JPH08268965A - Antitumor compound - Google Patents

Abstract

PURPOSE: To obtain an antitumor compound having a tumor cell multiplication- inhibiting activity and a P-glucoprotein function-inhibiting action and useful as an active ingredient for antitumor agents, etc., by extracting Taxus cuspidata Sieb. et. Zucc. of Japan, etc. CONSTITUTION: This compound of formula I, preferably a compound of formula II. The compound is obtained e.g. by extracting the branches, etc., of Taxus cuspidata Sieb. et. Zucc. with ethanol, adding a salt aqueous solution to the extract, extracting the aqueous solution with toluene, concentrating and drying the extracted toluene layer, dissolving the residue in hexane/acetone, and subsequently subjecting the solution to a column chromatographic separation treatment, etc. When used as a medicine, the compound is preferably orally administered at a daily dose of approximately 1-100mg.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel compound derived from a natural product, more specifically, a tumor cell growth inhibitory action,
Also, the present invention relates to a novel compound having a function-inhibiting effect of a P-glycoprotein involved in resistance to tumor cells. The present invention also relates to an antitumor agent containing the above compound as an active ingredient and a detolerizer for resistant tumor cells.

[0002]

2. Description of the Related Art Various compounds have been hitherto developed and studied as pharmaceuticals for cancer chemotherapy. Many of these compounds include natural products, and among them, taxol extracted from the Taxus plant is effective against melanoma and ovarian cancer, and clinically cross-linked with other antitumor agents. Since it is not resistant, it has been attracting attention in recent years (Latest Medicine, 46, 6, pp.1240-1244, 1991).

Although there are many antitumor agents that have been put to practical use so far, the resistance of cancer cells and tumor cells to these antitumor agents has become a major problem. The factor of this resistance acquisition is
It has been clarified that the P-glycoprotein overexpressed in cancer cells / tumor cells causes the antitumor agent to be excreted extracellularly. Therefore, a drug that inhibits the drug efflux function of P-glycoprotein may suppress the multidrug resistance of cancer cells and tumor cells. Such drugs include verapamil, cyclosporine A
(Cyclosporin A), FK506 and its derivatives have been studied, but all of these compounds have strong toxicity and side effects, and the emergence of new drugs has been desired.

[0004]

[Problems to be Solved by the Invention and Means for Solving the Problems] The present inventors have advanced the search for physiologically active substances derived from natural products, and as a result, Japanese yew (Taxus cuspidata)
From the branch extract of P. esculentum, and its inhibitory activity against tumor cells, and P-
The inventors have found a novel compound having a glycoprotein function-inhibiting action, and completed the present invention.

That is, the present invention has the following formula:

Embedded image The present invention provides a novel compound represented by

The compound of the present invention was prepared according to the method described in the examples of the present specification by using the Japanese yew (Taxus cuspidata,
Sieb. Et Zucc.) And was first isolated and purified by the present inventor. The compounds of the present invention are, for example, Taxusaceae (Taxacea).
e) plants belonging to the genus Taxus (Taxus), for example, Japanese yew (Taxus cuspidata, Sieb. et Zucc.), Taihei yew (Taxus brevifolia Nutt.), etc., preferably Japanese yew stems, leaves, etc. It can be extracted and purified from roots, flowers, fruits, seeds, etc., preferably branches etc., but the method for producing the compound of the present invention is not limited to extraction / purification from natural products, and can be carried out by those skilled in the art. It may be produced from a readily available production raw material by a chemical synthesis method.

The extraction operation from the natural product is not particularly limited,
It may be performed according to a method known to those skilled in the art. For example, ether, ethyl acetate, acetonitrile,
Acetone, methanol, ethanol, dichloromethane, using an organic solvent such as chloroform, extraction from the above raw material at any temperature from room temperature to the reflux temperature of the solvent,
About 1 M saline solution is added to the obtained extract, and extraction is further performed using an organic solvent such as toluene. The organic solvent layer is concentrated to dryness under reduced pressure, the residue is dissolved in an organic solvent such as pentane, hexane, heptane, benzene, and acetone,
Separation is performed by column chromatography packed with silica gel, alumina, chemically modified silica gel or the like. The compound of the present invention can be obtained by repeating the above-mentioned separation / purification steps as necessary.

The compound of the present invention can also be produced by extracting it from a culture obtained by culturing callus according to a conventional method. Such manufacturing methods include, for example, JP-A Nos. 5-244971, 5-336985, and Japanese Patent Publication No. 5-5076.
As described in No. 29, etc., callus obtained by culturing cells derived from tissues such as stems, leaves, branches, roots, flowers, fruits and seeds of plants belonging to the Taxus genus Taxus. Can be used.

The compound of the above formula has a plurality of asymmetric carbon atoms and a plurality of optical isomers based on these asymmetric carbon atoms. Optically pure optical isomers, any mixture of optical isomers, racemates, diastereomers based on two or more asymmetric carbons, any mixture of diastereomers, etc. are all within the scope of the present invention. Included in. Further, any hydrate is also included in the scope of the present invention.

Of these isomers, compounds having the following structural formulas are preferred as optical isomers:

[Chemical 4] (In the present specification, the optical isomer may be referred to as “taxapine D”).

The compound of the present invention has an action of suppressing the growth of tumor cells and an action of inhibiting the P-glycoprotein involved in the resistance of tumor cells. Without being bound to a particular theory, P-glycoprotein has an action of excreting an antitumor agent from the inside of tumor cells, and activation of P-glycoprotein causes tumor cells to respond to the antitumor agent. It is considered to be one of the causes of resistance. The compound of the present invention inhibits resistant tumor cells by inhibiting the anti-tumor agent efflux function of P-glycoprotein present in resistant tumor cells that have been made resistant to one or more antitumor agents. It has an action of detolerizing a tumor drug. Therefore, the medicament containing the compound of the present invention as an active ingredient is useful as a detolerizing agent for resistant tumor cells or an agent for enhancing antitumor activity of various antitumor agents. The above-mentioned taxa spine D is a preferable compound as an active ingredient of these medicines.

The dosage form of the medicament of the present invention is not particularly limited, and it can be administered orally or parenterally. As the drug of the present invention, the compound of the above formula which is the active ingredient may be used as it is, but a pharmacologically or pharmaceutically acceptable formulation additive is added to the compound of the above formula which is the active ingredient. It is preferred that the pharmaceutical composition be prepared by administration and administered to a patient.

The pharmacologically and pharmaceutically acceptable additives include, for example, excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases,
A solubilizer or solubilizer, an isotonicity agent, a pH adjuster, a stabilizer, a propellant, and an adhesive can be used. Examples of the formulation suitable for oral administration include tablets, capsules, powders, fine granules, granules, liquids, syrups and the like. Formulations suitable for parenteral administration include, for example, injections, drops, suppositories, inhalants, patches and the like.

The preparations suitable for oral administration, transdermal or transmucosal administration include pharmacologically and pharmaceutically acceptable additives such as glucose, lactose, D-mannitol,
Excipients such as starch or crystalline cellulose; disintegrants or disintegration aids such as carboxymethylcellulose, starch or carboxymethylcellulose calcium; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone or gelatin; magnesium stearate Or a lubricant such as talc; a coating agent such as hydroxypropylmethyl cellulose, sucrose, polyethylene glycol or titanium oxide; a base such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat be able to.

Further, propellants such as CFCs, diethyl ether, or compressed gas; adhesives such as sodium polyacrylate, polyvinyl alcohol, methyl cellulose, polyisobutylene, polybutene; and preparations such as cotton cloth or base cloth such as plastic sheet. The formulation may be manufactured using additives. Formulations suitable for injection or infusion include, for example, distilled water for injection, physiological saline, and solubilizers or solubilizers that can compose aqueous or injection-soluble injections such as propylene glycol; glucose, sodium chloride, D-mannitol. A tonicity agent such as glycerin; a pharmaceutical additive such as a pH adjusting agent such as an inorganic acid, an organic acid, an inorganic base or an organic base may be added.

The drug of the present invention can be used in combination with various antitumor agents, and may be used together with various drugs other than antitumor agents. Such agents are not particularly limited, but examples thereof include antibiotics, antibacterial agents, anti-inflammatory agents, hormone agents, and the like. Further, the dose of the drug of the present invention is not particularly limited and may be appropriately selected depending on the administration form, age, body weight, symptoms and the like. For example, about 0.1 to 500 mg, preferably about 1 to 100 mg, of the compound of the present invention can be orally administered to an adult per day. The above daily dose may be administered once a day, or may be administered twice or three times a day at appropriate intervals, or may be administered intermittently at a rate of several days to several weeks. When used as an injection, the compound of the present invention is administered to an adult in an amount of 0.01 to 100 mg, preferably 0.1 to 50 mg per day.
About mg can be administered.

[0017]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples. Production example Japanese yew (Taxus cuspidata Sieb. Et Zucc.) Branches (0.5 kg dry weight) were extracted with methanol three times (2 liters each), and 1 M saline (750 ml) was added to the extract. In addition, it was extracted three times with toluene (750 ml). The toluene layer was concentrated to dryness under reduced pressure. The residue was dissolved in a small amount of hexane / acetone mixed solvent and the silica gel (Wako Pure Chemical Industries, C-
Adsorbed on a column (4.0 × 37 cm) packed with 300), and hexane / acetone [7: 1 (800 ml) → 3: 1 (800 ml) → 1: 3 (800 m
l)]. 1650-2250 ml from the first fraction
The elution fraction of was concentrated to dryness under reduced pressure.

This fraction was dissolved in a small amount of acetonitrile and packed in a column packed with ODS (Develosil LOP ODS 24S,
It was adsorbed on Nomura Chemical Co., Ltd., 2.5 × 50 cm) and eluted with acetonitrile / water (80:20). The elution fraction with a retention time of 58 minutes was concentrated to dryness under reduced pressure, and the obtained residue was adsorbed on a column (0.6 × 15 cm) packed with silica gel and purified with chloroform / ethyl acetate (95: 5). The compound of the present invention (Taxpaine D) was obtained as a colorless amorphous substance (yield 0.0059).
%).

Properties: colorless amorphous substance; [α] ²¹ _D −32.2 ° (c 0.87, MeOH); UV (MeOH) λ _max 218 (ε 11600), 223 (sh), 277 nm (149
00) ； IR (film) ν _max 3540, 1750, 1710, 1640, 1580, 1250
cm ⁻¹ ; ¹ H NMR (CDCl ₃ δ ppm) 1.17 (3H, s), 1.49 (3H, s), 1.
53 (3H, s), 1.57 (3H, s), 1.89 (1H, m), 1.96 (3H, s),
2.00 (3H, s), 2.02 (3H, s), 2.06 (2H, m), 2.19 (3H,
s), 2.20 (3H, s), 2.36 (1H, d, J = 17.8 Hz), 2.57 (1
H, dd, J = 17.8, 6.8Hz), 3.69 (1H, d, J = 6.8Hz), 4.84
(2H, m), 5.08 (1H, s), 5.20 (1H, s), 5.34 (1H, dd, J
= 9.4, 6.2 Hz), 5.41 (1H, m), 5.60 (1H, d, J = 4.8 Hz),
5.80 (1H, d, J = 6.8 Hz), 6.46 (1H, d, J = 16.0 Hz), 7.
40 (3H, m), 7.54 (1H, m), 7.70 (1H, d, J = 16.0 Hz);

¹³ C NMR (CDCl ₃ , δ ppm) 11.6 (q), 14.8
(q), 20.7 (q), 21.2 (q), 21.4 (q), 24.1 (q), 25.8 (t),
31.3 (q), 32.7 (t), 40.8 (d), 41.3 (s), 43.1 (s), 50.4
(d), 67.4 (d), 68.3 (d), 70.4 (d), 74.6 (d), 77.3 (d),
78.7 (d), 110.9 (t), 117.8 (d), 124.3 (s), 128.1 (d), 1
28.9 (d), 130.4 (d), 134.2 (s), 142.6 (s), 144.2 (s), 1
45.1 (d), 165.2 (s), 168.5 (s), 169.3 (s), 170.2 (s), 1
70.4 (s), 170.6 (s); EIMS m / z (%) 707 (M ⁺ -OH, 0.3), 664 (2), 622 (2), 605
(4), 562 (2), 544 (2), 503 (2), 491 (2), 474 (3), 456
(2), 432 (2), 414 (5), 372 (7), 354 (13), 312 (15), 294
(10), 131 (100); HREIMS m / z 707, 3073 (M ⁺ -OH, calcd for C ₃₉ H
₄₇ O ₁₂ , 707, 3067)

Test Example 1: In Vitro Cytotoxicity Test for Mouse Leukemia Cultured Cells Rozewell Park Memorial Institute Medium 1610
In addition, a culture solution containing heat-inactivated fetal bovine serum at a concentration of 10% and a culture solution containing kanamycin at a concentration of 50 μg / ml were used. 0.6 with Taxaxpine D in various concentrations
% DMSO (dimethyl sulfoxide) solution in 1 ml of mouse leukemia cultured cells L 1210 (5 × 10 ⁴ ce
ll / ml) and added in a carbon dioxide incubator at 37 ℃ for 48 hours.
Cultured for hours.

The antitumor activity was determined by the value of IC ₅₀ [expressed by the concentration of the sample required to inhibit 50% of cell proliferation (μg / ml)]. IC ₅₀ values were obtained by plotting the logarithm of the concentration of compound against the growth rate of cells (percent of control). As a result, taxa spine D against the mouse leukemia cultured cell L 1210
The IC ₅₀ value was 3.0 μg / ml. Similarly, the IC ₅₀ value for human epithelial cancer cells KB was determined to be 1.8 μg /
ml.

Test Example 2: Tubulin Depolymerization Inhibitory Action The microtubule protein was analyzed by the method of Takahashi et al. (J. Antibiot., 40, p
p.66-72, 1987). Protein concentration uses bovine serum albumin as a standard,
The method of Raleigh et al. (J. Biol. Chem., 193, pp.265-275,
1951). Microtubule assembly assay is 100 mM 2-N-morpholinoethanesulfonic acid, 1 mM
Ethylene bis (oxyethylene nitrilo) -4 acetic acid, 0.5
It was carried out in a MES buffer (pH 6.5) containing mM MgCl ₂ , 1 mM mercaptoethanol and 1 mM guanosine-5′-3 phosphate-3-sodium salt.

The microtubule assembly was monitored on a temperature controlled spectrophotometer with a liquid circulator. Temperature is 37
The temperature was kept at ℃ and the turbidity at 400 nm was measured. Taxapine D 10 μM was dissolved in DMSO and added to 1 ml of buffer containing 2 mg of microtubule protein. The final concentration of DMSO was 1% or less. After incubating the mixture for 30 minutes, 4 mM CaCl ₂
Was added and incubated for another 30 minutes, during which the change in turbidity was measured. As a result, taxa spine D was found to have an inhibitory effect on tubulin depolymerization, and its strength was about half that of taxol.

Test Example 3: Inhibitory action of P-glycoprotein function Multidrug-resistant human uterine cancer-derived cell line 2780AD (Science, 22)
4, pp.994-996, 1984) with 5% fetal bovine serum and 100 μg /
The cells were cultured in RPMI-1640 medium containing ml kanamycin. 6-
1 million cells were plated on a well tissue culture plate (manufactured by Corning) and incubated at 37 ° C for 24 hours. Culture medium in each well with 10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid and [ ³ H] vincristine (7.5
pmole, was replaced with fresh medium 0.5 ml of which comprises a specific activity 259 GB _q / mmol), was added Takisasupain D dissolved or suspended in PBS. After incubating at 37 ° C for 2 hours, accumulation of vincristine in cells was confirmed by the method of Tsuruo et al. (Cancer Res., 41, pp. 1967-1972, 1981; Cancer Re
s., 43, pp.2905-2910, 1983; Cancer Res., 49, pp.14.
52-1455, 1989; and Cancer Res., 49, pp.5002-500.
6, 1989). As a result, taxaxpine D showed about half the accumulation of vincristine of verapamil.

[0026]

INDUSTRIAL APPLICABILITY The compound of the present invention has an activity of suppressing the growth of tumor cells (cell-killing activity) and is therefore useful as an active ingredient of an antitumor agent. Further, since it acts on the P-glycoprotein of multidrug-resistant tumor cells to promote the accumulation of antitumor agents, it is useful as an active ingredient of a tumor cell detolerizing agent.

 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Kuniko Koiso 374 Matsudo Nitta, Matsudo City, Chiba Prefecture (72) Inventor Naruo Iwasaki 2-31-12-402 Nishiikebukuro, Toshima-ku, Tokyo

Claims (5)

[Claims] 1. The following formula: A compound represented by the formula: 2. The following formula: The compound according to claim 1, which is represented by 3. An antitumor agent comprising the compound according to claim 1 or 2 as an active ingredient. 4. A detolerizing agent for resistant tumor cells, which comprises the compound according to claim 1 or 2 as an active ingredient. 5. An antitumor effect enhancer for an antitumor agent, which comprises the compound according to claim 1 or 2 as an active ingredient.