JPH0940567A - Production of taxol and taxane terpenoid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a compound useful as an antitumor agent, producible from a natural product such as Japanese Taxus cuspidata and also capable of being produced by tissue culture, which uses an easily induced callus. SOLUTION: Japanese Taxus cuspidata (e.g. grown in Miyagi prefecture or in Toyama prefecture) is used as a raw material. For instance, its leaves, core portions and barks are directly extracted with an alcohol such as methanol, several times, and the extract is condensed by an active carbon-celite treatment, etc. The condensate is further extracted with ethyl acetate. The ethyl acetate layer is extracted with a sodium bicarbonate water to obtain a carboxylic acid fraction, extracted with a dilute alkali to obtain a phenol fraction and extracted with a dilute hydrochloric acid to obtain an alkaloid fraction and a neutral fraction. The neutral fraction is charged on a silica gel chromatography column having an activated carbon layer on its top and eluted with an organic solvent to obtain taxol.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing taxol. More specifically, it relates to a method for producing taxol, which is characterized by using Japanese yew as a raw material.

[0002]

BACKGROUND OF THE INVENTION Taxus L. is one of the northern hemispheres.
0 species are distributed, and yew (onco) Taxus cusp in Japan
idata Sieb et Zucc. and the shrub caraboku (T.cuspidata
var.nana Hort. ex Rehder) and a yellow fruit-bearing chyme onion (T.cuspidata f.luteobaccata) are growing. Until now, research on Japanese yew components has been centered around Takagi's Onko. Ueda's report was first published in 1913, and then Kondo and Amano extracted alkaloids from Hida yew leaves as a raw material, and presumed that this was the same substance as European alkaloids, and called it "taxin". I named it. Then, around 1960, research was conducted by the Kyoto University group and Tohoku University Nakanishi group, and several diterpenes having a “taxane” skeleton were obtained.

On the other hand, taxol, whose structure was determined in 1971, is a yew from North America, the Pacific yew (Taxus brevifoli).
It has been attracting attention as a new type of anticancer drug because it is obtained from the bark of (a), and in addition to its specific chemical structure, it stabilizes microtubules during mitosis and suppresses their depolymerization. In particular, taxol is effective not only for leukemia but also for refractory ovarian cancer, and is characterized by having a wide therapeutic spectrum.

[0004]

As described above, taxol has a low content in plants and can be obtained only in a yield of 0.01% from bark. Therefore, approaches to synthesis are popular.

At present, two groups have been totally synthesized in the world (KCNicolaou et.al., Nature, Vol. 367,
630, 1994, RA Holton et.al., J. Am. Che
m.Soc., 116, 1599, 1994),
It is not always a practical method.

In addition, taxol is semi-synthesized from a precursor such as 10-deacetylbaccatin III (The Journal of Organic Chemistry, Vol. 56, page 6939, 1991) and a method for producing taxol by tissue culture. As JP-A-6-29
No. 2588 and JP-A-6-296493.
However, the present has been reached without an effective supply method to the market.

[0007]

[Means for Solving the Problems] In the process of investigating the composition of Japanese yew tree of the genus Taxus, the present inventors found that taxol can be obtained from yew bark in Sapporo city and based on the findings. The present invention has been completed.

That is, the present invention is a method for producing taxol, which is characterized by using Japanese yew as a raw material, and Japanese yew leaves, heartwood or skin are directly extracted with alcohol, and then a concentrated solution of the alcohol extract is obtained. Extract with ethyl acetate and wash with dilute aqueous alkaline solution,
A method for producing taxol and taxane terpenoids, characterized in that a crude extract is obtained.

The manufacturing method of the present invention will be described below.

Taxanes and terpenoids such as taxol contained in yew trees have alkali-labile ester bonds and are hydrolyzed by alkali. However, when the yew tree extract is dissolved in a lower fatty acid ester solvent such as ethyl acetate and washed with an alkali (NaOH or KOH aqueous solution), the taxanes / terpenoids are not decomposed and the phenols are contained in a high content that makes the separation operation difficult. Can be removed as an alkali salt.

When the concentration of the alkaline aqueous solution used for removing the phenols is higher than 1%, the extraction operation becomes difficult due to emulsification and precipitation of the phenol salt. In the operation using a salt-saturated to half-saturated aqueous solution, it is possible to easily obtain the taxane terpenoids in a neutral and basic mixture, which can prevent the emulsification and the precipitation of Na salt (prolonging the standing time causes the precipitation of salt). it can.

Conventionally, taxanes and alkaloids are strongly fat-soluble and it has been difficult to extract them with an aqueous mineral acid solution [J. Nat. Products, 56 (4) 5
14-520 (1993)], but the lower alcohols such as methanol were added to the extract excluding phenols in an amount of 5-10.
The taxanes and alkaloids are obtained by extraction (3 times) with a mineral acid (5-10% hydrochloric acid or sulfuric acid) aqueous solution containing 3% concentration, separating the aqueous layers and making the mixture alkaline and extracting with ethyl acetate.

Thus, the ethyl acetate layer from which the alkaloids have been removed is dried and concentrated, whereby neutral taxane-terpenoids containing taxol are separated. By subjecting the latter to column chromatography using silica gel, thin layer chromatography (TLC), and high performance liquid chromatography (HPLC), taxol and taxane terpenoids can be easily separated.

The manufacturing method of the present invention will be described in detail below.

Japanese yew bark is collected and extracted several times with alcohol. Alcohols include methanol, ethanol, isopropanol and the like, with methanol being preferred.

Then, the extract is treated with activated carbon-celite and concentrated. Then, the concentrate was extracted with ethyl acetate, the ethyl acetate layer was extracted with aqueous sodium hydrogen carbonate, extracted with carboxylic acid fraction, diluted alkali (half-saturated salt solution of KOH or NaOH of 1% or less), phenol fraction, extracted with diluted hydrochloric acid. Separate the alkaloid and neutral compartments. Taxol can be obtained by subjecting the neutral section to silica gel column chromatography with activated carbon laid on the upper layer and eluting and separating with an organic solvent such as benzene, toluene, hexane, and ethyl acetate. If necessary, preparative thin layer chromatography (PTLC) can also be used.

[0017]

INDUSTRIAL APPLICABILITY In the method of the present invention, a raw material of a yew plant can be used.

Further, since it can be easily induced into callus, taxol can be produced by tissue culture. Hereinafter, the effect of the manufacturing method of the present invention will be described in comparison with the conventional manufacturing method.

1. Differences between production areas Yew grows naturally in the subalpine zone in Honshu, but in Hokkaido, natural forest zones can be seen even in the lowlands. The common yew from Miyagi prefecture contained 0.012% of taxol. On the other hand, taxi was not contained in the yew bark from Toyama prefecture, but it was 0.0014% and 0.004% in the yew bark of Sapporo city of Hokkaido and Takayama of Gifu prefecture, respectively.
Included taxol.

[0020]

[Table 1]

(HPLC analysis) Column: Finepak SIL C18-5 (4.6 mm × 250 mm) Solvent: MeOH: H ₂ O: CH ₃ CN (40:82:78), flow rate 1.0 ml / min Taxol retention time t _R : 18min, Analyzer JASCO Tri
roter 2. Difference due to extraction method W. MIller et al. (J. Org. Chem., Vol. 46, p. 1469-p. 1474, 1981) dilute the yew dry powder ethanol extract with water, wash with petroleum ether to remove fats and oils, and add chloroform. Extracted. The concentrate of the extract was subjected to silica gel column chromatography to separate chloroform / methanol (95: 5) from the eluate by a drop counter current (DCC) to separate taxol.

In the method of the present invention, the raw material of the yew plant is directly extracted with methanol (three times), and the concentrated solution is extracted with ethyl acetate. When the ethyl acetate layer is washed several times (about 3 times) with a dilute alkaline aqueous solution (1% or less of KOH or NaOH half-saturated salt solution), the phenol fraction which hinders the separation operation can be almost completely removed. The organic solvent was distilled off, and the residue was chromatographed on silica gel with benzene (toluene, hexane): ethyl acetate (3: 1 to 1: 1).
Elution with 1: 1) gives the taxol compartment after the less polar taxane terpenoids.

As described above, the taxol and taxane terpenoids can be easily separated from the yew extract by the method of the inventor by removing the phenol segment which hinders the separation operation.

[0024]

EXAMPLES The present invention will be specifically described below with reference to examples and comparative examples.

Example 1 Yew (Takagi) bark (1.74 kg) from Hida Takayama was extracted with methanol.

The methanol extract was concentrated, dissolved in ethyl acetate, and extracted three times with 0.5% aqueous solution of half-saturated sodium chloride to remove the phenol fraction. The ethyl acetate layer is further 5%
It was extracted 3 times with 5% aqueous hydrochloric acid containing methanol. The diluted hydrochloric acid layers were combined and made alkaline to extract with ethyl acetate to obtain taxane alkaloids (350 mg).

TLC of 400 mg in the neutral category was carried out twice by TLC (hexane: ethyl acetate = 1: 1, R _f = 0.12).
0.17, hexane: ethyl acetate = 1: 2, R _f = 0.
60-0.65) and recrystallized from 80% methanol to give taxol, mp 214-216 ° C, [α] _D -4.
5 ° (MeOH), MS m / e853 (M ⁺ ) (about 5
mg) was obtained.

Separated by benzene: ethyl acetate (3: 1 to 1: 1) elution using silica gel column chromatography and recrystallized from methanol, taxol was obtained.

Example 2 Bark (2.49 Kg) of Takagi yew from Toyama prefecture was extracted with methanol, and the extract was treated with activated carbon and concentrated. The concentrated solution was extracted with ethyl acetate to obtain a crude extract (82.0 g). The ethyl acetate solution of the crude extract (65.0 g) was washed with 0.5% NaOH half-saturated with sodium chloride (3 times), the carboxylic acid and phenol fractions were removed, and the mixture was extracted 3 times with 5% hydrochloric acid containing 5% methanol. Then the alkaloid category (370
mg) and the neutral compartment (14.9 g) were obtained. The neutral fraction was subjected to silica gel column chromatography with activated carbon as an upper layer, and eluted with benzene-ethyl acetate (3: 1 to 1: 1) to give 1.721 g of glyceride and taxinine (m).
p. 270 mg to 730 mg, β-sitosterol 730 mg, followed by taxane terpenoids (63
0 mg) was obtained.

TLC (hexane: ethyl acetate =
1: 2), 134 mg of 4 (20), 11-
taxadiene-2,5,7,9,10,13-h
exanol, 5-cinnamoyl, 2, 7, 9,
10,13-pentaacetate

[0031]

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A small amount of taxinin B (10 mg) was obtained.

[0033]

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M. p. 265-266 ° C [α] _D +9
3.8 ° (CHCl ₃ ), and elution with benzene-ethyl acetate (1: 1) gave a taxol-corresponding section. This section was further fractionated by TLC (hexane: ethyl acetate = 1: 2) to obtain a section corresponding to taxol Rf value (90 mg).

It was an aromatic compound that did not show the presence of a taxane ring in ¹ H-NMR analysis, and did not contain taxol.

Example 3 360 mg of the neutral category containing alkaloids was obtained from a methanol: chloroform (3: 1) extract of 74.8 g of the female strain Kimino onco bark. This was extracted with dilute hydrochloric acid to obtain 136 mg of alkaloid category and 200 mg of neutral category. HPLC analysis of the latter showed the presence of taxol (0.012% content). From the methanol extract of 655 g of leaves of the same strain, 757 mg of alkaloids and 1.877 g of neutrals were obtained.

When the latter was subjected to HPLC analysis, the taxol content was 0.0014%.

Claims (5)

[Claims] 1. A method for producing taxol, which comprises using Japanese yew as a raw material. 2. A method for producing taxane terpenoids, which comprises using Japanese yew as a raw material. 3. The method for producing taxol according to claim 1, wherein the yew produced in Japan is produced in Miyagi prefecture. 4. The method for producing taxol according to claim 2, wherein the yew produced in Japan is produced in Toyama Prefecture. 5. A Japanese yew leaf, heartwood or hide is directly extracted with alcohol, then a concentrated solution of the alcohol extract is extracted with ethyl acetate and then washed with a dilute alkaline aqueous solution to obtain a crude extract. Claim 1 characterized by the above-mentioned.
A method for producing the described taxol.