Carboxy Prothrombin) in which Glu is not converted to Gla as in the case of vitamin K deficiency. It is known to have an antitumor effect and an inhibitory effect on portal vein invasion of hepatocellular carcinoma (JP 2004-107330, Non-Patent Document 3). Furthermore, menaquinone-4 is known to have an antitumor effect due to cell differentiation inducing action (Japanese Patent Laid-Open No. 6-305955). In addition, it is known that vitamin K3, which is a synthetic vitamin K, and derivatives thereof have an antitumor effect against hepatocellular carcinoma. However, it has been reported that the antitumor effect of natural vitamin K is very low compared to vitamin K3 and its derivatives (see Non-patent documents 1 and 2).

On the other hand, vitamin Ks are compounds that do not dissolve in water at all. In oral administration, solubility becomes the rate-determining process of bioavailability, and solubilization by adding a large amount of nonionic surfactant is used for preparing water-soluble preparations of vitamin Ks. However, addition of a large amount of nonionic surfactant may cause serious problems such as anaphylactic shock. Therefore, when it is administered repeatedly, its harmfulness cannot be completely eliminated.
As described above, natural vitamin K has an antitumor effect. However, it has been confirmed that the remarkable anticancer effect is limited to hepatocellular carcinoma, the anticancer effect is relatively low, and water dissolution. The present situation such as low bioavailability due to sex is an obstacle to effectively exerting anticancer action. Accordingly, there is a strong demand for the development of a drug that can effectively exert an anticancer effect by having an anticancer effect on various cancers, a high anticancer effect, and a high bioavailability.

The present inventors have produced a vitamin K hydroquinone which is an active vitamin K without undergoing a reduction process after administration of a vitamin K hydroquinone derivative having a specific structure, exhibiting high bioavailability and causing water insolubility of vitamin K Have already been disclosed (Patent No. 3088137, Non-Patent Documents 5 and 6). However, it is not clear whether vitamin K hydroquinone derivatives show an anticancer effect.
JP2004-107330 Japanese Patent No. 3088137 Wu et al., Life Sci., 52, 1797-1804 (1993). Wang et al., Hepatology, 22, 876-882 (1995). Otsuka et al., Hepatology, 40, 243-251 (2004). Nishikawa et al., J. Biol. Chem., 270, 28304-28310 (1995). Takata et al., Pharm Res., 12, 18-23 (1995). Takata et al., Pharm. Res., 12, 1973-1979 (1995).

  An object of the present invention is to provide an anticancer agent using a compound having a specific structure that is highly water-soluble and can be converted into vitamin K hydroquinone, which is an active vitamin K, without undergoing a reduction process after administration, and exhibit high bioavailability. It is to provide an agent for preventing cancer recurrence.

（式中、R_１およびR₂はそれぞれ水素原子、またはアミノ酸、N-アシルアミノ酸、N-アルキルアミノ酸、N,N-ジアルキルアミノ酸、ピリジンカルボン酸及びそれらのハロゲン化水素酸塩、アルキルスルホン酸塩または糖酸塩の残基から選ばれる窒素置換基を有するカルボン酸残基、またはジカルボン酸及びそのアルカリ金属塩の残基から選ばれるジカルボン酸残基を表し、R_１,
R₂の少なくとも一方は窒素置換基を有するカルボン酸残基、またはジカルボン酸残基である。R₃は水素原子または下記一般式（II）

もしくは下記一般式（III）

で示される基を表す。nは１〜１4の整数を意味する。）で表されるビタミンＫヒドロキノンのカルボン酸エステル類またはその塩。As described above, the present inventors converted the vitamin K hydroquinone derivative into vitamin K hydroquinone, which is an active vitamin K, without undergoing a reduction process after administration, and exhibited high bioavailability, resulting in water insolubility of vitamin K. Have already been reported to overcome the above-mentioned problems and to exhibit excellent effects on hypoprothrombinemia (Patent No. 3088137, Non-Patent Documents 5 and 6). As a result of further examination of the effectiveness against other diseases, the present inventors have found that vitamin K hydroquinone derivatives are effective as therapeutic agents for various cancers and preventive agents for recurrence, thereby completing the present invention. The vitamin K hydroquinone derivative is represented by the following general formula (I).
Formula (I)

Wherein R ₁ and R ₂ are each a hydrogen atom, or an amino acid, an N-acyl amino acid, an N-alkyl amino acid, an N, N-dialkyl amino acid, a pyridinecarboxylic acid, and their hydrohalides and alkylsulfonates. Or a carboxylic acid residue having a nitrogen substituent selected from residues of sugar salts, or a dicarboxylic acid residue selected from residues of dicarboxylic acids and alkali metal salts thereof, R ₁ ,
At least one of R ₂ is a carboxylic acid residue having a nitrogen substituent or a dicarboxylic acid residue. R ₃ is a hydrogen atom or the following general formula (II)

Or the following general formula (III)

Represents a group represented by n means an integer of 1 to 14. Vitamin K hydroquinone carboxylic acid esters or salts thereof.

  That is, this invention provides the anticancer agent and cancer preventive agent containing at least 1 sort (s) of the carboxylic acid ester of vitamin K hydroquinone represented by the said general formula (I), or its salt.

  As described above, according to the cancer disease drug according to the present invention, by applying the carboxylic acid ester of vitamin K hydroquinone or a salt thereof, it is possible to exert excellent therapeutic and preventive effects on various cancers. .

It is explanatory drawing which shows the growth inhibitory effect with respect to hepatocellular carcinoma (PLC / PRF / 5) by the vitamin K hydroquinone derivative concerning this invention. It is explanatory drawing which shows the growth inhibitory effect with respect to the lung cancer cell (A549) by the vitamin K hydroquinone derivative concerning this invention. It is explanatory drawing which shows the influence with respect to the caspase-3 / 7 activity of the leukemia cell (HL60) by the vitamin K hydroquinone derivative concerning this invention. It is explanatory drawing which shows the growth inhibitory effect with respect to gastric cancer cell (SDT4) by the vitamin K hydroquinone derivative concerning this invention. It is explanatory drawing which shows the growth inhibitory effect with respect to the mitomycin C resistant gastric cancer cell (ST4) by the vitamin K hydroquinone derivative concerning this invention. It is explanatory drawing which shows the growth inhibitory effect with respect to a colon cancer cell (HT29) by the vitamin K hydroquinone derivative concerning this invention. It is explanatory drawing which shows the effect | action enhancement effect with respect to the anticancer effect | action of the quinone type anticancer agent by the vitamin K hydroquinone derivative concerning this invention. It is explanatory drawing which shows the anticancer effect | action with respect to the mouse transplant human hepatocellular carcinoma by the vitamin K hydroquinone derivative concerning this invention.

Hereinafter, a preferred embodiment of the present invention will be described in detail.
The present invention relates to an anticancer agent and a cancer recurrence preventing agent containing the compound represented by the general formula (I) or a salt thereof. The compound represented by the general formula (I) can be contained alone in the preparation, or can be blended in the preparation as a salt thereof. In the present invention, a carboxylic acid residue R ₁ having a nitrogen substituent,
Include the following can be exemplified as R _2.
A hydrogen atom relative to a nitrogen atom;
1 or 2 alkyl groups for the nitrogen atom;
Acyl group for the nitrogen atom.
Examples of the alkyl group include straight-chain or branched alkyl groups having 1 to 6 carbon atoms, and the following are exemplified.
Methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, isopropyl group, isobutyl group, 1-methylpropyl group, tert-butyl group, 1-ethylpropyl group, isoamyl Group.
The alkyl group is particularly preferably a methyl group or an ethyl group. Moreover, the hydrocarbon chain in the case of having an acyl group can be similarly defined.

The amino group and the carbonyl group are preferably bonded with a linear, branched or cyclic alkylene group having 1 to 7 carbon atoms. Examples of the branched alkylene group include the following.
Derived from alkyl groups such as isopropyl, isobutyl, tert-butyl, 1-ethylpropyl and the like.
Examples of the cyclic alkylene group include the following.
Containing a cyclopentane ring, cyclohexane ring, or methylcyclohexane ring in the structure.
As the alkylene group, a methylene group or an ethylene group is particularly preferable.

As the hydrohalide, hydrochloride, hydrobromide and the like are preferable. In the present invention, the hydrohalide salt is often crystallized or solidified, and has the advantage that it is easy to handle during formulation.
Examples of other salts include the following.
Examples of the alkyl sulfonate include methane sulfonate, and examples of the saccharide include gluconate, glucoheptanoate, and lactobionate.

In the present invention, the dicarboxylic acid residue R ₁ ,
R ₂ is selected from the residues of dicarboxylic acids and their alkali metal salts. The carbonyl groups of the dicarboxylic acid residues are bonded by a linear alkylene group having 2 to 4 carbon atoms. Particularly preferred as the alkylene group is an ethylene group. Sodium salts and potassium salts are preferred as the alkali metal salts.

In the present invention, in the compound represented by the general formula (I), R ₁ and R ₂ are each selected from a hydrogen atom, a carboxylic acid residue having the nitrogen substituent, or the dicarboxylic acid residue. Group. R ₁ ,
At least one of R ₂ is the carboxylic acid residue having the nitrogen substituent or the dicarboxylic acid residue, more preferably a carboxylic acid residue having a nitrogen substituent.

In the present invention, various methods for producing the compound represented by the general formula (I) are conceivable. Typical methods are as follows.

Vitamin Ks represented by the general formula (IV) are reduced with a reducing agent to form vitamin K hydroquinone represented by the general formula (V), and the vitamin K hydroquinone and a carboxylic acid having a nitrogen substituent, or a reaction thereof. The target substance (I) of the present invention can be obtained by conducting an esterification reaction with a basic acid derivative or a hydrohalic acid salt thereof by a conventional method. The reducing agent used here reduces the naphthoquinone skeleton of vitamin Ks to the naphthohydroquinone skeleton, and the following are exemplified.
Sodium borohydride, sodium hydrosulfite, tri-n-butylphosphine, zinc chloride, stannous chloride.

  The esterification reaction of vitamin K hydroquinone follows a conventional method, but when esterifying an amino acid having a primary, secondary amino group or hydroxyl group or thiol group in the side chain, a tert-butoxycarbonyl group (hereinafter referred to as t-BOC group). Abbreviation), benzyloxycarbonyl group (hereinafter abbreviated as Z group), 9-fluorenylmethoxycarbonyl group (hereinafter abbreviated as FMOC group). It is preferable to carry out the reaction in the presence of an active esterification reagent such as dicyclohexylcarbodiimide (hereinafter abbreviated as DCC) or N, N-disuccinimide oxalate (hereinafter abbreviated as DSO) using a hydrohalide. Give the result. An anhydrous pyridine is preferable as a reaction solvent in the reaction. In the method using a reactive acid derivative, a method using acid halogenite, particularly acid chloride is particularly preferable. The reaction solvent in this case is preferably an anhydrous benzene-anhydrous pyridine mixture. Hydrohalates, alkyl sulfonates and saccharides are produced by reacting free vitamin K hydroquinone nitrogen-containing carboxylic acid ester with hydrohalic acid, alkyl sulfonic acid, and acidic sugar lactone by a conventional method. Further, after the N-acylamino acid ester is produced, the hydrohalide can be produced by deprotection with hydrohalic acid by a conventional method.

Hereinafter, although the more concrete Example of this invention is described, this invention is not limited to these.
Examples 1-28
Vitamin K hydroquinone derivatives shown in Tables 1 to 5 were produced by the methods shown in the following production methods A to G. Moreover, the mass spectrum (ionization method; FD method and FAB method) and ¹ H-NMR spectrum value of the obtained substance are shown in Tables 6-8.

[Production Method A]
0.1 mol of amino acid is dissolved in 100 ml of distilled water-dioxane (1: 1 v / v), 30 ml of triethylamine is added, di-tert-butyl dicarbonate is gradually added and stirred for 30 minutes at room temperature. Dioxane is distilled off under reduced pressure, 50 ml of aqueous sodium hydrogen carbonate solution (0.5 M) is added, and the mixture is washed with 100 ml of ethyl acetate. The ethyl acetate layer was washed with 50 ml of sodium bicarbonate solution, and the aqueous layers were combined and acidified (pH 3) with an aqueous citric acid solution (0.5 M) under ice-cooling, saturated with sodium chloride, and extracted with ethyl acetate. (100ml x 3 times). The extract is dehydrated with anhydrous sodium sulfate and the solvent is distilled off under reduced pressure. Isopropyl ether is added to the oily residue or crystallized by cooling to obtain Nt-BOC-amino acid. Dissolve 6.75 mmol vitamin K in 40 ml isopropyl ether, add 47 mmol sodium borohydride dissolved in 15 ml methanol and stir at room temperature until the yellow color of the solution is colorless. Add 60 ml of isopropyl ether and 100 ml of distilled water to the reaction solution, separate the isopropyl ether layer, extract 100 ml of isopropyl ether to the aqueous layer, extract the soluble fraction, combine the isopropyl ether layers and dehydrate with anhydrous sodium sulfate. Concentrate under reduced pressure. N-hexane is added to the residue to precipitate a white precipitate to obtain vitamin K hydroquinone.

  Vitamin K hydroquinone, N-t-BOC-amino acid (13.55 mmol) and DCC (13.55 mmol) are added to anhydrous pyridine (50 ml), and the mixture is stirred at room temperature for 20 hours. The solvent is distilled off under reduced pressure, and ethyl acetate is added to the residue to extract a soluble fraction (100 ml × 2 times). The extract is concentrated under reduced pressure, and the residue is separated and purified by silica gel column chromatography (eluent: n-hexane-isopropyl ether) to obtain vitamin K hydroquinone-1,4-bis-Nt-BOC-amino acid. Vitamin K hydroquinone-1,4-bis-Nt-BOC-amino acid is dissolved in a small amount of acetone, and hydrochloric acid-dioxane (2.5-4.0N) is added in an amount corresponding to the amount of hydrochloric acid about 20 times the amount of ester 1 After stirring for a period of time, the solvent is distilled off under reduced pressure. The residue is recrystallized with acetone-methanol system to obtain hydrochloride salt of vitamin K hydroquinone-1,4-bis-amino acid ester.

[Production Method B]
Dissolve 6.75 mmol vitamin K in 40 ml isopropyl ether, add 47 mmol sodium borohydride dissolved in 15 ml methanol and stir at room temperature until the yellow color of the solution is colorless. Add 60 ml of isopropyl ether and 100 ml of distilled water to the reaction solution, separate the isopropyl ether layer, add 100 ml of isopropyl ether to the aqueous layer, extract the soluble fraction, combine the isopropyl ether layers, dehydrate with anhydrous sodium sulfate, and then reduce the pressure. Concentrate underneath. N-hexane is added to the residue to precipitate a white precipitate to obtain vitamin K hydroquinone. Vitamin K hydroquinone, 13.55 mmol of N, N-dialkylamino acid hydrochloride and 13.55 mmol of DCC are added to 50 ml of anhydrous pyridine and stirred at room temperature for 20 hours. The solvent is distilled off under reduced pressure, the residue is suspended in distilled water, sodium bicarbonate is added to adjust the pH of the solution to 7-8, and the mixture is extracted with ethyl acetate (100 ml × 3 times). The extract was dehydrated with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was separated and purified by silica gel column chromatography (eluent: isopropyl ether-ethyl acetate) to give vitamin K hydroquinone-1,4-bis-N, N-dialkyl amino acid ester is obtained.

[Production Method C]
6.75 mmol of vitamin K is dissolved in 40 ml of isopropyl ether, 50 mmol of hydrosulfite sodium is dissolved in 50 ml of distilled water, and the mixture is stirred at room temperature until the isopropyl ether turns brown and becomes colorless. The isopropyl ether layer is separated, and 100 ml of isopropyl ether is added to the aqueous layer to extract a soluble fraction. The isopropyl ether layers are combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. N-hexane is added to the residue to precipitate a white precipitate to obtain vitamin K hydroquinone. 6.75 mmol of N, N-dialkylamino acid hydrochloride and 6.75 mmol of DCC are added to vitamin K hydroquinone and stirred in 50 ml of anhydrous pyridine for 20 hours. The solvent is distilled off under reduced pressure, the residue is suspended in distilled water, sodium bicarbonate is added to adjust the pH of the solution to 7-8, and the mixture is extracted with ethyl acetate (100 ml × 3 times). The extract was dehydrated with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was separated and purified by silica gel column chromatography (eluent: isopropyl ether-ethyl acetate, 3: 2) to give vitamin K hydroquinone-1-N, N-dialkyl amino acid esters and vitamin K hydroquinone-4-N, N-dialkyl amino acid esters are obtained.

[Production Method D]
Dissolve 6.75 mmol vitamin K in 40 ml isopropyl ether, add 47 mmol sodium borohydride dissolved in 15 ml methanol and stir at room temperature until the yellow color of the solution is colorless. Add 60 ml of isopropyl ether and 100 ml of distilled water to the reaction solution, separate the isopropyl ether layer, add 100 ml of isopropyl ether to the aqueous layer, extract the soluble fraction, combine the isopropyl ether layers, dehydrate with anhydrous sodium sulfate, and then reduce the pressure. Concentrate underneath. N-hexane is added to the residue to precipitate a white precipitate to obtain vitamin K hydroquinone. Vitamin K hydroquinone is dissolved in 30 ml of anhydrous benzene-anhydrous pyridine (1: 1, v / v), pyridine carboxylic acid chloride hydrochloride is added, and the mixture is stirred at room temperature for 3 hours. Insoluble materials are removed by filtration, and the filtrate is concentrated under reduced pressure. The residue is suspended in 100 ml of distilled water, sodium hydrogen carbonate is added (pH 7-8), and the soluble fraction is extracted in ethyl acetate (100 ml × 3 times). The extract is concentrated under reduced pressure, and the residue is separated and purified by silica gel column chromatography (eluent: isopropyl ether-ethyl acetate, 9: 1) to obtain vitamin K hydroquinone-1,4-bis-pyridinecarboxylic acid ester.

[Production Method E]
Vitamin K hydroquinone-1,4-bis-N, N-dialkylamino acid ester or 2 mmol of vitamin K hydroquinone-1,4-bis-pyridinecarboxylic acid is dissolved in 20 ml of acetone, and hydrochloric acid-dioxane (2.5-4.0N) is dissolved in hydrochloric acid. The amount corresponding to 10 times the molar amount of ester is added, the solvent is distilled off under reduced pressure, the residue is recrystallized with acetone-methanol, vitamin K hydroquinone-1,4-bis-N, N-dialkylamino acid or vitamin The hydrochloride of K hydroquinone-1,4-bis-pyridinecarboxylic acid is obtained.

[Production Method F]
2 mmol of vitamin K hydroquinone-1,4-bis-N, N-dialkylamino acid or vitamin K hydroquinone-1,4-bis-pyridinecarboxylic acid is dissolved in 20 ml of dichloromethane, and 2 mmol of alkylsulfonic acid is added and stirred. The precipitated crystals are collected by filtration to obtain an alkyl sulfonate of vitamin K hydroquinone-1,4-bis-N, N-dialkylamino acid ester or vitamin K hydroquinone-1,4-bis-pyridinecarboxylic acid ester.

[Production method G]
Vitamin K4.55mmol is dissolved in isopropyl ether 40ml, sodium borohydride 31.5mmol is methanol 15
Dissolve in ml and add at room temperature until the yellow color of the solution is colorless. Add 60 ml of isopropyl ether and 100 ml of purified water to the reaction solution, separate the isopropyl ether layer, add 100 ml of isopropyl ether to the aqueous layer, extract the soluble fractions, combine the isopropyl ether layers, and dehydrate with anhydrous sodium sulfate. The solvent is distilled off under reduced pressure. To the residue were added 8.97 mmol of dimethylaminopyridine and 18.0 mmol of succinic anhydride, and isopropyl ether-dioxane (6: 4,
v / v) Dissolve in 100 ml, stir at room temperature for 3 hours, react for 2 hours while heating to 50-60 ° C, and further react for 10 hours while allowing to cool at room temperature. 100 ml of purified water is added to the reaction solution, the isopropyl ether layer is separated, dehydrated with anhydrous sodium sulfate, and the solvent is distilled off under reduced pressure. The residue is suspended in isopropyl ether, and 100 ml of ethyl acetate and 100 ml of purified water are added to the precipitate obtained by centrifugation, and the ethyl acetate-soluble fraction is extracted, dehydrated with anhydrous sodium sulfate, and the solvent is distilled off under reduced pressure. The residue is suspended in isopropyl ether, and the insoluble material is recrystallized with ethyl acetate to obtain vitamin K hydroquinone-1,4-bis-succinate.

  Next, in order to specifically describe the present invention, application examples will be given below, but the present invention is not limited thereto.

In order to show the usefulness of the compound of the present invention as an anticancer agent and a cancer recurrence preventing agent, experimental examples of growth inhibition by various human cultured cell systems and experimental examples of the inhibitory effect on mouse transplanted human cancer in vivo are given.
Human cultured cancer cells used in the experiment were PLC / PRF / 5 (hepatocellular carcinoma), HepG2 cells (hepatocellular carcinoma), Hep3B cells (hepatocellular carcinoma), A549 (lung cancer), HL60 (leukemia), SDT4 cells (stomach cancer) ), ST4 cells (gastric cancer, mitomycin C resistant strain), HT29 cells (colon cancer), HT29 / MMC cells (colon cancer, mitomycin C resistant strain).

PLC / RPF / 5 cells, Hep3B cells, and HepG2 cells, which are human hepatocellular carcinomas, are Dulbecco's containing 10% fetal bovine serum, penicillin, and streptomycin.
Modified Eagle's medium (DMEM) medium is used, SDT4 cells, ST4 cells, HT29 cells, HT29 / MMC cells use 10% fetal bovine serum, RPMI1640 medium containing kanamycin, HL60 cells use 10% fetal bovine serum, penicillin, streptomycin And subcultured using RPMI1640 medium.

Evaluation method 1: Evaluation of proliferation inhibition effect by cell number evaluation using WST-8
PLC / RPF / 5 cells, Hep3B cells, HepG2 cells, and A549 cells are seeded at 0.5 × 10 ⁴ cells / well in a 96-well plate, cultured for 24 hours, and then the medium is menatetrenone, menadione, compound No.
Change to medium supplemented with 10, 11, 12, 24, 25, 29, remove the medium containing the drug after culturing at 37 ° C., 5% CO ₂ for 24 hours, 48 hours, 72 hours, and do not contain the drug After adding WST-8 reagent and culturing for 2 hours, 450 nm,
The number of cells was measured by measuring the absorbance at 655 nm, and the cell growth inhibitory effect was evaluated.

Evaluation method 2: Evaluation of growth inhibition effect by inhibition of ³ H-thymidine uptake
Hep3B cells and HepG2 cells were seeded at 2 × 10 ⁴ cells / well in a 24 well-plate, cultured for 24 hours, and then the medium was menatetrenone, menadione, compound no.
The medium was replaced with a medium supplemented with 10, 11, 12, 24, 25 and cultured for 3 days. The medium was replaced with a medium containing ³ H-thymidine 0.5mμCi / mL, and after culturing for 4 hours, the medium was removed, and the cells were washed twice with isotonic phosphate buffer, and then lysed.
Cells were lysed with 400 μL of Buffer. The cell lysate was transferred to a scintillation vial, a scintillation cocktail was added, the radioactivity was measured with a liquid scintillation counter, and the cell growth inhibitory effect was evaluated from the inhibition of ³ H-thymidine DNA uptake.

Evaluation method 3: Evaluation of growth inhibition effect by cell number evaluation using CellTiter-Glo Luminescent Cell Viability Assay reagent
HL60 cells are seeded at 1x10 ⁴ cells / well in a 96 well plate, and after adding drugs, 37 ° C, 5%
Celltiter-Glo Luminescent Cell after culturing under CO ₂ condition, 3, 6, 12, 24 hours after drug addition
Viability Assay reagent (Promega) was added to 100 μL of each well, and the amount of luminescence was measured with a 96-well plate luminometer to evaluate the cell growth inhibitory effect.

(Application example 1)
[Proliferation inhibitory effect of vitamin K hydroquinone derivative on hepatocellular carcinoma]
Cell proliferation of PLC / RPF / 5 cells, which are hepatocellular carcinoma, was suppressed in a dose-dependent manner by the addition of menaquinone-4 and menahydroquinone-4 derivatives (compounds 10, 11, and 12). However, the onset time of the growth inhibitory effect varies greatly depending on the drug, and a slight growth inhibitory effect was observed with the menahydroquinone-4 derivative (compounds 10, 11, 12) at 8 hours after addition, but at 10 hours after addition, compound 10, No. 12 showed a prominent growth inhibitory effect, and the proliferative inhibitory effect of Compound 11 was manifested 48 hours after addition. Menaquinone-4 showed no growth inhibitory effect until 48 hours after addition, and the growth inhibitory effect was expressed in 72 hours. As a typical example, FIG. 1 shows the growth inhibitory effect on the cell proliferation of PLC / RPF / 5 cells according to Evaluation Method 1. Table 9 shows the 50% growth inhibitory concentration (IC ₅₀ ) for PLC / RPF / 5 cells. As is clear from FIG. 1 and Table 9, it was revealed that the menahydroquinone-4 derivatives (compounds 10, 11, and 12) quickly exhibited a cell growth inhibitory effect as compared with menaquinone-4. Further, the IC _{50 at} 72 hours after addition of the menahydroquinone-4 derivative (compounds 10, 11, and 12) showed an excellent cancer cell growth inhibitory effect at a lower dose than menaquinone-4.

Cell proliferation of HepG2 cells and Hep3B cells was suppressed in a dose-dependent manner by the addition of menaquinone-4, menahydroquinone-4 derivatives (compounds 10, 11, 12, 29) and menadione (vitamin K3). The onset time of the growth inhibitory effect varies greatly depending on the drug, and the compound 10, 11, 12, 29 and menadione showed a remarkable growth inhibitory effect at 24 hours after addition, but menaquinone-4 showed a slight growth inhibition at 72 hours after addition. The effect was seen. Tables 10 and 11 show 50% growth inhibitory concentrations (IC ₅₀ ) for HepG2 cells and Hep3B cells according to Evaluation Method 1, respectively. Further, Table 12 shows menaquinone-4, menahydroquinone-4 derivatives (compounds 10, 11, 12), phylloquinone, phytohydroquinone derivatives (compounds 24, 25), menadione (vitamin K3) for HepG2 cells and Hep3B cells according to Evaluation Method 2. The 50% growth inhibitory concentration (IC ₅₀ ) was shown. It was revealed that menadione and menahydroquinone-4 derivatives (compounds 10, 11, 12, 29) on HepG2 and Hep3B cells rapidly exerted cell growth inhibitory effects compared to menaquinone-4. All ₅₀ showed an excellent cancer cell growth inhibitory effect at a low dose compared with menaquinone-4. It was revealed that DCP-positive HepG2 cells and DCP-negative Hep3B cells are effective against hepatocellular carcinoma at low concentrations. From Table 12, it is clear that the phyllohydroquinone derivative (Compound 24) exhibits a growth inhibitory effect superior to that of phylloquinone on Hep3B cells. However, the effect of the phyllohydroquinone derivative was lower than that of the menahydroquinone-4 derivative.

Compared with menaquinone-4, the vitamin K hydroquinone derivative exhibited a growth inhibitory effect faster than hena cell carcinoma, and the rate was Compound 10> Compound 12> Compound 11. Takada et al. Have already reported that the rate at which menahydroquinone-4 is produced from compounds 10-12 by enzymes in liver microsomes is compound 10 <compound 12 <compound 11 (Takata et al., Pharm Res., 12, 18-23 (1995)). That is, the slower the conversion from vitamin K hydroquinone derivative to menahydroquinone-4 (compound V), the faster the growth-inhibiting effect is exhibited. Vitamin K hydroquinone derivatives (compounds 10 to 12) are menahydroquinone-4. This suggests that the cancer cell proliferation inhibitory effect can be exhibited in the structure of the derivative without being converted into (Compound V).
Therefore, the vitamin K hydroquinone derivative itself and its secondary metabolite, vitamin K hydroquinone, also have a cancer cell growth inhibitory effect in specific liver cancers, and it is possible to provide a more efficient and safe cancer therapeutic agent. It is clear that

[Delivery to target organ after administration of vitamin K hydroquinone derivative]
It was shown in Application Example 1 that the vitamin K hydroquinone derivative has an excellent effect on hepatocellular carcinoma. In order to exhibit this excellent effect more efficiently, the vitamin K hydroquinone derivative is the target organ. Delivery to the liver has favorable results. Takada et al. Have shown that menahydroquinone-4 derivatives (compounds 10, 11, 12) migrate to the liver almost 15 minutes after intravenous administration in rats (Takata et al., Pharm. Res., 12 , 1973-1979 (1995)). That is, since menahydroquinone-4 derivatives (compounds 10, 11, 12) are selective delivery methods of menahydroquinone-4 derivatives (compounds 10, 11, 12) to the liver, menahydroquinone-4 derivatives are hepatocytes. It shows that an effective treatment for cancer can be provided. Takada et al. Have also shown that menahydroquinone-4 derivatives are converted to menahydroquinone-4 and menaquinone-4 in the liver (Takata et al., Pharm. Res., 12, 1973-1979 (1995). )), Menaquinone-4 has been shown to have an anticancer effect against hepatocellular carcinoma, and therefore menaquinone-4 can also function as an anticancer agent. Furthermore, menahydroquinone-4 derivatives are metabolized to menaquinone-4, and menaquinone-4 is a safe compound that has not been reported to have serious side effects in the treatment of osteoporosis. It is shown that it is possible to provide an effective treatment method for hepatocellular carcinoma, which can exert an excellent effect on the hepatocellular carcinoma, and is highly safe.

(Application example 2)
[Proliferation inhibitory effect of vitamin K hydroquinone derivative on lung cancer cells]
Cell proliferation of A549 cells, which are lung cancer cells, was suppressed in a dose-dependent manner by the addition of menaquinone-4 and menahydroquinone-4 derivatives (compounds 10, 11, 12, 14). No growth inhibition was observed with the addition of phylloquinone. The onset time of the growth inhibitory effect varies greatly depending on the drug. The menahydroquinone-4 derivative (compounds 10, 12, and 14) shows a remarkable growth inhibitory effect at 24 hours after addition, and the compound 11 shows a significant growth inhibition at 48 hours after addition. The effect was expressed. Menaquinone-4 showed no growth inhibitory effect until 48 hours after addition, and the growth inhibitory effect was expressed in 72 hours. As a typical example, the growth inhibitory effect on cell proliferation of A549 cells by Evaluation Method 1 is shown in FIG. Table 13 shows the 50% growth inhibitory concentration (IC ₅₀ ) for A549 cells according to Evaluation Method 1. As is clear from Table 13, it was revealed that menahydroquinone-4 derivatives (compounds 10, 11, 12, and 14) quickly exhibited a cell growth inhibitory effect as compared with menaquinone-4. In addition, the IC _{50 at} 72 hours after addition of the menahydroquinone-4 derivative (compounds 10, 11, 12, 14) showed an excellent lung cancer cell growth inhibitory effect at a lower dose than menaquinone-4.

(Application example 3)
[Proliferation inhibitory effect of vitamin K hydroquinone derivative on leukemia cells]
Cell proliferation of HL60 cells, which are leukemia cells, was suppressed in a dose-dependent manner by the addition of menahydroquinone-4 derivatives (compounds 10, 11, 12, 14). The expression time of the growth inhibitory effect varies greatly depending on the drug, and a significant growth inhibitory effect is observed with the menahydroquinone-4 derivative (compound 14) after 3 hours of addition, and the menahydroquinone-4 derivative (compounds 10 and 12) after 12 hours of addition A remarkable growth inhibitory effect was observed, and the remarkable growth inhibitory effect of Compound 11 was manifested 24 hours after the addition. Menaquinone-4 and phylloquinone were not observed to inhibit growth until 24 hours after addition. Table 14 shows the 50% growth inhibitory concentration (IC ₅₀ ) for A549 cells according to Evaluation Method 3. As is clear from Table 14, it was revealed that the menahydroquinone-4 derivative (compounds 10, 11, 12, 14) quickly exhibited a cell growth inhibitory effect as compared with menaquinone-4. Further, the IC _{50 at} 24 hours after addition of the menahydroquinone-4 derivative (compounds 10, 11, 12, 14) showed an excellent cancer cell growth inhibitory effect at a lower dose than menaquinone-4.

[Effect of vitamin K hydroquinone derivative on caspase-3 / 7 activity in HL60 cells]
HL60 cells are seeded at 1 × 10 ⁴ cells / well in 96 well plate, and after addition of drug, 37 ° C, 5%
Cultured in CO ₂ conditions, Caspase-Glo 3/7 Assay Reagent from drug addition after 4,12 h (Promega) was added to 100μL each well, and measuring the luminescence amount in a 96-well plate luminometer caspase / Seven activities were evaluated. Z-VAD-FMK was used as an inhibitor of caspase-3.
Caspase-3 / 7 of HL60 cells is activated by addition of menahydroquinone-4 derivatives (compounds 10 and 14), approximately 6 times in 4 hours after addition of 40 μM of compound 14, and 12 hours after addition of 80 μM of compound 10. It rose about 7 times (Fig. 3). The increase in caspase-3 / 7 activity by the derivative was completely suppressed by the addition of the caspase inhibitor Z-VAD-FMK. It was shown that the induction of apoptosis accompanied by caspase 3 activation is involved in the suppression of proliferation of HL60 cells by the menahydroquinone-4 derivative.

(Application example 4)
[Proliferation inhibitory effect of vitamin K hydroquinone derivative on gastric cancer cells]
Cell proliferation of SDT4 cells, which are gastric cancer cells, was suppressed in a dose-dependent manner by the addition of menaquinone-4, menadione, and menahydroquinone-4 derivatives (compounds 10, 11, and 12). As a typical example, the growth inhibitory effect on SDT4 cells by Evaluation Method 2 is shown in FIG. Table 15 shows the 50% growth inhibitory concentration (IC ₅₀ ). IC ₅₀ of menaquinone -4 respect SDT4 cells but was more than 5000MyuM, IC ₅₀ of Mena hydroquinone 4 derivatives (compounds 10, 11, 12) relative to this is a low concentration both, particularly compound 10 The concentration was about 1/100 or less, and showed excellent gastric cancer growth inhibitory effect. IC ₅₀ was similar to menadione (vitamin K3).

(Application example 5)
[Proliferation inhibitory effect of vitamin K hydroquinone derivative on mitomycin C (MMC) resistant gastric cancer cells]
The cell proliferation of ST4 cells, which are mitomycin C (MMC) resistant gastric cancer cells, was suppressed in a dose-dependent manner by the addition of menaquinone-4, menadione, and menahydroquinone-4 derivatives (compounds 10, 11, and 12). As a typical example, the growth inhibitory effect on ST4 cells is shown in FIG. Table 15 shows the 50% growth inhibitory concentration (IC ₅₀ ). Menahydroquinone-4 derivatives (compounds 10, 11, and 12) show growth inhibitory effects on ST4 cells, which are mitomycin C (MMC) resistant gastric cancer cells, similar to those on SDT4 cells, and are resistant to mitomycin C (MMC). It was also shown to be effective against.

(Application example 6)
[Proliferation inhibitory effect of vitamin K hydroquinone derivative on colon cancer cells]
Cell proliferation of HT29 cells, which are colorectal cancer cells, was suppressed in a dose-dependent manner by the addition of menaquinone-4, menadione, and menahydroquinone-4 derivatives (compounds 10 and 12). As a typical example, the growth inhibitory effect on HT29 cells is shown in FIG. Table 15 shows the 50% growth inhibitory concentration (IC ₅₀ ). IC ₅₀ of menaquinone -4 on HT29 cells was not less than 7000MyuM, IC ₅₀ of Mena hydroquinone derivative (Compound 10, 12) relative to this is a low concentration both, excellent colon cancer growth inhibitory effect Indicated. In particular, the IC _{50 of} compound 10 was 30 μM, which was similar to 20 μM of menadione (vitamin K3).

(Application example 7)
[Proliferation inhibitory effect of vitamin K hydroquinone derivative on mitomycin C (MMC) resistant colon cancer cells]
Cell proliferation of HT29 / MMC cells, which are mitomycin C (MMC) resistant colon cancer cells, was all dose-dependently suppressed by the addition of menaquinone-4, menadione, and menahydroquinone-4 derivatives (compounds 10 and 12). Table 15 shows the 50% growth inhibitory concentration (IC ₅₀ ). Menahydroquinone-4 derivatives (compounds 10 and 12) also show mitomycin C (MMC) resistance against HT29 and MMC cells, which are mitomycin C (MMC) resistant colon cancer cells, as well as HT29 cells. It was shown to be effective against strains.

(Application example 8)
[Effects of quinone-based anticancer drugs on the anticancer activity]
The effect of the vitamin K hydroquinone derivative (Compound 11) on the cell growth inhibitory effect of the quinone anticancer drug mitomycin C (MMC) on SDT4 cells, which are gastric cancer cells, was evaluated according to the above experimental method. The results are shown in FIG. The IC ₅₀ (0.25 μM) of MMC was reduced to about ３ to 0.8 μM by the addition of menahydroquinone-4 derivative, indicating that vitamin K hydroquinone derivative enhances the cell growth inhibitory effect of MMC.

(Application example 9)
[Anti-tumor effects of vitamin K hydroquinone derivatives on human transplanted human hepatocellular carcinoma in vivo]
Suspend PLC / PRF / 5 cells in BD Matrigel 1x10 ⁶
Cells were transplanted subcutaneously into the flank of 8-week-old male BALB-c nu / nu mice (Japan SLC). Nine days after transplantation, 6 mice / group of PLC / PRF / 5 transplanted mice were prepared, and menahydroquinone-1,4-bis-dimethylglycinate (Compound 10) was dissolved in 4% ethanol to give 400 μM and administered with drinking water. The control group received 4% ethanol by drinking water. The average drinking water is 4 mL / animal / day and the dose of compound 10 is 0.7 mg / animal / day. The same experimenter measured the major axis and minor axis of the tumor using a digital display caliper, and the major axis × (minor axis) ² × 0.52 was taken as the tumor volume. The anticancer effect was evaluated from suppression of tumor volume increase. FIG. 8 shows changes in tumor volume after transplantation of PLC / PRF / 5 cells. The increase in tumor volume in the group administered with Compound 10 is significantly lower than that in the control group,
It was revealed that it can exert an antitumor effect against hepatocellular carcinoma in vivo. Moreover, weight loss due to drug administration was not observed.

As described above, menaquinone-4 is effective in liver cancer as a cancer therapeutic agent and cancer recurrence preventing agent containing a carboxylic acid ester of vitamin K hydroquinone represented by the above general formula (I) according to the present invention. It has an excellent effect on both DCP (des-g-carboxy prothrombin) positive liver cancer and DCP negative liver cancer in which menaquinone-4 has a very low effect. In addition, the cancer therapeutic agent and cancer recurrence preventive agent containing the carboxylic acid ester of vitamin K hydroquinone represented by the above general formula (I) according to the present invention is epithelial such as lung cancer, gastric cancer, colon cancer and the like, where the effect of menaquinone-4 is low. Has an excellent effect on cancer. The cancer therapeutic agent and cancer recurrence preventive agent containing the carboxylic acid ester of vitamin K hydroquinone represented by the above general formula (I) is excellent also for epithelial cancers such as gastric cancer and colorectal cancer resistant to quinone anticancer agents. Has an effect. Furthermore, the cancer therapeutic agent and cancer recurrence preventive agent containing the carboxylic acid ester of vitamin K hydroquinone represented by the above general formula (I) according to the present invention enhances the action of the quinone anticancer agent. The cancer therapeutic agent and cancer recurrence preventive agent containing the carboxylic acid ester of vitamin K hydroquinone represented by the general formula (I) has an excellent effect on leukemia.
Furthermore, metabolites in the body of the compound according to the present invention are mainly vitamin Ks, and their safety is extremely high.
Further, it has been clarified that the carboxylic acid ester of vitamin K hydroquinone represented by the above general formula (I) itself exerts an anticancer action or a cancer recurrence preventing action, and it is also resistant to vitamin K which is a secondary metabolite thereof. Since it has a cancer action or a cancer recurrence prevention action, the present invention can provide a more efficient and safe cancer therapeutic agent and cancer recurrence prevention agent.

Claims (7)

The following general formula (I)

Wherein R ₁ and R ₂ are each a hydrogen atom, or an amino acid, an N-acyl amino acid, an N-alkyl amino acid, an N, N-dialkyl amino acid, a pyridinecarboxylic acid, and their hydrohalides and alkylsulfonates. Or a carboxylic acid residue having a nitrogen substituent selected from residues of sugar salts, or a dicarboxylic acid residue selected from residues of dicarboxylic acids and alkali metal salts thereof, R ₁ ,
At least one of R ₂ is a carboxylic acid residue having a nitrogen substituent or a dicarboxylic acid residue. R ₃ is a hydrogen atom or the following general formula (II)

Or the following general formula (III)

Represents a group represented by n means an integer of 1 to 14. The cancer therapeutic agent containing at least 1 sort (s) of the carboxylic acid ester of vitamin K hydroquinone represented by this, or its salt.   A quinone-based anticancer agent enhancing action adjuvant containing at least one of the compounds represented by formula (I) or a salt thereof.   A cancer preventive agent comprising at least one of the compound represented by the general formula (I) or a salt thereof.   The cancer preventive agent according to claim 3, which is a cancer recurrence preventive agent. By using the compound represented by the general formula (I) or a salt thereof, the general formula (V) (wherein R ₃ is a hydrogen atom, the general formula (II) or the general formula (III) N represents an integer of 1 to 14. This produces the vitamin K hydroquinone represented by general formula (V) and is anticancer or cancerous by the vitamin K hydroquinone represented by the general formula (V). The anticancer agent, the cancer preventive agent, or the action enhancement auxiliary agent according to any one of claims 1 to 4, which exhibits a recurrence prevention action and an action enhancement auxiliary action of the quinone anticancer agent.

By using the compound represented by the general formula (I) or a salt thereof, the general formula (IV) in the body is efficiently (wherein R ₃ is a hydrogen atom, the general formula (II) or the general formula ( III) represents a group represented by n), and n represents an integer of 1 to 14. The vitamin K represented by the formula (IV) is increased, and the anticancer effect and cancer recurrence by vitamin K represented by the general formula (IV) is represented. The anticancer agent, cancer recurrence preventive agent, or action enhancer according to any one of claims 1 to 4, which exerts a preventive action and an action enhancement of a quinone anticancer agent.

By using the compound represented by the general formula (I) or a salt thereof, the vitamin K hydroquinone derivative itself represented by the general formula (I) and the secondary product represented by the general formula (V) The anticancer agent according to any one of claims 1 to 4, which exhibits an anticancer effect, a cancer recurrence preventing effect, and an action enhancing effect of a quinone anticancer agent by vitamin K hydroquinone and / or vitamin K represented by the general formula (IV). Or cancer recurrence prevention agent, action enhancement adjuvant.

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