WO2014102889A1 - Antitumor agent and method for producing same - Google Patents

Abstract

The present invention provides: a very highly effective antitumor agent which has an extremely high effect especially on various leukemia, lymphoma and solid cancers including refractory ATL; and a method for producing the antitumor agent. A compound represented by general formula (I) (excluding a compound represented by formula (D)). (In formula (I), R₁ represents a phenyl group that is substituted by at least one group selected from among a hydroxyl group, a methoxy group, a methylenedioxy group and a phosphoric acid group; R₂ represents one group selected from among a hydrogen atom, a hydroxyl group, a methoxy group, an acetyl group, an anilino group and groups represented by formulae (II); R₃ represents one group selected from among a hydrogen atom, a hydroxyl group, a methoxy group and an acetyl group; and R₄ represents one group selected from among a hydrogen atom, a hydroxyl group, a methoxy group and an acetyl group.)

Description

Antitumor agent and method for producing the same

The present invention relates to an antitumor agent and a method for producing the same. In particular, the present invention relates to an anti-tumor agent derived from the Labiatae plant Hyptis verticillata and a method for producing the same. Specifically, the present invention relates to an antitumor agent comprising a compound and composition derived from Hiptis / Verticillata as an active ingredient and a method for producing the same.

Hyptis Vertisirata, a Lamiaceae plant, is native to tropical America and inhabits the tropical and subtropical regions of the Americas. In Mexico, the leaves of this plant, generally called “hierba maltina”, are used orally as drugs for headache, stomach pain, digestive disorders and the like. Moreover, what boiled down this whole plant is used as a coating agent, such as an insect bite, a rheumatism, and a skin infection, and is also widely used as an anthelmintic and a laxative.

Many compounds having a lignan skeleton have been isolated from Hyptis Verticillata by past research (Non-patent Documents 1 and 2). Non-Patent Document 2 describes a compound having a lignan skeleton as shown in FIG.

Adult T-cell leukemia (Adult T-cell Leukemia: hereinafter referred to as ATL) is a leukemia in which T cells that are responsible for cellular immunity are invaded by a virus called HTLV-1 and become cancerous. Some of the converted ATL cells have nuclei shaped like petals and are called “flower cells”. Examples of the infection route include vertical infection via breast milk, placenta and birth canal, and horizontal infection via sexual intercourse, blood transfusion and organ transplantation. The incubation period until the onset of ATL is 40 to 60 years, and almost no onset of ATL is seen from those who have been horizontally infected after adulthood. The carrier of HTLV-1 virus is 1.2 million in Japan and 10 to 20 million in the world. In Japan, there are many carriers in the Kyushu and Okinawa regions. Onset of symptoms often results in increased white blood cell count, lymphadenopathy, enlarged liver and spleen, skin lesions such as erythema and subcutaneous mass, and digestive symptoms such as diarrhea and abdominal pain. Moreover, when the calcium level in the blood rises (hypercalcemia) due to the worsening of the disease state, it causes symptoms such as general malaise and impaired consciousness. Furthermore, it becomes easy to get an infectious disease due to a decrease in immunity, and opportunistic infectious diseases caused by fungi, protozoa, parasites and viruses are frequently combined. ATL cells are less effective for anticancer drugs and have a high recurrence rate even if remission is obtained. For this reason, since the conventional treatment method is extremely refractory, a search for an effective therapeutic agent is required.

Non-Patent Document 3 describes etoposide (VP-16), a compound belonging to lignans:

Is described. Etoposide was synthesized using podophyllotoxin isolated from a plant podophyllum podophyllum (Podophyllum peltatum) as a raw material. Etoposide has a topoisomerase inhibitory action and is used as a drug for small cell lung cancer, malignant lymphoma, cervical cancer, acute leukemia, testicular tumor, bladder cancer, chorionic disease and the like. Although etoposide is also used for the treatment of ATL, there is no report to date that compounds belonging to lignans other than etoposide have antitumor activity against ATL cells.

Patent Document 1 describes that an extract from the Asteraceae plant safflower ragwort has antitumor activity against ATL cells.

However, it has not been reported so far that a compound or extract derived from the Labiatae plant Hyptis verticillata has antitumor activity against ATL cells.

JP 2008-105960 A

An object of the present invention is to provide an antitumor agent having a very high effect, particularly an antitumor agent having a very high effect on various leukemias, lymphomas and solid cancers including refractory ATL, and a method for producing the same. And

The present inventor has found that compounds and compositions derived from the Labiatae plant Hyptis Verticillata have high antitumor activity.

The present invention includes the following inventions.

(1) General formula (I):

[Where
R ₁ is a phenyl group substituted by at least one group selected from a hydroxyl group, a methoxy group, a methylenedioxy group and a phosphate group;
R ₂ represents a hydrogen atom, a hydroxyl group, a methoxy group, an acetyl group, an anilino group, and the following:

Selected from;
R ₃ is selected from a hydrogen atom, a hydroxyl group, a methoxy group and an acetyl group;
R ₄ is selected from a hydrogen atom, a hydroxyl group, a methoxy group and an acetyl group]
(Wherein the formula (D):

Except for compounds represented by

(2) The compound according to (1) above, wherein R ₃ is selected from a hydroxyl group, a methoxy group, and an acetyl group.

(3) Formula (A):

The compound as described in said (1) or (2) shown by these.

(4) A composition derived from Hiptis verticillata containing the compound described in (3) above.

(5) The composition has formula (B):

A compound represented by:
Formula (C):

A compound represented by:
Formula (D):

And a compound represented by formula (E):

The composition as described in said (4) containing at least 1 sort (s) chosen from the compound shown by these.

(6) The composition according to (4) or (5) above, wherein the composition is an extract of Hyptis vertisirata or a processed product thereof.

(7) When the ether layer obtained by partitioning the methanol extract of Hyptis verticillata with ether and water was partitioned with 85-95% aqueous methanol and hexane, the aqueous methanol extract was subjected to silica gel column chromatography. A composition obtained by treatment with

(8) The composition according to (7) above, which is a fraction eluted with 60% ethyl acetate / hexane when ethyl acetate / hexane-ethyl acetate is used as the mobile phase of silica gel column chromatography.

(9) The composition according to (7) above, which is a fraction eluted with 40% to 50% ethyl acetate / hexane when ethyl acetate / hexane-ethyl acetate is used as the mobile phase of silica gel column chromatography.

(10) An antitumor agent comprising, as an active ingredient, the compound according to any one of (1) to (3) above or the composition according to any one of (4) to (9) above.

(11) The antitumor agent according to (10) above, which is a therapeutic agent for adult T cell leukemia.

(12) The antitumor agent according to (10) or (11) above, which is administered to a patient having drug resistance.

(13) The antitumor agent according to any one of (10) to (12), which is a pharmaceutical product.

(14) The antitumor agent according to any one of (10) to (12), which is added to food.

(15) Extracting Hyptis vertisilata and purifying the obtained extract, the formula (A):

The manufacturing method of the compound shown by these.

According to the present invention, an antitumor agent having a very high effect, particularly an antitumor agent having a very high effect on various leukemias, lymphomas and solid cancers including refractory ATL, and a method for producing the same are provided. Can do.

FIG. 1 shows a compound previously isolated from Hiptis vertissirata. FIG. 2 is a diagram showing an extraction scheme from Hiptis vertisirata. FIG. 3 is a diagram showing a purification scheme of an extract of Hiptis vertisirata and an isolation scheme of compounds (A)-(E) from the extract. FIG. 4 is a diagram showing the structure, HMBC correlation, ¹ H NMR chemical shift value, and ¹³ C NMR chemical shift value of compound (A). FIG. 5 is a diagram showing a ¹ H NMR spectrum of the compound (A). FIG. 6 is a diagram showing a ¹³ C NMR spectrum of the compound (A). FIG. 7 is a diagram showing the structure of compound (B) and ¹ H NMR chemical shift value (ppm). FIG. 8 is a diagram showing the structure of compound (C) and ¹ H NMR chemical shift value (ppm). FIG. 9 is a diagram showing the structure of compound (D) and ¹ H NMR chemical shift value (ppm). FIG. 10 is a diagram showing the structure, HMBC correlation, ¹ H NMR chemical shift value, and ¹³ C NMR chemical shift value of compound (E). FIG. 11 is a graph showing the cytotoxic activity of compound (A) against cancer cell lines.

The compounds of the present invention have the general formula (I):

[Where
R ₁ is a phenyl group substituted by at least one group selected from a hydroxyl group, a methoxy group, a methylenedioxy group and a phosphate group;
R ₂ represents a hydrogen atom, a hydroxyl group, a methoxy group, an acetyl group, an anilino group, and the following:

Selected from;
R ₃ is selected from a hydrogen atom, a hydroxyl group, a methoxy group and an acetyl group;
R ₄ is selected from a hydrogen atom, a hydroxyl group, a methoxy group and an acetyl group]
(Wherein the formula (D):

Except for compounds represented by

In the compound of formula (I) of the present invention, the absolute configuration at the 1-position may be either R-form or S-form, or a racemate. The preferred absolute configuration is the R form. Also, the compound of formula (I) differs from etoposide in that there is a double bond at the 2-3 position. Furthermore, the compound of formula (I) is different from the above etoposide in the direction of the substituent at the 5-position and the lactone ring.

R ₁ is preferably:

Selected from.

R ₂ is preferably a hydrogen atom, a hydroxyl group or a methoxy group.

R ₃ is preferably a hydrogen atom, a methoxy group or a hydroxyl group, particularly preferably a methoxy group or a hydroxyl group.

R ₄ is preferably a hydrogen atom.

The compound of the formula (I) of the present invention has an extremely effective antitumor activity, and particularly has an extremely high effect on various leukemias, lymphomas and solid cancers including refractory ATL.

The composition of the present invention contains a compound of formula (I) as an active ingredient.

The compounds of the present invention particularly preferably have the formula (A):

It has the structure shown by. The absolute configuration at the 1-position of the compound (A) may be either R-form or S-form, or may be a racemate. The absolute configuration of the preferred compound (A) is the R form. Compound (A) is different from etoposide, which has been reported to have antitumor activity against ATL cells, in that it has a double bond at positions 2-3 and is not a glycoside. Further, the compound (A) is different from the above etoposide in the 4′-position and 5-position substituents and the direction of the lactone ring.

The composition of the present invention is derived from Hyptis verticillata belonging to the genus Crabaceae. The part of Hiptis vertisirata to be used is not particularly limited, and all plants can be used, but it is preferable to use leaves and stems.

The composition of the present invention preferably contains a compound (A).

The composition of the present invention preferably contains at least one selected from compounds (B)-(E).

The composition of the present invention preferably contains at least one selected from compound (A) and compounds (B)-(E).

The composition of the present invention is preferably an extract of Hyptis verticillata (hereinafter referred to as an extract) or a processed product thereof (hereinafter referred to as a processed product). The treated product is obtained by subjecting the extract to at least one of various treatments such as separation, purification, and isolation.

As an extraction solvent, water; aliphatic hydrocarbons such as hexane; alcohols such as methanol, ethanol, propanol and butanol; esters such as acetate such as ethyl acetate; ethers such as ethyl ether and dioxane; ketones For example, acetone etc. are mentioned. When the extract is once solvent-removed and used as a dried product, any of the above-described solvents can be used alone or in combination. On the other hand, when the extract is used in a state dissolved in a solvent, it is necessary to use a solvent that does not have a harmful effect on the human body. In this case, it is preferable to use water, ethanol, or a mixture thereof. . In the extraction, Hiptis vertissirata can be used as it is, or can be crushed or pulverized after drying to enhance contact with the solvent.

The extract of the present invention can be extracted with an extraction solvent of preferably 5 to 30 L, particularly preferably 10 to 15 L, per 1 kg of Hiptis vertissirata. The extraction temperature is preferably in the range of room temperature to the boiling point under pressure, and the extraction time varies depending on the extraction temperature and the like, but is preferably 24 to 60 hours, particularly preferably 36 to 48 hours.

The extract thus obtained may be filtered with a cloth, a stainless steel filter, a filter paper, a filter sterilization filter or the like as necessary to remove insoluble matters, impurities, and the like. Moreover, you may give processes, such as a spray-dry process, a freeze-dry process, a supercritical process, to the extract after filtration.

The extract thus obtained or its treated product is subjected to various types such as silica gel column chromatography, high performance liquid chromatography (HPLC), adsorption / reverse phase partition chromatography, ion exchange chromatography, gel filtration chromatography, dialysis and the like. It is good also as a processed material which processed by the refinement | purification means and raised activity further.

Examples of the extract of the present invention include, for example, a methanol extract of Hyptis verticillata, and an ether layer obtained by partitioning it with ether and water is distributed with 85 to 95%, preferably 90% methanol aqueous solution and hexane. And an aqueous methanol solution extract.

As the treated product of the present invention, for example, elution obtained by fractionating the above extract using silica gel column chromatography (for example, using ethyl acetate / hexane-ethyl acetate, methylene chloride / methanol / water and methanol sequentially as the mobile phase). Fraction, preferably 60% ethyl acetate / hexane elution fraction.

As the treated product of the present invention, the elution fraction is further purified by, for example, HPLC (for example, Cosmosil 5C18-MS-II, φ4.6 × 250 mm, φ10 × 250 mm, for example, 51% methanol / water, 50% methanol / water as a mobile phase). Elution fractions collected using water).

As the treated product of the present invention, the 60% ethyl acetate / hexane elution fraction is further separated using, for example, HPLC (for example, Cosmosil 5C18-AR, φ10 × 250 mm, for example, using 50% methanol / water as a mobile phase). The collected elution fraction is mentioned. Compounds (A) and (C) of the present invention are obtained from these elution fractions. The compounds (B), (D), and (E) of the present invention may be prepared using, for example, HPLC (for example, Cosmosil 5C18-MS-II, φ4.6 × 250 mm, φ10 × 250 mm, eg, 30% as a mobile phase). (Acetonitrile / water, 43% methanol / water).

The compounds of the formula (I) or the compounds (B) to (E) of the present invention may be produced by synthesis. The compound of the present invention can be synthesized based on the description of the synthesis method of lignans Eur. J. Org. Chem., 2007, 3815-3828.

The composition of the present invention is preferably 0.01 to 0.02 parts by mass, particularly preferably 0.015 to 0.017 parts by mass of the compound of formula (I) of the present invention, with the total amount of the composition being 100 parts by mass. Contains.

The composition of the present invention can be used as it is as an active ingredient of the antitumor agent of the present invention. The antitumor agent of the present invention may be a pharmaceutical product or may be added to food.

In the present invention, “tumor” includes solid tumors and hematopoietic tumors. Here, the solid tumor is, for example, brain tumor, head and neck cancer, esophageal cancer, thyroid cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, stomach cancer, gallbladder / bile duct cancer, liver cancer, pancreatic cancer, colon cancer, rectal cancer, ovary Cancer, choriocarcinoma, endometrial cancer, cervical cancer, renal pelvis / ureter cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, fetal cancer, viral tumor, skin cancer, malignant melanoma, neuroblastoma Osteosarcoma, Ewing's tumor, soft tissue sarcoma, etc., preferably non-small cell lung cancer and colon cancer. On the other hand, hematopoietic tumors include, for example, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), acute promyelocytic leukemia (APL), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL). Leukemia such as hair cell leukemia (HCL) and adult T cell leukemia (ATL), Hodgkin disease, lymphoma such as non-Hodgkin lymphoma (eg B cell lymphoma, T cell lymphoma, etc.), multiple myeloma, etc. Are acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia (APL), and adult T cell leukemia (ATL), particularly preferably adult T cell leukemia (ATL).

Furthermore, the antitumor agent of the present invention has antitumor activity also against tumor cells having drug resistance. Examples of the gene involved in drug resistance include MRP-1, P-glycoprotein gene, BCRP and the like. Thus, the antitumor agent of the present invention can be expected to have a therapeutic effect on cancer patients who have acquired resistance to conventional anticancer agents.

The antitumor agent of the present invention can be formulated in combination with a known food carrier or pharmaceutical carrier. The dosage form is not particularly limited and is appropriately selected as necessary. In general, tablets, capsules, granules, fine granules, powders, solutions, syrups, suspensions, emulsions, elixirs, etc. Used as an oral agent. In addition, the antitumor agent of the present invention may be used as a parenteral agent such as an injection, infusion, suppository, inhalation, transdermal absorption agent, transmucosal absorption agent, patch, ointment and the like. In addition, the antitumor agent of the present invention can be added to foods, chewing gums, beverages and the like to make so-called foods for specific health (for example, cancer preventive foods).

The dose of the antitumor agent of the present invention varies depending on the age, weight, degree of disease, and route of administration of the patient, but for oral administration, it is usually 1 day as a dry powder of the compound of formula (I) of the present invention. The dosage is usually 1 to 3 times a day for oral administration.

Oral preparations are produced according to a conventional method using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts.

In addition to the above-mentioned excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrances, and the like can be appropriately used in this type of preparation.

Specific examples of the binder include crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carmellose sodium , Ethylcellulose, carboxymethylethylcellulose, hydroxyethylcellulose, wheat starch, rice starch, corn starch, potato starch, dextrin, pregelatinized starch, partially pregelatinized starch, hydroxypropyl starch, pullulan, polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, aminoalkyl Meta Relay copolymers RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylamino acetate, polyvinyl alcohol, gum arabic, gum arabic powder, agar, gelatin, white shellac, tragacanth, purified sucrose, macrogol.

Specific examples of the disintegrant include crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, and partially pregelatinized. Starch, hydroxypropyl starch, sodium carboxymethyl starch, tragacanth can be mentioned.

Specific examples of surfactants include soybean lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate, sorbitan monostearate Sorbitan monopalmitate, sorbitan monolaurate, polysorbate, glyceryl monostearate, sodium lauryl sulfate, lauromacrogol.

Specific examples of lubricants include wheat starch, rice starch, corn starch, stearic acid, calcium stearate, magnesium stearate, hydrous silicon dioxide, light anhydrous silicic acid, synthetic aluminum silicate, dry aluminum hydroxide gel, talc, Examples thereof include magnesium aluminate metasilicate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, sucrose fatty acid ester, waxes, hydrogenated vegetable oil, and polyethylene glycol.

Specific examples of the fluidity promoter include hydrous silicon dioxide, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.

In addition, the antitumor agent of the present invention may contain a flavoring agent and a coloring agent when administered as a solution, syrup, suspension, emulsion, or elixir.

Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to these examples.

(Example 1)
1. Extract of Hiptis vertisirata, preparation of processed product thereof and isolation of compounds (A)-(E) Antitumor activity against leukemia cell line S1T cells derived from ATL patients was used as an index. Antitumor activity was measured by the method shown in 3 below.

Hyptis Verticillata (Angiospermia, Dicotyledonous Net, Chrysanthemum, Lamiaceae, Lamiaceae, Crab Genus) was collected in Jamaica.

Hyptis verticillata methanol extract (IC ₅₀ value for S1T cells 0.1 μg / mL) was partitioned into two layers with ether and water. The ether soluble part (IC ₅₀ value 0.125 μg / mL) was further partitioned between 90% aqueous methanol solution and hexane, and the obtained 90% methanol aqueous solution soluble part (IC ₅₀ value 0.03 μg / mL) was concentrated under reduced pressure. . This extraction scheme is shown in FIG.

Silica gel column chromatography (mobile phase: 5% ethyl acetate / hexane-100% ethyl acetate (fractions 1 to 18), methylene chloride: methanol: water = 7: 3 : 0.5 (fraction 19), and fractionated using 100% methanol (fraction 20).

Fifth fraction 53.2 mg (IC ₅₀ value 3 ng / mL) eluted with 60% ethyl acetate / hexane was further HPLC (Cosmosil 5C18-AR, φ10 × 250 mm, mobile phase: 50% methanol / water, 18 fractions) ). From the obtained sixth fraction, 1.6 mg (IC ₅₀ value: 2.5 ng) of Compound (B) was further obtained using HPLC (Cosmosil 5C18-MS-II, φ4.6 × 250 mm, mobile phase: 30% acetonitrile / water). / ML) and 2.2 mg (IC ₅₀ value 22 ng / mL) of compound (C) was isolated from the 10th fraction. From the twelfth fraction, 0.5 mg (IC ₅₀ value 0.15 ng / mL) of compound (D) was measured using HPLC (Cosmosil 5C18-MS-II, φ10 × 250 mm, mobile phase: 43% methanol / water). Compound (E) 1.3 mg (IC ₅₀ value 15.0 ng / mL) was isolated. Furthermore, 1.7 mg (IC ₅₀ value: 3.0 ng / mL) of compound (A) was isolated from the 17th fraction.

Fifth fraction 66.1 mg (IC ₅₀ value 0.35 μg / mL; silica gel TLC (50% ethyl acetate / hexane) Rf value approximately 0.4) eluted with 40% -50% ethyl acetate / hexane Fractionation using HPLC (Cosmosil 5C18-AR, φ10 × 250 mm, mobile phase: 51% methanol / water, 9 fractions). A fourth fraction (IC ₅₀ value 0.25 ng / mL; retention time about 80 minutes) and a seventh fraction (IC ₅₀ value 0.25 ng / mL; retention time about 140 minutes) were obtained.

The above purification and isolation scheme is shown in FIG.

2. Structure determination Compound (A) was obtained as an amorphous and yellowish solid. From analysis by FAB mass spectrum, the molecular weight 426, and a molecular formula _C 23 _H 22 _{O 8.} Further, it was found that the analysis by ^{1 H} NMR spectrum shows a lignan skeleton. Further, by analysis including ¹³ C NMR, ¹³ C DEPT, HMBC, and HMQC spectra, compound (A) was converted to 4-methoxy-9- (3,4,5-trimethoxyphenyl) -8,9-dihydrofuro [ 3 ′, 4 ′: 6,7] naphtho [2,3-d] [1,3] dioxol-6 (5H) -one. FIG. 4 shows the structure, HMBC correlation, ¹ H NMR chemical shift value, and ¹³ C NMR chemical shift value of compound (A). Further, ¹ H NMR spectrum and ¹³ C NMR spectrum of the compound (A) are shown in FIGS. 5 and 6, respectively. Compound (A) was the first isolated compound this time, with no reports of isolation to date.

Compound (B) was obtained as an amorphous colorless solid. From analysis by FAB mass spectrum, the molecular weight 414, and a molecular formula _C 22 _H 22 _{O 8.} Further, structural analysis by ¹ H NMR, ¹³ C NMR, ¹³ C DEPT, HMBC, HMQC spectra, and B. Konuklugil, Chem. Nat. Compd., 2005, 41, 306-307 and DE Jackson and PM Dewick, Phytochemistry, 1984 , 23, 1147-1152, the compound (B) was converted to β-peltin ((5R) -5,8,8aβ, β-tetrahydro-10-hydroxy-5- (3,4, 5-trimethoxyphenyl) furo [3 ′, 4 ′: 6,7] naphtho [2,3-d]-[1,3] -dioxol-6 (5aαH) -one). FIG. 7 shows the structure of the compound (B) and ¹ H NMR chemical shift value (ppm).

Compound (C) was obtained as an amorphous and yellowish solid. From analysis by FAB mass spectrum, the molecular weight 384, and a molecular formula _C 21 _H 20 _{O 7.} Further, by structural analysis by ¹ H NMR, ¹³ C NMR, ¹³ C DEPT, HMBC, HMQC spectrum, and comparison with DE Jackson and PM Dewick, Phytochemistry, 1984, 23, 1147-1152, compound (C) 4 4'-demethyldesoxypodophyllotoxin ((5R) -5β- [3,4,5-trimethoxyphenyl] -5aα-hydroxy-5,8,8aβ, 9-tetrahydrofuro [ 3 ′, 4 ′: 6,7] naphtho [2,3-d] -1,3-dioxol-6 (5aH) -one)). The structure of compound (C) and ¹ H NMR chemical shift value (ppm) are shown in FIG.

Compound (D) was obtained as an amorphous colorless solid. From analysis by FAB mass spectrum, the molecular weight 396, and a molecular formula _C 22 _H 20 _{O 7.} Furthermore, by structural analysis by ¹ H NMR, ¹³ C NMR, ¹³ C DEPT, HMBC, HMQC spectra, and comparison with M. Kuhnt et al, Phytochemistry, 1994, 36, 485-489, compound (D) was converted to hypotinin. ) (9α- (3,4,5-trimethoxyphenyl) -5,9-dihydrofuro [3 ′, 4 ′: 6,7] naphtho [2,3-d]-[1,3] -dioxole-6 (8H) -one). FIG. 9 shows the structure of compound (D) and ¹ H NMR chemical shift value (ppm).

Compound (E) was obtained as an amorphous colorless solid. From analysis by FAB mass spectrum, the molecular weight 398, and a molecular formula _C 22 _H 22 _{O 7.} Furthermore, by ¹ H NMR, ¹³ C NMR, ¹³ C DEPT, HMBC, structural analysis by HMQC spectrum, and comparison with Y. Inamori et al. Chem. Pharm. Bull., 1985, 33, 704-709 (E) is deoxypodophyllotoxin ((5R) -5,8,8aβ, 9-tetrahydro-5β- (3,4,5-trimethoxyphenyl) furo [3 ′, 4 ′: 6, 7] naphtho [2,3-d]-[1,3] -dioxol-6 (5aαH) -one). FIG. 10 shows the structure, HMBC correlation, ¹ H NMR chemical shift value, and ¹³ C NMR chemical shift value of compound (E).

3. Measurement of antitumor activity Antitumor activity was measured by measuring cytotoxicity against S1T cells, which are ATL cell lines, by the WST method.

Using RPMI-1640 10% FCS culture medium, placed in a 96-well flat-bottom multi-plate fractionation solution S1T cells 1 × ¹⁰ 4 / 100μL / well and 10-fold dilution series (100 [mu] L / well), 72 hours 37 ° C., The culture was performed in the presence of 5% CO ₂ .

During the last 4 hours, 20 μL / well of tetrazolium salt WST-8 reagent was added, and the culture was continued. The absorbance at 450 nm was then measured. The cell viability was evaluated based on the ratio of each concentration fraction to the control with the control group as 100%. As an expression of antitumor activity was the concentration of compound causing 50% of the cytopathic effect as IC _50. IC ₅₀ was expressed in μg / mL and nM.

The results of the antitumor activity test for compound (A) are shown in Table 1 and FIG. S1T for cytotoxicity against the leukemia cell lines K3T (ATL cells), Jurkat (acute lymphocytic leukemia cells), HL60 and K562 (myeloid leukemia cells), and A549 (lung cancer cells) and SW480 (colon cancer cells). Measurement was performed in the same manner as the cells.

Furthermore, Table 2 shows the results of antitumor activity tests conducted on S1T (ATL cells) and ActT (activated T cells) for compound (A). The antitumor activity test was performed by measuring cytotoxicity by the WST method. Compound (A) was diluted from a final concentration of 10 μg / mL to a 10-fold dilution series using a 96-well multiplate, S1T cells or ActT cells 1 × 10 ⁵ / well were added thereto, and the mixture was incubated at 37 ° C., 5% CO ₂ . The cells were cultured for 72 hours under the same conditions, and colored by the WST method in the same manner as described above to measure the cytotoxic activity. The concentration causing 50% cell cytotoxicity was _{IC 50.}

Table 2 shows that compound (A) has 12 times the antitumor activity of ActT against S1T, which is an ATL cell.

From the above, it was found that the compound (A) of the present invention has very high antitumor activity against cancer cells, particularly ATL cells which are leukemia cells.

4). Measurement of antitumor activity against drug resistant cell lines Table 3 shows the results of antitumor activity tests of compound (A), etoposide and mitoxantrone on drug resistant cell lines. Drug resistant cells include epithelioid carcinoma KB3-1 and cell KB / MRP transferred with the drug resistant gene MRP-1, KB-G2 similarly transferred with the drug resistant gene P-glycoprotein gene, myeloid leukemia cell line K562 and K562 / BCRP into which the drug resistance gene BCRP was transferred were used. MRP-1, P-glycoprotein gene and BCRP are all involved in various types of anticancer drug resistance. Etoposide was used as a control agent for MRP-1 and P-glycoprotein gene transfer strains, and mitoxantrone was used as a control agent for BCRP transfer strains. The antitumor activity test was performed by measuring cytotoxicity by the WST method.

Using RPMI-1640 10% FCS culture medium, placed fraction solution of each cell 1 × ¹⁰ 4 / 100μL / well and 10-fold dilutions in 96-well flat-bottomed multi plates (100 [mu] L / well), 72 hours 37 ° C., The culture was performed in the presence of 5% CO ₂ .

After adding 20 μL / well of tetrazolium salt WST-8 reagent and culturing for 4 hours under the same conditions, absorbance at 450 nm was measured. The cell viability was evaluated based on the ratio of each concentration fraction to the control with the control group as 100%. The concentration causing 50% cell cytotoxicity was _{IC 50.}

KB / MRP cells are 28.6 times more resistant to etoposide than KB3-1, but only 1.17 times more resistant to compound (A). KB-G2 cells are 20.8 times more resistant to etoposide than KB3-1, but not to compound (A) at all. K562 / BCRP cells are about 15 times more resistant to mitoxantrone than K562, but only about 1.9 times less resistant to compound (A).

From the above, it has been clarified that the compound (A) acts on cancer cell lines beyond the drug resistance involving MRP-1, P-glycoprotein genes and BCRP, which are multidrug resistance genes.

All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.

Claims (15)

1. Formula (I):
   

   

   [Where
   R ₁ is a phenyl group substituted by at least one group selected from a hydroxyl group, a methoxy group, a methylenedioxy group and a phosphate group;
   R ₂ represents a hydrogen atom, a hydroxyl group, a methoxy group, an acetyl group, an anilino group, and the following:
   

   

   Selected from;
   R ₃ is selected from a hydrogen atom, a hydroxyl group, a methoxy group and an acetyl group;
   R ₄ is selected from a hydrogen atom, a hydroxyl group, a methoxy group and an acetyl group]
   (Wherein the formula (D):
   

   

   Except for compounds represented by
2. The compound according to claim 1, wherein R ₃ is selected from a hydroxyl group, a methoxy group and an acetyl group.
3. Formula (A):
   

   

   The compound of Claim 1 or 2 shown by these.
4. A composition derived from Hiptis verticillata containing the compound according to claim 3.
5. Said composition is represented by formula (B):
   

   

   A compound represented by:
   Formula (C):
   

   

   A compound represented by:
   Formula (D):
   

   

   And a compound represented by formula (E):
   

   

   The composition of Claim 4 containing at least 1 sort (s) chosen from the compound shown by these.
6. The composition according to claim 4 or 5, wherein the composition is an extract of Hiptis vertisirata or a processed product thereof.
7. When the ether layer obtained by partitioning the methanol extract of Hyptis verticillata with ether and water was partitioned with 85-95% aqueous methanol and hexane, the aqueous methanol extract was treated by silica gel column chromatography. Composition obtained.
8. The composition according to claim 7, which is a fraction eluted with 60% ethyl acetate / hexane when ethyl acetate / hexane-ethyl acetate is used as a mobile phase of silica gel column chromatography.
9. The composition according to claim 7, which is a fraction eluted with 40% to 50% ethyl acetate / hexane when ethyl acetate / hexane-ethyl acetate is used as a mobile phase of silica gel column chromatography.
10. An antitumor agent comprising as an active ingredient the compound according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 9.
11. The antitumor agent according to claim 10, which is a therapeutic agent for adult T cell leukemia.
12. The antitumor agent according to claim 10 or 11, for administration to a patient having drug resistance.
13. The antitumor agent according to any one of claims 10 to 12, which is a pharmaceutical product.
14. The antitumor agent according to any one of claims 10 to 12, which is added to food.
15. Extracting Hiptis vertissirata and purifying the resulting extract, formula (A):
    

    

    The manufacturing method of the compound shown by these.

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