JPH0657674B2 - Antitumor substance Akimyl acids - Google Patents

Description

Detailed Description of the Invention a. OBJECT OF THE INVENTION The present invention relates to an antitumor agent of the formula (I), akimyl acid (A).
chimilic acid).

[Wherein R and R ¹ are different from each other and are methyl or hydroxy, R ² is hydrogen or lower alkyl, and the wavy line is Z or E form. ] Conventional Technology As an antitumor higher plant component, trypdiolide (tri
pdiolide) [J.Amer.Chem.Soc., 94 , 7194, (1972)] and bruceantin [J.Org.Chem., 38 , 178, (1973)] Vinblastine of vinca alcaride
[The Merck Index, 10th Edition 9784], vincristine [[Production-Active Natural Substances] 431 (1
978)] and the like. As a structurally similar substance,
Secotanapartholide A and B (Phytoc) whose ester moiety is lactonized
hemistry 21 , 2543 (1982)], but no antitumor effect has been reported.

Problems to be Solved by the Invention Conventionally, various natural products and compounds derived from natural products have been tested for antitumor effects, and among alkaloids and protein-bound polysaccharides, they have been put to practical use as antitumor agents. Some of them are natural products, but none of them are in practical use because they have many problems in terms of efficacy and toxicity.

B. Structure of the Invention Means for Solving the Problems The inventors of the present invention have been researching natural product polyoxides having an antitumor activity. As a result, Achillea millefolium L, Composita
An active substance having an antitumor effect on the polyoxides contained in the plant of e) was found and named as akimyl acid. formula
The akimyl acids represented by (I) (R ₂ = H) can be separated from Achillea millefolium by the following method, for example. That is, the flower part of the plant is extracted with a polar organic solvent at room temperature for several days, and then the extract is distributed with water using a lower alkyl alcohol immiscible with water, for example, n-butanol or pentanol, and further, In order to obtain an acidic substance, it is first extracted with a basic solution, and then the aqueous layer is acidified and extracted with a suitable organic solvent immiscible with water, for example, ethyl acetate, dichloromethane, n-butanol and the like. Furthermore, by separating the obtained acidic substance by silica gel column chromatography, P3
The active substance for 88 can be obtained. Examples of the polar organic solvent used for extraction include lower alcohols such as methanol, ethanol, isopropanol, n-butanol, sec-butanol or tret-butanol,
Acetone, acetonitrile, etc. are mentioned. As the basic solution, a medium base such as a saturated aqueous solution of sodium hydrogen carbonate, sodium carbonate or sodium acetate is preferable.

In the present invention, (I) (R
² = Alkyl) can be further separated into crude products for each isomer by subjecting it to silica gel column chromatography. R ³ represents linear or branched C ₁ -C ₅ alkyl, specifically methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tret-
Such as butyl, pentyl, isopentyl or neopentyl. This esterification can be accomplished by a method of reacting an alkyl halide with a salt of a carboxylic acid, a method of using a diazoalkane, a method of refluxing an organic acid and an alcohol in the presence of a catalyst, and the like. As the carboxylic acid salt, sodium, potassium or silver salt may be used. As the halogen moiety of the alkyl halide, chlorine, bromine or iodine may be used, and as the alkyl moiety, an alkyl corresponding to the alkyl moiety of the ester may be used.

A typical combination is, for example, methyl iodide,
Examples include ethyl bromide and n-propyl iodide. As the diazoalkane, for example, one having a linear or branched lower alkane such as diazomethane, diazoethane, diazoisopropane may be used. Alcohols having a linear or branched lower alkyl, for example, methanol, ethanol, isopropanol, n
-Butanol or the like may be used. An acid such as concentrated sulfuric acid, hydrochloric acid or benzenesulfonic acid may be used as the catalyst. Alternatively, the ester can be obtained by using the Schotten-Baumann method in which the carboxylic acid moiety is used as an acid chloride and reacted with an alcohol. As the alcohol used in this reaction, any alkyl alcohol corresponding to the alkyl portion of the ester may be used. Further, as a deoxidizing agent for removing the hydrochloric acid generated in the reaction, it is preferable to use a base such as magnesium metal, dimethylaniline, pyridine or sodium hydroxide. The reaction solvent used in the esterification reaction may be the alcohol itself involved in the esterification, or may be any solvent that does not react with the raw material, for example, ether, dichloromethane or acetone is used. .

The crude product can be further purified by recrystallization or column chromatography. As the recrystallization solvent, pentane, n-hexane, heptane, benzene, ether, tetrahydrofuran or the like may be used alone or in combination. Chromatographic elution solvents include lower alkyl alcohols such as methanol, ethanol, propanol, isopropanol or butanol, lower alkyl esters such as ethyl acetate or butyl acetate, alkyl ketones such as acetone, methyl ethyl ketone or methyl isobutyl ketone, chloroform or dichloromethane. Chlorinated hydrocarbons such as, aromatic hydrocarbons such as benzene or xylene, and water may be used alone or in combination depending on the polarity of the substance.

In the general formula of the present invention, the Z-form or E-form is determined by the coordination of hydrogen of the exo double bond of the five-membered ring, and there are two kinds of isomers depending on the coordination of hydroxy. I have a body.

Examples and physical constants will be shown below to clarify the embodiments of the present invention, but the scope of the present invention is not limited to these.

Examples Example 1. Extraction of active substances from the flower part of Yarrow Achillea milleum (Scientific name: Achillea millefo
44 g of lium L.) in 3 parts of methanol (500 ml)
Soak and extract at room temperature for a day. This operation was repeated 3 times, and methanol was distilled off under reduced pressure to obtain 9.6 g of a methanol extract extract as a brown oily substance. This methanol extract was added with n-butanol (100 ml) and water (5
(0 ml) and the n-butanol layer is extracted with 5% sodium hydrogen carbonate solution (20 ml × 6). The solution was adjusted to pH 3 by adding dilute hydrochloric acid to the alkaline solution layer, and extracted with ethyl acetate (20 ml × 5). The solvent layer is washed with water and dried, and then the solvent is distilled off under reduced pressure to obtain 324 mg of an acidic substance. The acidic substance is subjected to adsorption chromatography using 20 g of Merck silica gel (0.063-0.200 mm). When developed and eluted with 80 ml of chloroform-methanol-water (85: 15: 1.5) and then with 30 ml of a similar solvent (80: 20: 2), the fraction eluted in the latter half of the fraction gave a slightly yellow oil, P388.
70 mg of active substance are obtained.

Example 2. Synthesis of methyl ester of akimyl acid A, B and C 200 mg of active substance is dissolved in 10 ml of acetone, 160 mg of potassium carbonate and 1 ml of iodomethyl are added and refluxed. After adding 1 ml of iodomethyl every 1 hour and reacting for a total of 3 hours, 50 ml of water was added to the reaction solution and the mixture was extracted with ethyl acetate (20 ml × 4). The solvent layer is washed with water, dried and the solvent is distilled off under reduced pressure to obtain crude akimyl acids A, B,
131 mg of a mixture of methyl esters of C are obtained. The mixture is subjected to adsorption chromatography on 30 g of silica gel (same as above). Chloroform-acetone (4: 1) (60 ml) was added to the eluate (35 ml) to obtain crude akimyl acid C methyl ester (30 ml).
get mg. Then, 28 mg of crude achymyl acid B methyl ester is obtained from 25 ml of the same solvent which is subsequently eluted. Then chloroform-
54 mg of crude achymyl acid A methyl ester are obtained from the fraction, which is eluted with 40 ml of acetone (7: 3).

(1) Akimyl acid A methyl ester 54 mg of a crude product was recrystallized from ether-n-pentane and then benzene to obtain 17 mg of colorless crystals. mp.7
6-77 °, [α] _D -23.6 ° (CHCl ₃ ), IR: νmax (CCl ₄ ) 3450,171
8,1663,1627,1370,1265, 1220,1140,1050,950cm ^-1 , MS: 292 [M ⁺ ], ¹³ C NMR: δppm (CDC
l ₃ ) 209.7,195.3,166.7,164.2,141.5, 141.3,136.0,132.9,126.2,76.1,52.1,41.1,38.3,29.7,2
9.0, 26.0. Elemental analysis: C ₁₆ H ₂₀ O ₅ Actual value: C, 65.71; H, 6.71% Calculated value: C, 65.74; H, 6.90% (2) Akimyl acid B methyl ester 28 mg of crude product was added to 10 g of silica gel (same as above). Subject to adsorption chromatography, eluting with benzene-ethyl acetate (1: 1). 12 mg are obtained as a colorless oil. [α] _D -78.2 °
(CHCl ₃ ), IR: νmax (CCl ₄ ) 3440, 1725, 1707, 1655, 1628, 1370, 1265, 1200, 1142, 1052, 950 cm ^-1 , MS: 292 [M ⁺ ], ¹³ C NMR: δ ppm (CDCl ₃ ) 20
8.3,195.7,166.9,161.5,141.7,141.5,139.6,134.8,126.
6,76.2,51.9,41.3,38.1,30.0,29.0,26.6. (3) Achimic acid C methyl ester 30 mg of the crude product was subjected to adsorption chromatography using 10 g of silica gel (same as above) to obtain benzene-ethyl acetate (1: 1).
Elute with. 19 mg are obtained as a colorless oil.
[α] _D -87.3 ° (CHCl ₃ ), IR: νmax (CCl ₄ ) 3480,1717,1662,1
626,1663,1255,1220,1145,1050,955cm ^-1 ,, MS: 292 [M ⁺ ], ¹³
CNMR: δppm (CDCl ₃ ) 207.3,195.2,167.9,164.6,141.2,14
0.4,135.9,132.3,126.8,75.7,52.6,40.7,38.2,29.9,27.
0,26.6. C. EFFECTS OF THE INVENTION The compound of the present invention was tested for its activity against P388 leukemia mice. 5 or 6 weeks old BDF1 as test animal
Six or seven mice (male) were used for each administration. P388 leukemia was used as the test cancer type, and 10 ⁶ cancer cells were intraperitoneally administered to each test animal. As a suspension, one day after the cancer transplantation, each sample was administered once, or in the case of continuous administration, a predetermined dose was administered for 5 days. Only the solvent of the suspension was intraperitoneally administered to the control group. After the test, ILS% (Increased life spa) at each dose
n%, life extension rate) was calculated. ILS of 30% or more was judged to be effective.

S _T : Number of days of survival of experimental animals S _C : Number of days of survival of control animals When 2 mg / kg and 5 mg / kg of akimyl acid A methyl ester were administered once, ILS was 30% and 34%, respectively.
%, The ILS was 44 when continuously administered at 4 mg / kg.
It was in%. 1 mg of akimyl acid C methyl ester 5 mg / kg
The ILS after repeated administration was 39%. Also, 50 mg of a mixture of about 3: 1 of akimyl acid A and B of akimyl acid.
In each case, the ILS was 33% and 39%, respectively. From the above results, it is proved that the akimyl acids of the present invention have a remarkable antitumor effect.

Claims (1)

[Claims] 1. A compound represented by the following general formula. [Wherein R and R ¹ are different from each other and are methyl or hydroxy, R ² is hydrogen or lower alkyl, and the wavy line is Z or E form. ]