WO2004066994A1

WO2004066994A1 - Anticancer agent

Info

Publication number: WO2004066994A1
Application number: PCT/JP2004/000680
Authority: WO
Inventors: Shinya Kimura; Taira Maekawa
Original assignee: Kansai Technology Licensing Organization Co. Ltd.
Priority date: 2003-01-28
Filing date: 2004-01-27
Publication date: 2004-08-12
Also published as: JPWO2004066994A1

Abstract

An anticancer agent which contains as an active ingredient a compound represented by the general formula (1): (1) (wherein R1 to R9 are the same as defined in the description) or a pharmaceutically acceptable salt thereof.

Description

Specification

Anticancer drug

Technical field

The present invention relates to an anticancer agent and a method for treating cancer.

Background art

It has been known that some substances extracted from plants have an anti-cancer effect and a carcinogenesis-preventing effect, and many studies have been made on components that can be obtained from plant extracts. For example, it has been reported that a substance obtained from a plant called St. John's wort exhibits an antitumor effect (Christoph et al., Oncogene, 2002, 21, 1242-1250). In addition, among substances obtained from the genus Carophilium, there are substances that are effective in preventing cancer (Ito et al, Journal of Natural Products, 2002, 65 (3), 267-272), and substances that have cytotoxicity (David et al., Phytochemistry, 2001, 58, 571-575) and the presence of substances with an inhibitory effect on AIDS (Claude et al., Bioorganic & Medicinal Chemistry Letters, 1998, 8, 3475-3478). It has been reported. The structures of the substances reported in these documents vary, and their relation to function or action is not clear.

On the other hand, in the field of chemical synthesis, various drugs useful as anticancer agents have been developed. Until now, various anticancer agents and the like exhibiting a high cell growth inhibitory action have been developed. However, some of these were also toxic to normal cells, and many did not have sufficient suitability for actual administration to patients. In cancer, the relationship between the onset mechanism and p53 has been pointed out, and many anticancer drugs that exert their effects by inducing p53 have been developed, but they have not been expected to have any degree of effect. Many did not show a broad effect on cancer of different types, or to the extent desired. Furthermore, in order to reduce synergistic effects and side effects, multidrug therapy using a combination of multiple anticancer drugs has been performed. However, P-glycoprotein (P-glycoprotein) is induced, and cells become multidrug-resistant ( The administration of an anticancer drug to such a patient, who often acquires MDR), has a problem in that the drug concentration in the cancer cells is reduced and an effective effect cannot be obtained.

Based on these facts, it has a mechanism different from conventional ones, is effective for multidrug-resistant cancer cells, has a large safety margin, and is expected to develop new anticancer agents. An object of the present invention is to provide an anticancer agent having a mechanism different from the conventional one, effective for multidrug-resistant cancer cells, and having a large safety margin.

Another object of the present invention is to provide a method for treating cancer. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structures of 15 compounds obtained from Calophyllmn Brasiliensis.

FIG. 2 shows the results of examining the inhibitory effect of 15 compounds shown in FIG. 1 on the growth of BV173 cells at concentrations of ΟμΜ, M, 5μΜ, ΙΟμΜ, and 30Μ! .

FIG. 3 shows the results of measuring the cytostatic effect of compound 7 (GUT-70) on five types of leukemia cell lines (BV173, SEM, NALM6, HL60, and K562). (♦) indicates 0 μM, (translation) is 0.5 μΜ, (mouth) is 1.0 μΜ, (5.0) is 5.0 μΜ, and (X) is the measurement result at 10 10. .

FIG. 4 shows the results of morphological observation of apoptosis by compound 7. FIG. 4Α shows the observation results before treatment with compound 7, and FIG. 4Β shows the observation results after treatment with compound 7.

FIG. 5 shows the results of measuring the relationship between cell cycle and apoptosis using TUNEL Atssei. Fig. 5Α shows the results for the induction of apoptosis when GUT-70 was not treated and Fig. 5Β shows the results when GUT-70 was treated for 5 μΜ for 24 hours.

Figure 6 shows the leukemia cell line NALM6 cells and BV173 cells treated with compound 7, was examined the temporal change in the _P21 WAF./CIP.^ _P27 K _IPL, _p53 and _p57 results.

FIG. 7A shows the results of comparing the resistance of daunorubicin (DNR) to the P-glycoprotein high-expressing cell K562 / D1-9 and the parent strain 562 cells that do not express P-glycoprotein. FIG. 7B shows the results of comparing the resistance of compound 7 to the P-glycoprotein overexpressing cell K562 / D1-9 and the parent strain K562 cell not overexpressing P-glycoprotein.

FIG. 8 shows the results of examining the toxicity of compound 7 on normal cells using normal hematopoietic progenitor cells CFU-GM and BFU-E.

FIG. 9 shows the results of examining the effect of compound 7 (GUT-70) on human small cell lung cancer.

Disclosure of the invention

The inventor of the present invention has made intensive studies with the main object of solving the above problems. As a result, a compound having a specific structure has a remarkable inhibitory effect on the growth of cancer cells. They also found that they are effective against drug-resistant cancer cells, and that they have a large safety margin. The present inventors have made further studies and completed the present invention.

That is, the present invention relates to the following anticancer agents and methods for treating cancer.

1.The following general formula (1)

(Wherein RR ² independently represents a hydrogen atom, an alkyl group, a cycloalkyl group or an aryl group which may be substituted, or CR ² represents C = 〇;

R ³ and R ⁴ each independently represent a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, an alkyl group, a cycloalkyl group, an optionally substituted aryl group, an alkoxy group, an acyl group, a propyloxyl group, an alkoxy group, A carboxyl group, an amino group, a monoalkylamino group, a dialkylamino group or a quinoline: a diamino group;

R ⁵ , R ⁸ and R ⁹ each independently represent a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, an alkyl group, a cycloalkyl group, an optionally substituted aryl group, an alkoxy group, an acyl group, a carboxyl group , Alkoxycarbonyl group, optionally substituted aryloxy group, thiol group, alkylthio group, optionally substituted arylthio group, alkylsulfonyl group, optionally substituted arylsulfur group, alkylaminocarbonyl Group, an optionally substituted arylaminosulfonyl group, an alkylaminosulfoyl group, an optionally substituted arylaminosulfonyl group, an alkoxycarbonyl group, an optionally substituted aryloxycarbonyl Group, amino group, monoalkylamino group, dialquinoleamino group, acylamino , Alkylsulfonyl § amino group or a substituted been, even! /, Arino Resnore Honinoreamino Represents a group;

R ⁶ represents an alkyl group, a cycloalkyl group, an optionally substituted aryl group, an alkoxy group, an acyl group, a carboxyl group, an alkoxycarbol group, or a compound represented by the following general formula (2)

Represents a structure represented by;

R ⁷ represents 0, NH or NR ¹³ ;

R ^1. Represents a hydrogen atom, an alkyl group, a cycloalkyl group or an aryl group which may be substituted;

R ^u and R ¹² each independently represent a hydrogen atom, an alkyl group, a cycloalkyl group, or a substituted or unsubstituted aryl group;

R ¹³ represents an alkyl group, a cycloalkyl group or an aryl group which may be substituted) or a pharmaceutically acceptable salt thereof as an active ingredient.

2. The anticancer agent according to item 1, wherein R ⁷ is an oxygen atom.

3. anticancer agent according to claim 1 which is R ⁵ force S methoxy group.

4. The anticancer agent according to item 1, wherein R ⁹ is an alkyl group or an optionally substituted aryl group.

5. The anticancer agent according to item 1, wherein R ⁹ is an n-propyl group.

6. R ⁶ is the following general formula (2)

Wherein R ^1G , R ¹¹ and R ¹² are as defined above. The anticancer agent _{c according} to item 1, which is a group represented by

7. The following general formula (3)

Item 4. The anticancer agent according to item 1, represented by the formula:

8. A method for treating cancer, which comprises administering an effective amount of the compound according to any one of Items 1 to 7.

9. The cancer is acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, choriocarcinoma, multiple myeloma, soft tissue tumor, small cell lung cancer, chronic myelogenous leukemia, medullary thyroid carcinoma, osteosarcoma, head and neck Item 9. The method according to Item 8, which is selected from the group consisting of cancer, esophageal cancer, non-small cell lung cancer, colon cancer, stomach cancer, biliary tract cancer, brain tumor, malignant melanoma, kidney cancer, knee cancer, and liver cancer.

10. The method according to item 9, wherein the cancer is selected from the group consisting of acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, chronic myeloid leukemia, and small cell lung cancer.

11. The method according to item 8, wherein the cancer is a multidrug resistant (DR) cancer.

The present invention relates to an anticancer agent preferably containing a compound represented by the general formula (3) as an active ingredient, and a method for treating cancer by administering the compound to a cancer patient.

More preferably, acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, choriocarcinoma, multiple myeloma, soft tissue tumor, small cell lung cancer, chronic myelogenous leukemia, medullary thyroid carcinoma, osteosarcoma, head and neck cancer Esophageal cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, biliary tract cancer, brain tumor, malignant melanoma, renal S tumor, knee cancer, liver cancer. The present invention relates to an anticancer agent containing a compound represented by the formula (3) as an active ingredient. Further, the present invention preferably relates to a method for treating cancer, which comprises administering an anticancer effective amount of a compound represented by the general formula (3).

More preferably, acute antimyelogenous leukemia, acute lymphocytic leukemia, malignant lymphoma, choriocarcinoma, multiple myeloma, characterized in that an anticancer effective amount of the compound represented by the general formula (3) is administered. Soft tissue tumor, small cell lung cancer, chronic myeloid leukemia, medullary thyroid cancer, osteosarcoma, head and neck cancer, esophageal cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, biliary tract cancer, brain tumor, malignant melanoma, kidney The present invention relates to a method for treating a disease selected from the group consisting of cancer, victory cancer, and liver cancer.

Hereinafter, the present invention will be described in detail.

Structure of anticancer compound

The active ingredient of the anticancer agent of the present invention has a structure represented by the following general formula (1).

-General formula (1)

(In the formula, R ¹ to are as defined above.)

In to ³ , the alkyl group includes a substituted or unsubstituted linear or branched one, and usually has 1 to 18, preferably 1 to 8, and more preferably 1 to 3, for example, Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutynole, tert-butynole, n-pentyl, isopentyl, hexyl, heptyl, octyl, nonyl and decyl.

The cycloalkyl group may be substituted or unsubstituted, and the number of carbon atoms is not particularly limited, but is usually 3 to 10, preferably 3 to 7. Specific examples of the cycloalkyl group include cyclopropanol, cyclobutyl, cyclopentyl, cyclohexynole, and cycloheptyl. As the aryl group, the term "aryl group" means a monocyclic or polycyclic group composed of a 5- or 6-membered aromatic hydrocarbon ring, and specific examples include phenyl, naphthyl, fluorenyl, and anthrenyl. , Biphenyl, tetrahydronaphthyl, chromanyl, 2,3-dihydro-1,4-dioxanaphthalenyl, indanyl and phenanthryl.

The number of substituents of the aryl group having a substituent is 1 to 3, preferably 1 or 2, and the substituent includes methyl, ethyl, methoxy, ethoxy, amino, methylamino, dimethinoleamino, cyano, nitro, Examples include fluorine, chlorine, bromine, trifluoromethyl, hydroxy, carboxyl, methoxycarbonyl, ethoxycarbol, propoxylproponyl, butoxycarbonyl, CONH2, acetyl. Examples of the substituted aryl group include phenyl, 2-, 3- or 4-fluorophenyl, 2-, 3- or 4-chlorophenol, 2_, 3- or 4-bromophenyl, 2,4- Difluorophenyl, 2, 3_, 2, 4-, 3, 4-, 3, 5- or 2,6-dichlorophenyl, 3-chloro-1-phenylphenyl, 4-isopropylphenyl, 2,6- Dimethynophenyl, 2-, 3- or 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-trifluoromethoxyphenyl, 2-, 3- or 4-dimethylaminophenyl, 2_ , 3- or 4-1-trophenyl, and 4-sulfamoylphenyl.

Examples of the alkoxy group include groups represented by 〇R ′ (R ′ is the above-mentioned alkyl group). For example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy , T-butoxy.

Examples of the acyl group include a group represented by one COR ′ ′ (R ″ is the above-mentioned alkyl group or the above-mentioned optionally substituted aryl group), and examples thereof include acetyl, propionyl, and benzoyl. Is done.

Examples of the halogen atom include fluorine, chlorine, bromine, and iodine.

Examples of the asinoleamino group include a group represented by one NHCOR ′ ′ (R ″ is the above-mentioned alkyl group or the above-mentioned optionally substituted aryl group). Can be

Examples of the alkoxycarbonyl group include a group represented by one CO〇R ′ (R ′ is the aforementioned alkyl group), for example, methoxycarbyl, ethoxycarbonyl, n-propoxycarbinole, isopropoxycarbonyl, Examples are n-butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl and t-butoxycarbonyl. You may be replaced! Examples of the / aryloxy group include a group represented by -0- (substituted or substituted aryl group), and examples include phenyloxy and naphthyloxy.

Examples of the alkylthio group include a group represented by -S- (alkyl group), and examples thereof include methylthio, ethylthio, _n -propynolethio, isopropylthio, n-butylthio, isobutylthio, and tert-butylthio.

You can be replaced! Examples of the / and arylthio groups include groups represented by -S- (substituted or substituted aryl groups), such as phenylthio and naphthylthio.

Examples of the monoalkylamino group include an amino group mono-substituted with the above alkyl, such as methylaminocarbonyl, ethylaminocarbonyl, n-propylaminocarbonyl, isopropylaminocarbonyl, n-butylaminocarbonyl, and isobutylamino. Carbonyl, tert-butylaminocarbonyl, n-pentylaminophenol, isopentylaminocarbonyl and hexylaminocarbonyl.

Examples of the dianolequinole amino group include amino groups di-substituted with the above alkyl, such as dimethylaminocarbonyl, getylaminocarbonyl, di-n-propylaminocarbonyl, diisopropylaminocarbonyl, di-n-butylaminoamino, Examples thereof include diisobutylaminocarbonyl, ditert-butylaminocarbonyl, di-n-pentylaminocarbonyl, diisopentylaminocarbonyl, and dihexylaminocarbonyl.

The alkylsulfonyl group, - S0 ₂ - include groups represented by (alkyl), for example, methylcarbamoyl Roh less Honoré Honi Honoré, E Chino less Honoré Honi Honoré, n- pro Pinot less Honoré Honi Honoré, isopropylidene Roh-less And norfoninole, n-butylsulfonyl, isoptylsulfonyl, and tert-butylsulfonyl. The optionally substituted § reel sulfonyl group, _S0 ₂ - include groups represented by (optionally substituted § aryl group), for example Fuenirusuruho - Le include naphthylsulfonyl El.

Examples of the alkylaminocarbo group include an aminocarbonyl group mono- or di-substituted with the alkyl. Examples of the amino group mono-substituted with the alkyl include methylaminocanoleponinole, ethylaminocarbonyl, and the like. n-propylaminocarbonyl, isopropylaminocarboel, n-butylaminocarbonyl, isobutylaminocarbonyl, tert-butylaminocarbonyl, n-pentylaminocarbonyl, isopentylaminocarbonyl, and hexinoleaminocarbonyl. As the amino-disubstituted di-substituted alkyl, Dimethylaminopropyl, diethylaminocarbonyl, di-n-propylaminocarbonyl, diisopropylaminocarbonyl, di-n-butylaminocarbonyl, diisobutylaminocarbonyl, di-tert-butylaminocarbonyl, di-n-pentyla Examples include minocarbonyl, diisopentylaminocarbonyl, and dihexylaminocarbonyl.

Examples of the optionally substituted arylaminocarbonyl group include a group represented by -CONH- (optionally substituted arylylcarbonyl), such as phenylaminoaminocarbonyl and naphthylaminocarbonyl. No.

Examples of the alkylaminosulfonyl group include an aminosulfonyl group mono- or di-substituted with the alkyl. Examples of the amino group mono-substituted with the alkyl include methylaminosulfonyl, ethylaminosulfonyl, and n-propylamino. Sulfonyl, isopropylamino sulfoel, n-butylaminosulfonyl, isobutylaminosulfonyl, tert-butynoleaminosulfonyl, n-pentylaminosulfonyl, isopentylaminosulfonyl, hexylaminosulfonyl, and the aminosulfonyl di-substituted with the alkyl. Examples of the methyl group include dimethylaminosulfonyl, getylaminosulfonyl, di-n-propylaminosulfonyl, diisopropylaminosulfonyl, di-n-butylaminosulfonyl, Two Honoré, di tert- heptyl aminosulfonyl, di n- pentyl aminosulfonyl, Jiisopenchirua Minosuruhoniru include carboxymethyl Honoré aminosulfonyl to di.

The substituents on these § reel aminosulfonyl group, - S0 ₂ NH- include groups represented by (substituted Moyoi Ariru group), like for example phenylalanine amino sulfonyl, naphthyl aminosulfonyl Can be

The substituted or unsubstituted aryloxycarbonyl group includes a group represented by -COO- (substituted or unsubstituted aryl group), for example, phenylcarboxy group. And naphthyloxycarbonyl.

Alkylsulfonyl § amino group, - NHS0 ₂ - et include groups represented by (alkyl) is, for example, methylsulfonyl § Mino, E chill sulfonyl § amino, n- propylsulfonyl amino, isopropylsulfonyl - Ruamino, n-butylsulfonylamino-containing isobutylsulfonylamino, tert-butylsnolephonylamino, n-pentylsulfonylamino, isopentylsulfonynonamino, hexylsulfonylamino, Substituted by Les, be good Le, as a § reel sulfonyl § amino group, - NHS0 ₂ - include groups (! Substituted /ヽalso good I Ariru group) represented by, e.g., phenylalanine sulfonyl Examples include naphthylsulfonylamino with amide.

In the compound represented by the general formula (1) of the present invention, R ⁷ is preferably an oxygen atom (〇). R ⁵ is preferably a methoxy group (_OCH ₃ ).

R ⁹ is preferably an alkyl group or an aryl group, particularly preferably an n-propyl group.

R ⁶ is a general formula (2)

Wherein R ^{1 (3} , R ¹¹ and R ¹² are as defined above)

The group represented by is preferred.

In the present invention, the general formula (3)

(5-methoxy-2,2-dimethyl_6- (2-methyl-oxo-2-butenyl) -10-prop-pyr-2H, 8H-benzo [1,2-b; 3,4 -b,] dipyran-8-one (5-methoxy-2,2-dimethy 6- (2- methtl-l-oxo-2-butenyl) -10-propyl-2H, 8H-benzo [l, 2-b; 3,4-b '] dipyran-8-one)) is preferred as an anticancer agent.

The compound of the general formula (3) has a particularly excellent cancer cell growth inhibitory action.

Pharmaceutically acceptable salts of the compounds represented by the general formula (I) of the present invention may be any one of -R ⁶ , R ⁸ , R ⁹ in which a carboxyl group (COOH) or an amino group, a mono- or di-alkylamino group. If the compound has a phenyl group, a pharmaceutically acceptable salt can be prepared. Examples of pharmaceutically acceptable salts of carboxy groups include alkali metal salts (sodium salt, potassium salt, lithium salt), and pharmaceutically acceptable salts of amino, mono- or dialkylamino groups. Inorganic salts such as sulfate, hydrochloride, hydrobromide, lead nitrate and phosphate, fumarate, maleate, succinate, methanesulfonate, toluenesulfonate And other organic acid salts. Such pharmaceutically acceptable salts can be obtained by reacting a compound of the present invention with an alkali metal carbonate or carbonate, an alkali metal hydroxide, or an inorganic or organic acid. Monkey

Method for producing compound of general formula (1)

The above compounds can be appropriately prepared by various methods, may be chemically synthesized, or may be separated from an appropriate plant by extraction or the like.

(i) Chemical synthesis method

As a method of chemically synthesizing, a known method can be appropriately used.

For example, the following method can be used.

The following general formula (4)

(Wherein is as defined above)

Or a compound represented by the following general formula (5)

(Wherein, to 1 ^5, wherein is as defined. Z represents a hydrogen atom or R ¹³ (R ¹³ is as defined above).)

And a compound represented by the following general formula (6)

Wherein R ⁸ and R ⁹ are as defined above.

The compound represented by is heated and reacted using concentrated sulfuric acid,

Each of the following general formula (7)

Wherein R ^ R ⁵ , R ⁸ and R ⁹ are as defined above.

Or a compound represented by the following general formula (8)

(Wherein, R ⁸ , R ⁹ and Z are as defined above)

Is obtained.

Next, the compound represented by the general formula (7) or (8) is added to, for example, the following general formula (9) R ⁶ — C1 (9)

Is reacted with a Friedel Crafts catalyst to obtain a compound represented by the following general formula (1)

(Wherein is as defined above)

Is obtained.

A compound having a structure wherein R ⁶ is represented by the general formula (2) is a compound represented by the following general formula (10) as a compound represented by the general formula (9).

(Wherein, R ^{1Q to} R ¹² are as defined above)

Can be obtained by an acylation reaction using a Friedel-Crafts catalyst.

The Friedel 'Crafts (Friede preparative Crafts) catalysts include, for example, _{_{_{A1C1 3, BF 3, ZnCl 2}}} , Sn Cl 4.

All of the compounds represented by the general formulas (4) to (10) can be obtained by a general-purpose method, and when there is a commercially available product, it may be used as it is.

The compound of the present invention thus obtained is usually dissolved, extracted, separated, gradient, filtered, concentrated, thin-layer chromatography, column chromatography, gas chromatography, high-performance liquid chromatography, distillation, sublimation, crystal Purification is carried out as appropriate by using a method such as chemical conversion alone or in combination. In the same manner as described above, various conjugates can be obtained by using an intermediate having a desired substituent or by appropriately introducing an appropriate functional group.

For example, in the general formula (4), a compound in which R ¹ and R ² are methyl groups, R ³ and R ⁴ are hydrogen, and R ^{5 is} —OCH ₃ group, and in the general formula (6), R ⁸ is hydrogen When a compound in which R ^{9 is a} —CH ₂ CH ₂ CH ₃ group is used, and in the general formula (10), a compound in which R ^1C) is hydrogen, R ″ and R ¹² is a methyl group,

The following general formula (3)

Can be obtained.

(ii) Extraction method from plants

The compound serving as an active ingredient of the pharmaceutical composition of the present invention can be obtained by extracting an appropriate plant power. Examples of the plant include a plant of the family Cucurbitaceae (Guttiferae). For plants of the family Tortaceae, those belonging to the genus Calophyllum, such as Calophyllum Brasiliensis or Calophyllum inophyllum, are preferred, and Calophyllum Brasiliensis is particularly preferred.

The method for obtaining the compound by extracting the compound from the plant can be appropriately selected from known methods, and is not particularly limited. For example, a method in which the bark of a plant is powder-framed and extracted with an organic solvent is used. Can be.

Specifically, it can be performed as follows. After extracting the dried bark of Calophyllum Brasiliensis or Calophyllum inophyllum with an appropriate organic solvent, for example, a lower alcohol such as acetone, hexane, methanol, ethanol, etc., at appropriate temperature, extract at room temperature, and then extract under low pressure. Evaporate the solvent. Further, the desired compound is obtained by performing appropriate separation and purification such as chromatography, filtration and extraction.

For example, the compound of the general formula (3) can be obtained by separating and purifying a plant extract of Calophyllum Brasiliensis (Guttiferae).

Manufacturing method of pharmaceutical composition (pharmaceutical formulation) The compound of the general formula (1) or a salt thereof is mixed with a pharmaceutically acceptable carrier, and solid preparations such as tablets, capsules, granules and powders; liquid preparations such as syrups and injections, and patches It can be appropriately formulated as a transdermal absorbent such as an ointment and a plaster, an inhalant, and a suppository. The pharmaceutical composition of the present invention is orally or parenterally administered, and the above compounds may be used alone or in combination of two or more.

As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials can be used. Specifically, excipients, lubricants, binders, disintegrants in solid preparations, solvents, dissolution aids, suspending agents, isotonic agents, buffers, soothing agents, etc. in liquid preparations Can be blended. If necessary, pharmaceutical additives such as preservatives, antioxidants, coloring agents and sweeteners can also be used.

Preferred examples of the excipient include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light caffeic anhydride and the like.

Preferred examples of the lubricant include, for example, magnesium stearate, calcium stearate, talc, colloidal silica and the like.

Suitable examples of the binder include, for example, crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylsenorellose, hydroxypropylmethylcellulose, polyvinylinolepyrrolidone and the like.

Preferred examples of the disintegrant include starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethyl starch sodium and the like.

Preferred examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like.

Preferred examples of the dissolution aid include, for example, polyethylene glycol, propylene dalicol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. .

Preferable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; polybutyl alcohol , Polyvinylpyrrolidone, sodium carboxymethinolate cellulose, methinoresenololose, hydroxymethylsenorelose, Examples include hydrophilic polymers such as droxicetyl cellulose and hydroxypropyl cellulose.

Preferable examples of the tonicity agent include sodium chloride, glycerin, D-mannitol and the like.

Preferred examples of the buffer include buffers such as phosphate, acetate, carbonate, and citrate.

Preferable examples of the painless darting agent include, for example, benzyl alcohol. Preferable examples of the preservative include, for example, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.

Preferable examples of the antioxidant include, for example, sulfite, ascorbic acid and the like. The anticancer agent of the present invention can be applied to the treatment of various cancers.

The type of cancer is not particularly limited, for example, acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, choriocarcinoma, multiple myeloma, soft tissue tumor, small cell lung cancer, chronic myeloid leukemia, medullary thyroid cancer Osteosarcoma, head and neck cancer, esophageal cancer, non-small cell lung cancer, colorectal cancer, stomach cancer, biliary tract cancer, brain tumor, malignant melanoma, kidney cancer, knee cancer, and liver cancer.

The anticancer agent of the present invention is particularly effective for acute myeloid leukemia, acute lymphocytic leukemia, and small cell lung cancer. Among them, it is suitably used for acute myeloid leukemia, acute lymphocytic leukemia, and small cell lung cancer.

The administration conditions and administration method of the anticancer agent of the present invention can be appropriately set according to the type of the disease, the condition of the patient, the application site, and the like.

Specifically, the dose is appropriately adjusted according to the type, size (or weight), symptoms, etc. of the animal. When administered to humans or non-human animals, about 0.1 to 100 mg / kg (body weight), preferably about 1.0 to 10 mg / kg (body weight), more preferably about 1.0 to 3. Administer Omg / kg (body weight). The administration method can also be adjusted appropriately to obtain the optimal therapeutic effect.For example, it can be administered in several divided doses daily, and if a dangerous condition occurs during treatment, the dose may be adjusted according to the condition. And the number of administrations can be reduced as appropriate.

The anticancer agent of the present invention can be administered alone to obtain a desired effect. It can also be used in appropriate combination with an anticancer agent, a chemotherapeutic agent, an anti-inflammatory agent or an immunotherapeutic agent.

Other anticancer agents used in combination with the anticancer agent of the present invention include, for example, antimetabolites, alkylating agents, platinum-based anticancer agents, topoisomerase inhibitors, anticancer antibiotics, tyrosine kinase inhibitors, human Antibodies.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to Examples and Test Examples, but the present invention is not limited to these Examples.

'' Preparation of compounds

10 Using Calophyllum Brasiliensis collected from Brassinole, several compounds were obtained by the following procedure. First, the dry bark (630 g) of Calophyllum Brasiliensis was pulverized and then subjected to cold immersion extraction three times with acetone (3 L). The obtained acetone extract was concentrated under reduced pressure, and the residue was used as an acetone extract (19.26 g). This acetone extract was subjected to Si gel column chromatography using hexane-acetone as eluent.

At around 15: Compound 7 (GUT-70) (137.8 mg) was obtained from the eluate at 85:15. The same treatment was carried out using hexane-acetone, acetone, CH ₂ C ₁₂ -MeOH, and methanol as eluents. As a result, 15 kinds of compounds including compound 7 were obtained. Figure 1 shows the structures of the obtained 15 compounds.

Compound 7 (GUT-70) was identified based on Palmer, C.J .; Josephs, J.L. Tetrahedron Lett. 1994, 35, 20 5363-5366, and references therein. The structures of other compounds were also identified based on known literature.

Cell preparation

Human leukemia cells BV173, SUPB15, NAL6, HL60 and SEM were obtained from the American Type Culture Collection. In addition, human small cell lung cancer cell lines SBC-1, SBC-3, SBC_5, 25N427, N69, LUT_130, H82, NCI345, LUT134B were used in the experiments. Human leukemia cell lines BV173, SUPB15, NALM6, HL60 and SEM, and human small cell lung cancer cell lines SBC-1, SBC-3, SBC-5, N427, N69, LUT-130, H82, NCI345, LUT134B are non- inactivated the 10% FCS (Hyclone) RPMI- 1640 medium supplemented with in (GibcoBRL), and maintained at 37 ° C, in 5% C0 ₂ moisture culture Yoshita. SEM is an Iscove culture (GibcoBRL) supplemented with inactivated 10% FCS. Cultured in P-glycoprotein high expression multidrug resistance (MDR) K562 / D1-9 cells were maintained in RPMI-1640 culture medium supplemented with inactivated 10% FCS and 0.1 μM daunorubicin (DNR).

In vitro cell proliferation activity measurement

Cell proliferation was determined using a count of cells using the trypan blue dye exclusion method and a modified MTT assay using a SF reagent (Nacalai Tesque). Leukemia cells were cultured in flat-bottom 96-well plates (HGreiner labortechnik) at 2 × 10 ⁴ cells / well in 100 μl medium and cultured for 72 hours with various concentrations of compound.調べる In order to examine the effect of GUT-70 (digestion compound 7) on cells expressing high levels of glycoprotein, K562 / D1-9 cells were cultured at various concentrations for GUT-70 or DNR for 72 hours under the same conditions. Cultured. The average of each of the six data was used. There was a linear relationship between TT assay and cell number.

Western plot analysis

Protein samples were separated by SDS / PAGE and nitrocellulose membrane (Amersham

Bioscience). The membrane was saturated with a solution of 5% non-fat dry milk in TBST (25 mM Tris-HCl pH 7.8, 140 mM NaCl, 0.1% (vol / vol) Tween20) and persimmon polyclonal _p21 w _AFI / _CIP1 , _p27Kipl , Incubated overnight with _p53 antibody (diluted to ₁₀₀₀ min). Then, the plate was washed thoroughly with TBST and incubated for 1 hour with donkey anti-Peacock IgG conjugated with horseradish peroxidase (Santa Cruz Biotech.). Detection was performed at Amersham Biosciences;

Apoptosis induction analysis

Apoptosis induction was examined for untreated NALM6 and NALM6 cells treated with GUT-70 for 12 hours and 24 hours. May-Giemsa staining was used to study the overall morphology, and a terminal deoxynucleotidyl transferase (TUNEL) assay was used to detect apoptosis. After drug treatment, the 5 X 10 ⁴ cells were washed with PBS _(P H7.3), and resuspended in the same buffer. The cytospin preparation of the cell suspension was fixed and stained with May-Giemsa stain. Cell morphology was determined by light microscopy. TUNEL assay was performed using an apoptosis detection kit (R & D systems, Wiesbaden, Germany) according to the instructions. Briefly, NAL 6 cells were resuspended in PBS containing 3.7% formaldehyde on ice for 10 minutes and rinsed with PBS. Then fixed fine The cells were incubated in cytonin for 30 minutes at room temperature and washed with labeling buffer. Cells are labeled using a 25 1 labeling mix (dNTPMix, Mn ²⁺ , TdT, TdT labeling buffer).

After resuspending at 37 ° C for 1 hour, the reaction was stopped with a stop buffer. Next, the cells were resuspended in a dark room at room temperature for 10 minutes using 25 μl of a solution containing streptavidin. Cells

The cells were rinsed with PBS, counterstained with 2 μg / ml propidium iodide for 30 minutes, and subjected to flow analysis using flow cytometry (FACScan; Becton Dickinson. NJ).

Toxicity evaluation of GUT-70 on normal hematopoietic progenitor cells

The sensitivity of normal hematopoietic progenitor cells to GUT-70 was examined by a standard methylcellulose culture assay containing GM-CSF, IL_3, and G-SCF. Normal hematopoietic progenitor cells used were bone marrow cells from a healthy volunteer with informed consent. The cells (1 × 10 ⁵ ) were cultured in methylcellulose for 12 days at 37 ° C. in a 5% CO ₂ incubator at a GUT-70 concentration of 0.20 or 50. The number of CFU-GM and BFU-E colonies was counted under a microscope. Three tissues were tested for each gnorape and the average was determined.

Statistical analysis

Statistics used Student's st test. P values less than 0.05 were considered statistically significant and were drawn from two-sided tests.

Cell growth inhibition evaluation

Fifteen natural extracts from C. Brasiliensis, namely six Xanthones (1-6) and nine 4-phenylcoumarins (which are two tricyclic coumarinides) 7,8), six tetracyclic coumarins (9-14) and one dimethylcyclopropyl (15) were obtained. Compound 8 is a compound previously identified as Calophyllolide (Spino) or Oblongulide (Dharmaratan). In addition, the anti-leukemia activity of five leukemia cells was examined using MTT Atsey. The proliferation of BV173 cells was suppressed by a 4-phenylcoumarin conjugate containing a tricyclic coumarin structure (i-Dai-conjugate 7 (GUT-70) and 8). This growth inhibitory effect was concentration dependent at concentrations ranging from M to 30 μM. On the other hand, the other 13 compounds did not show a growth inhibitory effect.

While both Compound 7 and Compound 8 showed an inhibitory effect on the growth of BV173 cells, Compound 7 having a propyl group (GUT_70) showed a more remarkable effect (FIG. 2). The compounds Ί and The IC50 value (halHnaximal inhibitory concentrations) of the compound 8 was 3 μM and 9 μM, respectively. Furthermore, Compound 7 also exhibited concentration-dependent and time-dependent cell growth inhibitory effects on the other five leukemia cell lines (K562, HL60, SEM, NALM6, and SUPB15) (FIG. 3). GUT-70 also steadily inhibited the growth of human small cell lung cancer cell lines SBC-1, SBC-3, LUT134B, and H82 (IC50 value of 5 μΜ to 8 μΜ, FIG. 9).

-As is evident from the above results, a compound with a tricyclic coumarin structure extracted from the stem of Calophyllum Brasiliensis collected from Brassinole has a significant cytostatic effect on leukemia cells and small cell lung cancer cells. It became clear.

Tricyclic coumarin compounds (I), including compounds 7 and 8, suppressed cell growth in a concentration-dependent manner. On the other hand, 13 compounds with other structures did not inhibit growth. Among them, some tetracyclic coumarindi conjugates have been reported to have a carcinogenesis-preventing action, but have no growth-suppressing action. Also, the effect of Compound 7 was more remarkable than Compound 8.

These findings indicate that the tricyclic coumarin structure is an essential structure for the growth inhibitory effect, and that the propyl group in the side chain increases the anti-leukemia effect. The reason that compound 7 having a propyl group in the side chain exhibits a more effective action is because of its stronger affinity for the membrane [I]. The knowledge gained about the importance of this side chain is expected to improve the action of 4-phenylcoumarin compounds, including tricyclic coumarins.

Cell proliferation suppression by inducing apoptosis

Then compound 7 (GUT- 70) of exposing the _c NALM-6 cells examined the relationship between leukemia cytostatic and apoptosis by changing the concentration of the compound 7 24 hours, morphological techniques and flow in the induction of apoptosis Measured by cytometry. After 24 hours of exposure to compound 7 at a concentration of 5 μΜ, significant cell apoptosis was observed morphologically (FIG. 4). Compound 7 induces TUNEL-positive (i.e., apoptosis) cells significant number, reduced the percentage of cells in G ₂ / M phase. Compound 7 was accumulated in Gi-phase cells by TUNEL-Assy using double-stained piumumoxide staining, and apoptotic cells mainly appeared near the G-S phase (FIG. ⁵ ).

As described above, cell apoptosis was evaluated using several different methods for the effect of compound 7, which showed an antiproliferative effect on five different human leukemia cells in vitro. As a result, it is clear that leukemia cells treated with compound 7 can cause DNA cleavage and induce apoptosis, and induce apoptosis mainly at the Gi / S boundary. became. Effect of compound 7 (GUT-70) on cell cycle related proteins

The time course of p21 WAFI ^{/ CIP1} , p27 ^Kipl , p53 and p57 was examined in NALM6 cells and BV173 cells. Compound 7 did not induce the p21 which induced the tumor suppressor gene product p53 and the cyclin-dependent kinase inhibitors p27 and p57 in NALM6 cells. In BV173 cells, induced a p27 and p57, _P 21及Pi p53 did not induce (Figure 6). In addition, as described above, Compound 7 also showed a growth inhibitory effect on human leukemia cell line HL60 lacking wild-type p53. From these results, it was found that Compound 7 was p53-independent, that is, the cell growth inhibitory effect of Compound 7 was not an effect of p21 expression through p53 induction.

Moreover, in all cells, the expression of p27 ^Kipl increased, ^whereas the expression of p53 increased only in NALM6. Therefore, it was found that the main signaling pathway after the treatment of Compound で is the p27 pathway that blocks G1 / S translocation rather than the p53-p21 pathway.

Effect of compound Ί (GUT-70) on P-glycoprotein 髙 -expressing cells K562 / D1-9

Daunorubicin (DNR) is generally used as an anticancer drug, and is expressed by P-glycoprotein in contrast to K562 / D1-9, which is a cell that expresses P-glycoprotein. It was about 75 times more resistant than the parent strain K562. On the other hand, the sensitivity of compound 7 was not reduced even for K562 / D1_-9, which overexpresses P-glycoprotein, as compared with the parent strain K562, which does not express P-glycoprotein (Fig. 7). ). These findings indicate that compound 7 (GUT-70) is not involved in the P-glycoprotein-related MDR system. Compound 7 (GUT-70) appears to be a good candidate component to overcome the induction of MDR. Patients with relapse have MDR that induces P-glycoprotein, which is an anticancer drug efflux pump, and clinical trials of compound 7 as an anticancer drug that often shows multidrug resistance are initially applied to patients with relapsed or refractory disease It is thought that it is done.

Safety evaluation of compound 7 on normal hematopoietic progenitor cells

Normal hematopoietic progenitor cells treated with 20 μM and 50 μM Compound 7 (GUT-70) should result in 75% ± 10% and 5% ± 2% CFU-G colonies, respectively, of controls Became evident. The number of BFU-II colonies was found to be 80% ± 10% and 3% ± 1% of the control, respectively (Fig. 8). These results revealed that compound 7 (GUT-70) had a very low inhibitory effect on proliferation of normal hematopoietic progenitor cells up to a concentration of 20 μM. From this, it was found that compound 7 (GUT-70) was a compound having a large safety margin. As shown in the above results, the pharmaceutical composition of the present invention has a remarkable inhibitory effect on cancer growth, and can exert an excellent effect on cancer treatment, particularly on leukemia and small cell lung cancer. .

The anticancer agent of the present invention has an action mechanism different from the conventional one, and suppresses cancer cell growth in a p53-independent manner. Therefore, the present invention can be expected to have an effective effect on cancers and the like which could not be expected with conventional anticancer agents. In addition, it is possible to treat a wider variety of cancers and reduce side effects by using them in combination with drugs having different mechanisms.

Furthermore, the pharmaceutical composition of the present invention maintains an excellent effect on multidrug-resistant cancer cells in which P-glycoprotein has been induced. Therefore, it can be effectively used for patients whose other anticancer drugs have stopped responding or for patients who need multiple drug combination therapy.

Also, the present invention is suitable for clinical use with a large safety margin.

As described above, the present invention has various requirements that have been eagerly sought in the treatment of cancer and provides an excellent means for treating those diseases.

Claims

The scope of the claims

1. The following general formula (1)

(Wherein RR ² independently represents a hydrogen atom, an alkyl group, a cycloalkyl group or an aryl group which may be substituted, or CR ² represents C == 0;

RR ⁴ independently represents a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, an alkyl group, a cycloalkyl group, an aryl group which may be substituted, an alkoxy group, an acyl group, a propyloxyl group, an alkoxycarboyl group , An amino group, a monoalkylamino group, a dialkylamino group or an acylamino group;

R ⁵ , R ⁸ and R ⁹ each independently represent a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, an alkyl group, a cycloalkyl group, an optionally substituted aryl group, an alkoxy group, an acyl group, a carboxyl group An alkoxycarbonyl group, an optionally substituted aryloxy group, a thiol group, an alkylthio group, an optionally substituted arylthio group, an alkylsulfonyl group, an optionally substituted arylarylsulfonyl group, an alkylaminocarbonyl group, An optionally substituted arylaminosulfonyl group, an alkylaminosulfonyl group, an optionally substituted arylaminosulfonyl group, an alkoxycarbonyl group, an optionally substituted aryloxycarbonyl group , Amino group, monoalkylamino group, dialkylamino group, acylamino group It represents alkylene Honoré sulfonyl § amino group or an optionally substituted § reel sulfonyl § amino group; R ⁶ represents an alkyl group, a cycloalkyl group, an optionally substituted aryl group, an alkoxy group, an acyl group, a carboxyl group, an alkoxycarbol group, or a compound represented by the following general formula (2)

Represents a structure represented by;

R ⁷ represents 〇, NH or NR ¹³ ;

R ⁿ and R ¹² each independently represent a hydrogen atom, an alkyl group, a cycloalkyl group, or a substituted or unsubstituted aryl group;

2. The anticancer agent according to claim 1, wherein R ⁷ is an oxygen atom.

3. anticancer agent according to claim 1 R ⁵ is a methoxy group.

4. The anticancer agent according to claim 1, wherein R ⁹ is an alkyl group or an optionally substituted aryl group.

5. The anticancer agent according to claim 1, wherein R ⁹ is an n-propyl group.

6. R ⁶ is the following general formula (2)

Wherein R ^1D , R ¹¹ and R ¹² are as defined above. 2. The anticancer agent according to claim 1, which is a group represented by the formula:

7. The following general formula (3)

The anticancer agent according to claim 1, which is represented by:

8. A method for treating cancer, which comprises administering an effective amount of the compound according to any one of claims 1 to 7.

9. The cancer is acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, choriocarcinoma, multiple myeloma, soft tissue tumor, small cell lung cancer, chronic myelogenous leukemia, medullary thyroid carcinoma, osteosarcoma, head and neck 9. The method according to claim 8, wherein the method is selected from the group consisting of cancer, esophageal cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, biliary tract cancer, brain tumor, malignant melanoma, kidney cancer, knee cancer, and liver cancer.

10. The method according to claim 9, wherein the cancer is selected from the group consisting of acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, chronic myelogenous leukemia, and small cell lung cancer.

11. The method according to claim 8, wherein the cancer is a multidrug resistant (MDR) cancer.