JPH0769893A - Carcinostatic agent - Google Patents

Abstract

PURPOSE:To obtain a carcinostatic agent containing a specific carbostyryl derivative, cytokine, etc., as active components and exhibiting strong action to suppress the proliferation of cancer cell. CONSTITUTION:This carcinostatic agent contains, as active components, (A) a compound selected from (i) a carbostyryl derivative of formula I [R is a (substituted)benzoyl], (ii) a carbostyryl derivative of formula II [R<5> is a (substituted)phenyl-lower alkyl, benzoyl or naphthoyl; R<6> is a (lower alkoxy- containing) benzoyl], (iii) a carbostyryl derivative of formula III (R<7> is OH, amino, a lower alkoxy, etc.; R<8> is H or a lower alkyl; R<9> is H), (iv) a carbostyryl derivative of formula IV [R<13> is a phenyl-lower alkyl; R<14> is O-A-R<15> (A is a lower alkylene; R<15> is group of formula V)], (v) 6-[4-(3,4- dimethoxybenzoyl)-1-1,2,3,4-tetrahydropyrazyl]-3,4-dihydrocarbostyryl and (vi) 7-methoxy-2-oxo-2,3,4,5-tetrahydro-1H-benzoazepine and (B) a cytokine and/or a retinoid.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a carcinostatic agent, and more particularly to a carcinostatic agent comprising a combination of a specific carbostyril derivative and the like, a cytokine such as human tumor necrosis factor and / or a retinoid such as retinoic acid as an active ingredient.

[0002]

DISCLOSURE OF THE INVENTION The anticancer agent of the present invention is represented by the following general formula (1):
The compound represented by the general formula (4) (hereinafter referred to as "compound (1)" to "compound (4)") and (5) 6-
[4- (3,4-dimethoxybenzoyl) -1-1,
2,3,4-Tetrahydropyrazyl] -3,4-dihydrocarbostyril (hereinafter referred to as “compound (5)”) and a salt thereof, and (6) 7-methoxy-2-oxo-2, 3,4,5-tetrahydro-
1H-benzazepine (hereinafter referred to as "compound (6)")
And at least one selected from salts thereof and cytokines and / or retinoids as active ingredients.

(1) General formula

[0004]

[Chemical 6]

[Wherein R represents a benzoyl group which may have 1 to 3 groups selected from the group consisting of a lower alkoxy group, a halogen atom, a lower alkyl group and a lower alkylthio group on the phenyl ring as a substituent. The carbon-carbon bond between the 3-position and 4-position of the carbostyril skeleton represents a single bond or a double bond. ] The carbostyril derivative and its salt represented by these, (2) General formula

[0006]

[Chemical 7]

[In the formula, R ⁵ represents a phenyl lower alkyl group which may have a lower alkanoyloxy group or a hydroxyl group on the phenyl ring, a benzoyl group or a naphthoyl group. R ⁶ represents a benzoyl group which may have a lower alkoxy group on the phenyl ring. ] The carbostyril derivative and its salt represented by these, (3) General formula

[0008]

[Chemical 8]

[In the formula, R ⁷ represents a hydroxyl group, an amino group, a lower alkoxy group, a lower alkyl group or a piperazinyl group.
R ⁸ represents a hydrogen atom or a lower alkyl group. R ⁹ represents a hydrogen atom. The bonds at the 3rd and 4th positions of the carbostyril skeleton represent a single bond or a double bond. ] The carbostyril derivative and its salt represented by these, (4) General formula

[0010]

[Chemical 9]

[In the formula, R ¹³ represents a phenyl lower alkyl group. R ¹⁴ is a group —O—A—R ¹⁵ [A is a lower alkylene group, R ¹⁵ is a group

[0012]

[Chemical 10]

Is shown. ] The carbostyril derivative and its salt represented by these.

Regarding the above-mentioned compounds (1) to (5) (carbostyryl derivatives) and the method for producing the same, for example, JP-A-58-83677 and JP-A-61-26755.
6, JP-A-1-6271 and JP-A-1-34.
963 bulletin, Proceedings of the 111th Annual Meeting of the Pharmaceutical Society of Japan 29
It can be produced according to the method described in TD11-1 (issued March 5, 1991) or the like. It is also known that each carbostyril derivative is useful as a cardiotonic agent, an antiarrhythmic agent, a platelet adhesion inhibitor, and the like.

As a result of further research on the above compounds (1) to (6), the present inventor has found that each compound has an anticancer activity which is difficult to predict from the above known effects that they have. It was found that they have the action and the ability to induce the differentiation of cells.

On the other hand, conventionally, stimulated lymphocytes, monocytes, macrophages, etc. produce various protein factors having biological activity, and the immune system response is regulated (start → It has been clarified that (proliferation → suppression → termination) is modified, and protein factors that control the expression of biological functions such as immune response, inflammatory response, and hemostatic response in living organisms have been generically called cytokines [Balkwill , FR, et al., Imm
unol.Today, 10 , 299 (1989)]. The cytokines are divided into several groups based on their similarities of action, for example, interferon (IFN), interleukin (IL), colony stimulating factor (CSF), tumor necrosis factor (TNF), etc., and these are further subdivided. However, because of their common antitumor activity, various clinical uses as antitumor agents are being studied. However, all of them have strong side effects such as chills, shivering, fever and lowering of blood pressure, and it is difficult or impossible to administer them in an amount that can be expected to have an antitumor effect.

As a result of subsequent studies, the inventors of the present invention utilize the specific compounds (1) to (6) having the above-mentioned carcinostatic action and cell differentiation-inducing ability in combination with the above-mentioned cytokine and / or the above retinoid. In some cases, surprisingly, even at low doses where the anti-tumor activity of cytokines alone does not appear, excellent tumor growth-suppressing effects and cell differentiation-inducing promoting effects are obtained, and yet strong early on. It was found that a carcinostatic effect appears.

The present invention has been completed based on these findings.

That is, the present invention relates to an anticancer agent containing at least one selected from compounds (1) to (6) and salts thereof, and a cytokine and / or retinoid as active ingredients.

In each of the general formulas representing the above compounds (1) to (4) as one of the active ingredients of the anticancer agent of the present invention, each defined group is more specifically as follows.

Halogen atom is fluorine, chlorine, bromine or iodine atom.

The benzoyl group which may have 1 to 3 groups selected from the group consisting of a lower alkoxy group, a halogen atom, a lower alkyl group and a lower alkylthio group on the phenyl ring as a substituent includes benzoyl and 2-chloro. Benzoyl, 3-chlorobenzoyl, 4-chlorobenzoyl, 2-fluorobenzoyl, 3-fluorobenzoyl, 4-fluorobenzoyl, 2-bromobenzoyl,
3-bromobenzoyl, 4-bromobenzoyl, 2-iodobenzoyl, 4-iodobenzoyl, 3,5-dichlorobenzoyl, 2,6-dichlorobenzoyl, 3,4
-Dichlorobenzoyl, 3,4-difluorobenzoyl, 3,5-dibromobenzoyl, 3,4,5-trichlorobenzoyl, 2-methylbenzoyl, 3-methylbenzoyl, 4-methylbenzoyl, 2-ethylbenzoyl, 3 -Ethylbenzoyl, 4-ethylbenzoyl, 3
-Isopropylbenzoyl, 4-hexylbenzoyl,
3,4-dimethylbenzoyl, 2,5-dimethylbenzoyl, 3,4,5-trimethylbenzoyl, 2-methoxybenzoyl, 3-methoxybenzoyl, 4-methoxybenzoyl, 2-ethoxybenzoyl, 3-ethoxybenzoyl, 4- Ethoxybenzoyl, 4-isopropoxybenzoyl, 4-hexyloxybenzoyl, 3,4
-Dimethoxybenzoyl, 3,4-diethoxybenzoyl, 3,4,5-trimethoxybenzoyl, 2,5-dimethoxybenzoyl, 3-methyl-4-chlorobenzoyl, 2-chloro-6-methylbenzoyl, 2-methoxy -3-chlorobenzoyl, 2-methylthiobenzoyl,
3-methylthiobenzoyl, 4-methylthiobenzoyl, 2-ethylthiobenzoyl, 3-ethylthiobenzoyl, 4-ethylthiobenzoyl, 3-isopropylthiobenzoyl, 4-hexylthiobenzoyl, 3,4-
A straight-chain or branched alkoxy group having 1 to 6 carbon atoms as a substituent on the phenyl ring such as dimethylthiobenzoyl, 2,5-dimethylthiobenzoyl, 3,4,5-trimethylthiobenzoyl group, a halogen atom, A benzoyl group which may have 1 to 3 groups selected from the group consisting of a straight-chain or branched alkyl group having 1 to 6 carbon atoms and a straight-chain or branched alkylthio group having 1 to 6 carbon atoms It can be illustrated.

Examples of the lower alkyl group include linear or branched alkyl groups having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl and hexyl groups.

The phenyl lower alkyl group is benzyl, 2-phenylethyl, 1-phenylethyl, 3-phenylpropyl, 4-phenylbutyl, 1,1-dimethyl-2-phenylethyl, 5-phenylpentyl, 6-.
Examples thereof include a phenylalkyl group which is a linear or branched alkyl group having 1 to 6 carbon atoms in the alkyl moiety such as phenylhexyl and 2-methyl-3-phenylpropyl group.

Examples of the lower alkylthio group include linear or branched alkylthio groups having 1 to 6 carbon atoms such as methylthio, ethylthio, propylthio, isopropylthio, butylthio, tert-butylthio, pentylthio and hexylthio groups.

The lower alkanoyloxy group is a straight chain having 1 to 6 carbon atoms such as formyloxy, acetyloxy, propionyloxy, butyryloxy, isobutyryloxy, pentanoyloxy, tert-butylcarbonyloxy and hexanoyloxy groups. Alternatively, a branched alkanoyloxy group can be exemplified.

Examples of the phenyl lower alkyl group which may have a lower alkanoyloxy group or a hydroxyl group as a substituent on the phenyl ring include, in addition to the above-mentioned unsubstituted phenyl lower alkyl group, 2-, 3- or 4-hydroxybenzyl, 2- (3,4-dihydroxyphenyl) ethyl,
1- (3,4-dihydroxyphenyl) ethyl, 2-
(3-hydroxyphenyl) ethyl, 3- (4-hydroxyphenyl) propyl, 6- (3,4-dihydroxyphenyl) hexyl, 3,4-dihydroxybenzyl,
3,4,5-trihydroxybenzyl, 2-formyloxybenzyl, 3-acetyloxybenzyl, 3- (2
-Acetyloxyphenyl) propyl, 4- (4-acetyloxyphenyl) butyl, 2-propionyloxybenzyl, 3- (3-butyryloxyphenyl) propyl, 4- (4-isobutyryloxyphenyl) butyl,
5- (2-tert-butylcarbonyloxyphenyl) pentyl, 6- (3-pentanoyloxyphenyl) hexyl, (2,4-diacetyloxy) benzyl and the like having 1 to 6 carbon atoms as a substituent on the phenyl ring. 1 to 3 linear or branched alkanoyloxy groups or hydroxyl groups
An example thereof is a phenylalkyl group having one or more, and the alkyl moiety is a linear or branched alkyl group having 1 to 6 carbon atoms.

Examples of the lower alkoxy group include linear or branched chain alkoxy groups having 1 to 6 carbon atoms such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentyloxy and hexyloxy groups. it can.

The benzoyl group which may have a lower alkoxy group as a substituent on the phenyl ring is, for example, benzoyl, 2-methoxybenzoyl, 3-methoxybenzoyl, 4-methoxybenzoyl, 2-ethoxybenzoyl, 3-ethoxybenzoyl. , 4-ethoxybenzoyl, 3-isopropoxybenzoyl, 4-butoxybenzoyl, 2-pentyloxybenzoyl, 3-hexyloxybenzoyl, 3,4-dimethoxybenzoyl,
It may have 1 to 3 linear or branched alkoxy groups having 1 to 6 carbon atoms as a substituent on the phenyl ring such as 2,5-dimethoxybenzoyl and 3,4,5-trimethoxybenzoyl groups. A benzoyl group can be illustrated.

Examples of the lower alkylene group include methylene, ethylene, trimethylene, 2-methyltrimethylene, 2,2-dimethyltrimethylene, 1-methyltrimethylene, methylmethylene, ethylmethylene, tetramethylene, pentamethylene and hexamethylene. 1 to 1
The linear or branched alkylene group of 6 can be exemplified.

The other active ingredient of the antitumor agent of the present invention may be any of various known cytokines. Examples of the cytokine include TNF (TNF-α,
TNF-β), IFN (IFN-α, IFN-β, IF
N-γ), CSF (M-CSF, G-CSF, GM-C
SF), IL (IL-1, IL-2, IL-3, IL-
4, IL-5, IL-6, IL-7, IL-8, IL-
9, IL-10, IL-11, IL-12) and the like. Each cytokine is not particularly limited as long as it is an active substance having each biological activity, and may be a natural type derived from human cells or the like or a recombinant type obtained by a gene recombination technique. The above-mentioned natural type includes, for example, known in vitro-derived culture supernatants containing desired cytokines and purified products thereof, and the recombinant type has the same amino acid sequence as the natural type obtained by gene recombination technology. In addition to the recombinant type having the above-mentioned, the same effect product in which a part of the amino acid sequence is modified (deleted, substituted, added) is also included.
The production of these materials can be carried out according to various known methods.

Further, retinoic acid is a derivative of vitamin A (retinol), which is produced from vitamin A in the body. Vitamin A derivatives containing retinoic acid are collectively called retinoids. Retinoids include retinoic acid as well as retinol, retinal, retinyl esters and known synthetic homologs of vitamin A. The ring in the homologue may be an aromatic ring or a heteroaromatic ring, and the side chain and the terminal group may be optionally substituted, oxidized or reduced,
The retinoid also includes alkali metal and alkaline earth metal salts of retinoic acid. Specific examples thereof include etretinate, isotretinoin, 13-cis retinoic acid, polyprenic acid, polyprenic acid derivative (E-5116), all-trans-retinoic acid (tretinoin) and the like. it can. A method for producing the retinoid is known, and for example, retinoid derivatives are disclosed in JP-A-61-275265, JP-A-61-275266, and JP-A-64-5001.
It can be manufactured with reference to Japanese Patent Publication No. 90, etc.

It was already found in 1925 by Wolbach & Howe that the deficiency of vitamin A causes abnormal differentiation. Bollag made some vitamin A derivatives,
Retinoic acid has been shown to suppress chemical carcinogenesis in mouse skin [Bollag, W., Eur. J. Cancer., 8, 689]
(1972)]. The retinoid exhibits a vitamin A-like action without causing toxic symptoms unlike oral administration of vitamin A, and is expected to have an effect on various skin diseases and cancer, and many cancer cells such as HeLa can be treated by retinoic acid. It has been reported that it has a certain level of differentiation ability against cells, malignant melanoma cells, etc. and has an anticancer effect. As described above, retinoic acid, which has the ability to differentiate cancer cells, is one of the molecules that suppresses development, and research on cancer treatment and research on the relationship with retinoic acid are remarkable and many studies have been made. , And clinical trials have also been made [Iwao Hiratsuka et al., Kawakubo Hospital, Ikuho, 12 (3) 109-112 (1989); Nobuo Mitsui, Chubu Japan Orthopedic Surgery Journal, 33 (1) 26-
27 (1990) ； Ninomiya Sansei, Gifu University School of Medicine Bulletin, 35 (6)
836-848 (1987)]. As a result, even with only retinoic acid, some therapeutic effect on cancer was observed [Menger,
H., et al., Blood, 12 (2) 567-572 (1988); Castaign
e, S., et al., Blood, 76 (8) 1704-1709 (1990), etc.], but this is associated with the side effects such as teratogenesis, hypervitaminosis A, and bone abnormalities associated with its large dose. And its use criteria are being considered [Kamihiko Sawada, Modern Medicine, 22 (increase
3) 428-432 (1990)].

The antitumor agent of the present invention is prepared and used so that at least one of the compounds (1) to (6) and salts thereof and the above-mentioned cytokine and / or retinoid are contained in a single preparation. Alternatively, it is also possible to separately formulate each of these and use both formulations. In either case, one of the cytokine and / or retinoinde and the compound (1) to the compound (6) and their salts are mutually prone to the growth of tumor cells in the other, as is clear from the results of the pharmacological tests described below. To enhance the suppressive effect,
The combined ratio (mixing ratio) of both can be appropriately selected from a wide range.

For example, in clinical use of the anticancer drug of the present invention, the amount of cytokine used as an active ingredient is usually about 0.1 to 1000 μg / body weight, preferably about 1 to 100 μg / body weight as protein amount per adult per day. The amount of the retinoid is usually selected from the range of about 0.01 to 10 mg / kg per day. In addition, compound (1) to compound (6) (and salts thereof) are usually about 3 per adult per day.
~ 1800 mg / body weight, preferably about 10-300
The amount is preferably selected from the range of about mg / body weight.

The compounds (1) to (6) of the present invention
And an anticancer agent containing at least one of these salts and the above-mentioned cytokine and / or retinoid, or a preparation containing one of the active ingredients, particularly a preparation containing compounds (1) to (6) or a salt thereof Can be formulated by an ordinary general method. Such a preparation is prepared using a diluent or an excipient such as a filler, a filler, a binder, a moisturizer, a disintegrant, a surface active agent, a lubricant and the like which are usually used. Various forms of this pharmaceutical preparation can be selected according to the therapeutic purpose, and typical examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, and injections ( Liquid medicine,
Suspension agents, etc.) and the like.

In the case of molding into tablets, those conventionally known in this field can be widely used as carriers, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid. Excipients such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose,
Potassium phosphate, polyvinylpyrrolidone sugar binder, dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, etc. Disintegrants, sucrose, stearin, cocoa butter, disintegration inhibitors such as hydrogenated oil, quaternary ammonium bases, absorption promoters such as sodium lauryl sulfate, glycerin, moisturizers such as starch, starch, lactose, kaolin,
Examples thereof include adsorbents such as bentonite and colloidal silicic acid, refined talc, stearates, boric acid powders, and lubricants such as polyethylene glycol. Further, the tablet may be a tablet coated with a usual coating, if necessary, such as a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multi-layer tablet.

In the case of molding in the form of pills, various carriers conventionally known in this field can be widely used, for example, glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil,
Examples include excipients such as kaolin and talc, gum arabic powder, tragacanth powder, binders such as gelatin and ethanol, and disintegrating agents such as laminaranthantene.

In the case of molding in the form of suppositories, conventionally known carriers can be widely used, and examples thereof include polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, and semisynthetic glycerides. it can.

When prepared as an injection, the solution and suspension are preferably sterilized and isotonic with blood, and when formed into a solution, emulsion or suspension, they are diluted. As the agent, all agents commonly used in this field can be used, and examples thereof include water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of sodium chloride, glucose or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a usual solubilizing agent, buffer, soothing agent, etc. may be added. May be.

If desired, the anticancer agent of the present invention may contain colorants, preservatives, fragrances, flavors, sweeteners and other pharmaceuticals.

The amount of the active ingredient compound contained in the above-mentioned pharmaceutical preparation is not particularly limited and is appropriately selected within a wide range, but it is usually 1 to 70% by weight, preferably 1 to 30% by weight in the whole composition. Is appropriate.

The administration method of the above-mentioned pharmaceutical preparation is not particularly limited, and it is determined according to various preparation forms, age of the patient, sex and other conditions, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered. In the case of an injection, it may be administered alone or mixed with a normal replenisher such as glucose or amino acid, and then intravenously administered. Further, if necessary, it may be administered intramuscularly, intracutaneously, subcutaneously or intraperitoneally. In the case of suppositories, it will be administered rectally.

A preparation containing a cytokine is more preferably prepared in the form of an injection, and the preparation may contain human serum albumin, L-type amino acids, saccharides, a surfactant, a sulfur-containing reducing agent, etc., if necessary. Additives can be added and compounded, which can improve the stability of cytokines.

Examples of the above amino acids include glycine,
Examples include cysteine and the like. Examples of sugars include monosaccharides such as glucose, mannose, galactose and fructose, sugar alcohols such as mannitol, inositol and xylitol, sucrose, maltose, disaccharides such as lactose, dextran and polysaccharides such as hydroxypropyl starch. Of these, sucrose, maltose, mannitol, inositol, dextran and the like are preferable. The surfactant may be either an ionic or nonionic surfactant, and among them, polyoxyethylene alkyl ether type, sorbitan monoacyl ester type, fatty acid glyceride type and the like are preferable. The amount of each additive added is not particularly limited, but is generally about 0.01 mg or more, preferably about 0.1 mg or less for saccharide per 1 μg of cytokine.
10 mg, about 0.00001 mg or more for a surfactant, preferably about 0.001 to 1 mg, about 0.0001 mg or more for human serum albumin, preferably about 0.001 to 1 mg, and about 0. 0
It is suitable to set the amount to about 01 to 10 mg. Specific examples of the sulfur-containing reducing agent include cysteine, N-acetylhomocysteine, thioctic acid, thioglycolic acid and salts thereof such as thioethanolamine, thioglycerol,
Sodium thiosulfate, thiolactic acid, dithiothreitol,
Glutathione acid and the like can be exemplified, and generally, it is suitable that the amount is about 0.001 mg or more, preferably about 0.01 to 10 mg per 1 μg of cytokine.

Furthermore, it is preferable that the anticancer agent of the present invention containing the above-mentioned cytokine is made isotonic with a buffer to give a stable isotonic preparation. As the buffer, for example, citric acid-sodium citrate is typically used. PH of citric acid-sodium phosphate, acetic acid-sodium acetate, citric acid-borax, etc.
Various buffer solutions having a pH of about 4 to 8, preferably about 5 to 6, can be exemplified.

The dose of the above-mentioned pharmaceutical preparation is appropriately selected depending on the usage, the age and sex of the patient, other conditions, the degree of disease and the like.

Further, a preparation containing a compound (1) to compound (6) or a salt thereof as an active ingredient of the antitumor agent of the present invention is treated with cancer chemistries regardless of its preparation form and administration route. It can be used in combination with various other anticancer agents known as therapeutic agents and radiation therapy, and can also be used in combination with conventionally known radiation therapy, hyperthermia and the like. In this case, in addition to the excellent anticancer effect of the active ingredient of the present invention, the effects of the chemotherapeutic agent, radiation therapy, hyperthermia, etc. used in combination can be further promoted to exert a synergistic effect. Therefore, even when the amount of the chemotherapeutic agent used in combination is considerably smaller than the normally used amount, a sufficient cancer treatment effect can be obtained, and thereby side effects due to the combined chemotherapeutic agent or treatment method can be reduced. it can. Examples of such chemotherapeutic agents include 5-fluorouracil (5-F)
U, manufactured by Kyowa Hakko Kogyo Co., Ltd., mitomycin (Mito)
mycin-C, manufactured by the same company, Futrafur (FT-207,
Taiho Pharmaceutical Co., Ltd., Endoxan,
Shionogi Pharmaceutical Co., Ltd., Toyomicin (Toyomicin,
Takeda Pharmaceutical Co., Ltd.) and the like.

[0049]

According to the present invention, cytokines, especially T
Anti-tumor effects of various cytokines including NF-α and / or retinoids such as retinoic acid and a compound selected from compounds (1) to (6) and salts thereof, in combination. There is provided a carcinostatic agent which can remarkably enhance each other and exert an effect of promoting cell differentiation induction. The carcinostatic agent can exert a remarkably excellent tumor suppressive effect and cell differentiation induction promoting effect even by utilizing a low dose of cytokine which cannot exert an antitumor effect by itself, and accordingly, the cytokine dose can be increased. Not only a large reduction, but side effects due to the cytokine can be reduced.

[0050]

[Examples] Formulation examples of the anticancer agent of the present invention and the results of pharmacological tests of the active ingredient compounds of the anticancer agent are listed below.

[0051]

[Formulation Example 1] 6- [4- (3,4-dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril 150 g Avicel (trademark, manufactured by Asahi Kasei Corporation) 40 g corn starch 30 g magnesium stearate 2 g Hydroxypropyl methylcellulose 10 g Polyethylene glycol-6000 3 g Castor oil 40 g Methanol 40 g After mixing and polishing the above active ingredient compounds, Avicel, corn starch and magnesium stearate, sugar coating R10 mm
Tablet with the key. The obtained tablets are hydroxypropylmethyl cellulose, polyethylene glycol-600.
A film-coated tablet consisting of 0, castor oil and methanol is coated to produce a film-coated tablet.

[0052]

[Formulation Example 2] 6- [4- (3,4-dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril 150.0 g Citric acid 1.0 g Lactose 33.5 g Dicalcium phosphate 70.0 g Pluronic F-68 30.0 g Sodium lauryl sulfate 15.0 g Polyvinylpyrrolidone 15.0 g Polyethylene glycol (Carbowax 1500) 4.5 g Polyethylene glycol (Carbowax 6000) 45.0 g Corn starch 30.0 g Dry sodium lauryl sulfate 3.0 g Dry Magnesium starate 3.0 g Ethanol Appropriate amount The above active ingredient compound, citric acid, lactose, dicalcium phosphate, Pluronic F-68 and sodium lauryl sulfate are mixed.

The above mixture was added to No. Sifted with 60 screen, polyvinylpyrrolidone, carbowax 1500
And wet granulation with an alcoholic solution containing Carbowax 6000. Alcohol is added as needed to make the powder into a pekato mass. Add corn starch and continue mixing until uniform particles are formed. No. Pass through 10 screens, place in trays and dry in oven at 100 ° C for 12-14 hours. The dried particles were Sieve through a 16 screen, add dry sodium lauryl sulfate and dry magnesium stearate, mix and compression mold to desired shape in tablet press.

The core is treated with a varnish and talc is sprinkled to prevent moisture absorption. The undercoat layer is coated around the core. Apply varnish a sufficient number of times for internal use. Further subbing layers and smooth coatings are applied in order to make the tablets completely round and smooth. Color coating is applied until the desired shade is obtained. After drying, the coated tablets are polished to prepare tablets of uniform gloss.

Compounds (1) to (6) shown below were tested compound Nos. A to W, the following retinoic acid as a retinoid, and the following TNF- as a cytokine:
The following pharmacological tests were carried out using α respectively.

Test compound No. A. 6- [4- (3,4-dimethoxybenzoyl)-
1-Piperazinyl] -3,4-dihydrocarbostyril B.I. 6- [4- (4-Ethylbenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyryl C.I. 8-methyl-6- (1-piperazinyl) -3,4
-Dihydrocarbostyril D. 7- [4- (3,4,5-Trimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyryl E. 7- [4- (3,4-dimethoxybenzoyl)-
1-Piperazinyl] -3,4-dihydrocarbostyryl F.I. 6- [4- (3,4-dimethoxybenzoyl)-
1-1,2,3,4-Tetrahydropyrazyl] -3,4
-Dihydrocarbostyryl G. 6-Hydroxy-3,4-dihydrocarbostyril H. 7-Hydroxy-3,4-dihydrocarbostyryl I. 6- [4- (4-methylthiobenzoyl) -1-
Piperazinyl] -3,4-dihydrocarbostyril J. 1-Benzyl-6- [4- (3,4-dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril K.I. 6- [4- (4-Ethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril L.I. 6- [4- (4-chlorobenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril M.I. 6- [4- (3-chlorobenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyryl N.V. 6-methoxycarbostyryl O. 6-Methylcarbostyril P.I. 8-Methyl-7-aminocarbostyryl hydrochloride Q.I. 7-Hydroxycarbostyryl R. 7-Methoxy-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine S.I. 1-Benzyl-6- [3- (1,2,4-triazol-1-yl) propoxy] -3,4-dihydrocarbostyril T.I. 1- (4-hydroxybenzyl) -6- [4-
(3,4-Dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril U.V. 1- (4-acetyloxybenzyl) -6- [4
-(3,4-Dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril V.I. 1-benzoyl-6- [4- (3,4-dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyryl W.I. 1-naphthoyl-6- [4- (3,4-dimethoxybenzoyl) -1-piperazinyl] -3,4-dihydrocarbostyril In the pharmacological test example 1 described later, each of the above test compounds was treated with 1N.
After dissolving in hydrochloric acid, it is diluted with FCS (fetal calf serum, manufactured by Gibco) to prepare a 1 mg / ml solution, and the solution is 10% F
After addition to CS-added RPMI-1640 medium (manufactured by Flow Co.) and neutralization with 1N NaOH, the medium alone was 10 and 3.
Each concentration of 0 μg / ml was prepared and used.

In the pharmacological test example 2 described below, each test compound was dissolved in dimethylsulfoxide (DMSO) to prepare a 10 mg / ml solution, and the solution was added with 10% FCS-added RPMI-1640 medium (manufactured by Flow Co.). Add to
Each concentration of 1.88, 7.5 and 30 μg / ml was prepared and used.

Retinoids Retinoids include retinoic acid (all trans.
Retinoic acid, made by Sigma) RPM with 10% FCS
It was diluted with I-1640 medium and adjusted to 10 ^-6 and 10 ^-7 M concentration and used.

Cytokine As cytokine, TNF-α (recombinant human TNF-α obtained by expression in Escherichia coli, specific activity: 2 × 10 5
1000 units / m by diluting ⁷ units / mg, manufactured by Zenzyme Co., Ltd. with RPMI-1640 medium containing 10% FCS.
It was adjusted to 1 concentration and used.

[0060]

[Pharmacological Test Example 1] Human promyelocytic leukemia cells (HL-6
0) Effect test on human promyelocytic leukemia cells (HL-60)
A human leukemia cell line established by rt Gallo) et al., whose cell properties are described in the literature [Gallo, RC, et al., Blood, 54 , 713].
(1979)] and the American Type Culture Collection (ATCC), "ATCC No. CC.
It has been deposited with a deposit number of "L-240".

RPM of the above HL-60 with 10% FCS
I-1640 medium (manufactured by Gibco) at 37 ° C, 5% C
The cells were cultured under O ₂ and prepared so that the cell number became 1 × 10 ⁵ cells / ml.

Next, only the medium prepared above was added to each well of a 6-well microplate (manufactured by Coaster) (control group), and the test compound No. A is 10 or 30μ
g / ml was added (AZ group) and all-trans-retinoic acid (ATRA) was added at 10 ^-7 M or 10 ^-6 M (ATRA group), and the plate was cultured at 37 ° C under 5% CO ₂ for 5 days. did. After culturing, the cell suspension in each hole was taken out, taken out into an Eppendorf tube, and the number of viable cells was measured with a Coulter counter (manufactured by Coal Tar Co.).

The above cells were prepared at 1 × 10 ⁶ cells / ml,
After staining with fluorescein isothiocyanate (FITC) -labeled anti-human CD11 antibody (Mo1, manufactured by Coulter),
The fluorescence intensity was measured by profile II (manufactured by Coulter) by the flow cytometry method.

The inhibitory effect on cell proliferation in each group of the AZ group, the ATRA group and the combination group of both obtained as described above was determined based on the result of the control group as a percentage of increase / decrease in cell proliferation inhibition (0) with respect to the reference value (0). %) In Table 1.

The results obtained for the CD11 expression level are shown in Table 2 as a relative value with respect to the CD11 expression level of the control group as 100%.

[0066]

[Table 1]

[0067]

[Table 2]

From Tables 1 and 2 above, according to the anticancer agent of the present invention (that is, the combined use of AZ and retinoic acid), the retinoic acid (ARTA group) has further enhanced cancer cell growth inhibitory ability and differentiation inducing ability. It is clear that

[0069]

[Pharmacological Test Example 2] HL-60 was placed in each well of a 96-well plate and cultured in 10% FCS-added RPMI-1640 medium at 37 ° C. under 5% CO ₂ to give a cell concentration of 1
It was prepared to be × 10 ⁵ cells / ml.

Next, each test compound was added to each well at a predetermined concentration, and 10 wells containing TNF-α of 1000 units / ml were added.
% FCS-supplemented RPMI-1640 medium (labeled +) or not (labeled-), 37 ° C, 5%
Cultured under CO ₂ for 3 days. As a control group, DMS
O (no test compound added) was added, and 1 of TNF-α was added.
Groups were similarly cultivated with or without the addition of (000) units / ml (+).

After the above culture, MTT reagent [3-
(4,5-Dimethyl-2-thiazoyl) -2,5-diphenyl-2H.terazolium bromide (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 2.5 mg / ml in Dulbecco's medium (PBS ^(-) , (Manufactured by Nissui Pharmaceutical Co., Ltd.) and sterilized by filtration] 10 μl was added, the mixture was incubated at 37 ° C. in the presence of 5% CO ₂ for 3 hours, and then 0.01 N containing 10% SDS.
Add 100 μl of HCl solution to each well and add 3 more
It was cultured overnight at 7 ° C. in the presence of 5% CO ₂ .

After culturing, the absorbance (OD580 nm) of each well was measured using Titer Tech Multiscan (manufactured by Titer Tech Co., Ltd.).

The obtained results are shown in Tables 3 and 4.

[0074]

[Table 3]

[0075]

[Table 4]

The above results (values at each concentration of each compound to be tested) are the DMS in the absence (-) of TNF-α.
The value of the O-added group (control) was used as a standard (0), and the percentage of increase and decrease in growth inhibition (%) was shown.

From each table, each test compound was tested for TNF-α.
When used in combination with (the anticancer agent of the present invention), TNF-
A stronger inhibitory effect on cancer cell growth was observed as compared with the case of α alone. From this, it was revealed that each of the above test compounds further promotes the cancer cell growth inhibitory effect of TNF-α.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication location A61K 38/00 C07D 215/22 (72) Inventor Kazuyoshi Kawai Mitsuho Mitsuho, Matsushige-machi, Itano-gun, Tokushima Prefecture Development 130-2-3 Heights Soreiyu No.502 (72) Inventor Michiaki Tominaga 310-6 Takaiso, Kamiita-cho, Itano-gun, Tokushima Prefecture (72) Inventor Shoichi Adachi 34-9-9 Ishihara-cho, Takasaki-shi, Gunma Prefecture

Claims (1)

[Claims] 1. A general formula: [In the formula, R represents a benzoyl group which may have 1 to 3 groups selected from the group consisting of a lower alkoxy group, a halogen atom, a lower alkyl group and a lower alkylthio group on the phenyl ring as a substituent. The carbon-carbon bond between the 3-position and 4-position of the carbostyril skeleton represents a single bond or a double bond. ] A carbostyril derivative represented by the formula and a salt thereof, (2) the general formula: [In the formula, R ⁵ represents a phenyl lower alkyl group which may have a lower alkanoyloxy group or a hydroxyl group on the phenyl ring, a benzoyl group or a naphthoyl group. R ⁶ represents a benzoyl group which may have a lower alkoxy group on the phenyl ring. ] The carbostyril derivative and its salt represented by the formula (3): [In the formula, R ⁷ represents a hydroxyl group, an amino group, a lower alkoxy group, a lower alkyl group or a piperazinyl group. R ⁸ represents a hydrogen atom or a lower alkyl group. R ⁹ represents a hydrogen atom.
The bonds at the 3rd and 4th positions of the carbostyril skeleton represent a single bond or a double bond. ] The carbostyril derivative and its salt represented by the following formula (4): [In the formula, R ¹³ represents a phenyl lower alkyl group. R ¹⁴ is a group —O—A—R ¹⁵ [A is a lower alkylene group, R ¹⁵ is a group Indicates. ] The carbostyril derivative represented by these, and its salt, (5) 6- [4- (3,4-dimethoxybenzoyl) -1-1,2,3,4- tetrahydropyrazyl]-
3,4-Dihydrocarbostyril and its salt, and (6) at least one compound selected from 7-methoxy-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine and its salt, and cytokine And / or a retinoid as an active ingredient.