JP2000053668A - Purification of taxane compound-including composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a purifying method in which purifying procedure is simplified and the loss of taxane compound on purifying is reduced for the purpose of efficiently obtaining a taxane compound-including ingredient and to provide a convenient method for purifying cephalomanine. SOLUTION: This method for purifying taxane compounds-including ingredients is to adsorb taxane compounds-including ingredients with a porous material having 25-120 Å minute hole diameter from a mixture including at least one kind of taxanes and then to elute the ingredients. This method for purifying cephalomanine is to adsorb cephalomanine with a porous material having 25-120 Å minute hole diameter from a mixture including cephalomanine and then to elute cephalomanine with an aqueous methanol solution including 30-40 wt.% methanol.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying a taxane compound-containing component. More specifically, a taxane compound-containing component is purified from a mixture containing a taxane compound such as 10-deacetylbaccatin III, in particular, a plant of the Taxus family or the plant extract using an adsorbent, and further selectively separated. , A purification method.

[0002]

2. Description of the Related Art Taxane compounds are diterpene compounds that have been used in the field of medicine, and can be used as medicines or as raw materials for medicines. Such taxane compounds include, for example, 10-deacetylbaccatin II
I, baccatin III, cephalomannine, 7-β-xylosyltaxol, 10-deacetyltaxol, taxinin and the like.

[0003] The production of taxol and taxane compounds has also been studied by an organic chemical synthesis method, but the number of steps is 40 to 50.
Many processes and low yields make it inefficient at present for industrialization by organic chemical synthesis (Nature, 367, 630).
(1994), J. Am. Chem. Soc., 116, 1597, 1599 (1994),
Kuwashima: Preprints of the 41st Lectures on Spices, Terpenes and Essential Oil Chemistry S3 page (1997)).

At present, a relatively easy method of obtaining taxol and taxane compounds is a method of solvent extraction from the leaves of a yew tree. Taxol, which is currently sold worldwide, can be obtained by solvent extraction from Taxus trees or purified by taxi, or semi-synthesized from 10-deacetylbaccatin III, a precursor of Taxol also contained in Taxus trees. It is a thing.

[0005] As methods for extraction and purification from Taxus trees, methods such as column chromatography, liquid chromatography, and recrystallization have been mainly reported. For example, Phar
maceutical Research, Vol. 12, No. 12, 1003 (1995)
And Japanese Unexamined Patent Publication No. 8-127574, and a method of supercritical extraction (Japanese Unexamined Patent Publication No. 5-202016).

However, extracts from Taxus trees contain more than 50 low-molecular compounds, lignin and lignin-sugar compounds, viscous substances called tar, and the like. In the method of separating a taxane compound by column chromatography, the separation is difficult because the filler is clogged with tar or lignin.
Unless tar and lignin are completely removed, the expensive column of the preparative liquid chromatograph used in the latter half of the purification is contaminated and the life of the column is shortened. for that reason,
It is necessary to remove a large amount of tar and lignin, and a very complicated operation is performed for the removal.

[0007] Even if an extract is obtained using a solvent that does not readily dissolve lignin or tar as the extraction solvent, the extraction efficiency of the taxane compound is poor when a low-polarity solvent is used. Moreover, the extract still contains various kinds of low-molecular compounds other than the taxane compound, which leaves problems in purification efficiency and operability.

In these operations, since the taxane compound is contained only in a very small amount, it is dispersed and adsorbed in the filler during the separation of tar and lignin, and only a small amount can be recovered. Or it does not come out by being adsorbed on a column or the like.

Further, in the method of supercritical extraction using CO ₂ gas, tar components and lignins are hardly extracted, but the recovery of taxane compounds from plants is lower than in methanol extraction and the like, and it must be performed at high pressure. And that large-scale industrial supercritical extraction equipment is very expensive.

Although taxol and taxane compounds are very expensive reagents or pharmaceuticals, the amount of these compounds contained in trees is as small as several tens to several hundred ppm, which is insufficient for administration to cancer patients worldwide. It is. Therefore, the economic loss due to the loss at the time of purification is large.

Further, cephalomannine remained as an impurity during purification of the taxane compound, and was not easily separated from other taxane compounds even by using preparative liquid chromatography. As a conventional separation method, a mixture of taxane compounds is subjected to an ozone oxidation reaction at −70 ° C. to oxidize cephalomannine into a derivative that can be easily separated (KV Rao eta).
l., Pharmaceutical Research, Vol. 12, 1003 (1995)
) Or by reacting bromine with the mixture and brominating cephalomannine into a derivative that can be easily separated (JM Rimo
ldi et al., J. Nat.Prod., Vol. 59, 167-168 (199
6)) is known. However, ozone oxidation at −70 ° C. is complicated for a large apparatus. Further, in the bromination reaction, a side reaction in which another taxane compound is mutated by bromine occurs, so that there is a problem that control of the reaction is difficult.

[0012]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a purification method in which a taxane compound-containing component is efficiently obtained, thereby simplifying a purification operation and further suppressing loss of the taxane compound during purification. It is in. It is a further object of the present invention to provide a simple method for purifying cephalomannine.

[0013]

That is, the gist of the present invention is as follows.
[1] 10-deacetylbaccatin III, baccatin
III, from a mixture containing one or more taxane compounds selected from the group consisting of cephalomannine, 7-β-xylosyltaxol, 10-deacetyltaxol and taxinine, a porous material having a pore diameter of 25 to 120 ° as an adsorbent. A method for purifying a taxane compound-containing component, wherein the taxane compound-containing component is purified by adsorbing and eluting the taxane compound-containing component using a conductive material; [2] further comprising: A method for purifying a taxane compound, which comprises a purification step of purifying and isolating the taxane compound, [3] adsorbing cephalomannine from a mixture containing cephalomanine using a porous material having a pore diameter of 25 to 120 ° as an adsorbent. And then
The present invention relates to a method for purifying cephalomanine, characterized by elution with a methanol aqueous solution having a methanol concentration of 30 to 40% by weight.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The process for purifying a taxane compound-containing component of the present invention comprises the steps of:
The present invention is characterized in that a taxane compound-containing component is adsorbed and eluted using a porous material having a pore diameter of 25 to 120 ° as an adsorbent.

In the present specification, the "taxane compound-containing component" is selected from the group consisting of 10-deacetylbaccatin III, baccatin III, 7-β-xylosyltaxol, 10-deacetyltaxol, cephalomannine and taxinine. And a mixed component of one or more taxane compounds.

The purification method of the present invention can be roughly divided into the following steps. 1) A step of adsorbing a mixture containing a taxane compound on a porous material. 2) a step of eluting the taxane compound-containing component from the porous material on which the taxane compound-containing component is adsorbed. Hereinafter, the present invention will be described step by step.

1) Step of adsorbing a mixture containing a taxane compound onto a porous material The mixture containing a taxane compound used in the method of the present invention is not particularly limited as long as it contains a taxane compound. Examples include, but are not limited to, a plant or an extract derived from a plant, a partially purified product obtained by partially purifying the extract by a known method, and a mixture containing by-products and the like obtained by chemical synthesis. In the present invention,
Plant leaves, bark, roots, twigs, seeds, crushed whole seedlings, etc. may be used directly as a mixture containing a taxane compound, but from the viewpoint of purification efficiency, the plant It is preferable to use an extract or the like extracted from leaves or the like as a mixture containing a taxane compound.

Further, the mixture containing the taxane compound may be washed with an alkaline aqueous solution and / or an acidic aqueous solution, if necessary. Such a washing operation is preferable because carboxylic acids and alkaloids can be effectively removed. For such a washing operation, an operation generally performed in the field of organic chemistry can be employed.

The plant is not particularly limited as long as it contains a taxane compound.
Since the taxane compound is contained in a relatively large amount, plants of the Taxus family and the like can be mentioned. As a plant of the Taxus family, a plant of the genus Taxus is preferred. Brevifolia, T.M. Baccata, T .; Media, T. Wallichiana, T.W. Canadensis, T.C. Cuspidata
, T .; Sumatrana (sumatorana) etc. are mentioned.

The method for extracting a mixture containing a taxane compound from such a plant is not particularly limited.
A known method such as a solvent extraction method can be used. The method for extracting with a solvent is not particularly limited as long as it is a commonly used method.For example, a portion containing a taxane compound such as the above-mentioned plant leaf, bark, root, twig, seed, or whole seedling is used. A method of pulverizing and extracting with a solvent may be mentioned. The solvent used for the extraction is not particularly limited as long as it can dissolve the taxane compound, but methanol, ethanol, chloroform,
Examples include dichloromethane, methyl acetate, ethyl acetate, acetone and the like. Such solvents may be used alone or as a mixture of two or more. The extract thus obtained may contain components such as lignin and tar in addition to the taxane compound.

The adsorbent used in the present invention is not particularly limited as long as it is a porous material having a pore size of 25 to 120 °.

The porous material has a pore diameter of 25-60.
Å is preferred, more preferably 30 to 50Å, and particularly preferably 30 to 40Å. It is preferably at least 25 ° from the viewpoint of the ease with which the molecules of the taxane compound enter the pores, and preferably at most 120 ° from the viewpoint of the selectivity of the component to be adsorbed.

The pore diameter in the above range is larger than other porous materials conventionally used as adsorbents on a daily basis. A taxane compound having a diameter can be captured in the pores of the porous material used in the present invention.

The material of the porous material is not particularly limited as long as it has a capability of adsorbing and eluting the taxane compound. Preferably, inorganic porous silica gel is used.

The silica gel is not particularly limited as long as it can adsorb and elute the taxane compound. Mesoporous silica gel and mesopore silica gel having a desired pore diameter are preferred. In particular, mesoporous silica gel, which has been successfully synthesized by the present inventors as a result of intensive studies, is more preferable because the width of the pore size distribution is narrow.

The method for synthesizing the mesoporous silica gel used in the present invention includes, for example, kanemite (NaHS).
_{i 2 O 5 · 3H 2 O} ) in the general formula (1)

[0027] _{C n H 2n + 1 (CH} 3) 3 N + Cl - (1)

(T. Yanagisawa, T. Shimizu,
K. Kuroda, C .; Kato, Bull. C
hem. Soc. Jpn. , 63, 988 (199
0)).

The alkyltrimethylammonium chloride used in the synthesis of the mesoporous silica gel is not particularly limited, but the alkyltrimethylammonium chloride represented by the general formula (1) wherein n is in the range of 12 to 18 is used. It is preferably from 14 to 18, more preferably from 16 to 18. When the carbon number of the alkyl group is small, the pore diameter becomes small, so that the carbon number is preferably 12 or more from the viewpoint of the ease of entry of the taxane compound into the pores, and when the carbon number of the alkyl group is too large, the pore diameter is too large. Because other compounds enter
From the viewpoint of maintaining selectivity and because it is difficult to obtain those having an alkyl group having 18 or more carbon atoms, the number of carbon atoms is 1
It is preferably 8 or less.

When a mesoporous silica gel is prepared by using a compound having n of 12 to 18 in the general formula (1), the mesoporous silica gel has many honeycomb-shaped pores having a pore diameter of about 20 to about 40 ° and a specific surface area of Value is 90
0 to 1200 m ² / g.

Mesoporous silica gel is a porous silica gel material having a pore size distribution wider than that of mesoporous silica gel, and those having various pore sizes are commercially available. For example, Kasei gel (manufactured by Tokyo Kasei) has a pore diameter in a wide range of 10 to 80 °, but the number of pores is smaller than that of mesoporous silica gel.
Therefore, the specific surface area value is as small as 250 to 500 m ² / g. Daisogel (manufactured by Daiso Corporation) is also mesopore silica gel having a pore diameter of about 60 ° and a specific surface area of about 500 m ² / g.

Among the above-mentioned mesoporous silica gels or mesoporous silica gels, particularly, the former is one which has succeeded in synthesizing one having a desired pore diameter as a result of intensive studies by the present inventors, and has a molecular diameter which cannot be adsorbed by ordinary porous materials. Large taxane compounds can be trapped in the pores in such gels.

The method of purifying the taxane compound-containing component is not particularly limited as long as the above adsorbent is used.

The amount of the porous material used in the present invention is not particularly limited, and can be freely adjusted by the content of the taxane compound-containing component contained in the mixture. Specifically, the amount is preferably from 1/100 to about 50 times the weight of the concentrate of the mixture containing the taxane compound, more preferably 1/20 to 20 times, particularly preferably 1/10 to 10 times. It is twice.

Here, the “concentrate” of the mixture containing the taxane compound is obtained by concentrating the mixture containing the taxane compound at a water bath temperature of 35 to 40 ° C. using an evaporator.

The solvent used for adsorption to the porous material used in the present invention is a solvent having a smaller polarity than the solvent for elution and capable of dissolving most of the mixture containing the taxane compound. There is no particular limitation, and examples thereof include dichloromethane, toluene, benzene, and chloroform. These may be used alone or as a mixture.

When the solvent used for extracting the taxane compound from the plant can be used as the solvent used for adsorption, the extract may be brought into contact with the porous material as it is. When the solvent at the time of extraction cannot be used as the solvent at the time of adsorption, after removing the solvent from the extract, an appropriate adsorption solvent may be added.

The amount of the adsorption solvent is not particularly limited, but is preferably about 3 to 1000 times, more preferably 5 to 800 times, particularly preferably 5 times the weight of the concentrate of the mixture containing the taxane compound. It is 5 to 50 times.

The method for adsorbing the taxane compound-containing component on a porous material having a predetermined pore size is not particularly limited. For example, an embodiment in which the porous material used in the present invention and the mixture containing the taxane compound are stirred together for an appropriate time, or the porous material used in the present invention is filled in a column, and the mixture is loaded on the column. And the like.

By bringing the mixture containing the taxane compound into contact with the porous material in this manner, a compound having a polarity in the pores of the porous material or a medium-polar one having a molecular weight of about 450 to 1,000 (Including taxane compounds) are adsorbed. The compounds that have not been adsorbed are removed by washing with the same solvent as the solvent used at the time of adsorption.

Before eluting the taxane compound-containing component from the porous material on which the taxane compound-containing component is adsorbed, the porous material may be washed. As a solvent used for washing, water is mentioned as a preferable one. When water is added to the porous material on which the taxane compound-containing component is adsorbed, many polar compounds other than the taxane compound-containing component are eluted. Subsequently, when eluted with a 30 to 40% by weight aqueous methanol solution, cephalomannine and 10-deacetylbaccatin III of the taxane compound are selectively dissolved in the washing solution,
It can be separated from other components. That is, cephalomannine can be efficiently purified by eluting with a methanol aqueous solution having such a concentration. The concentration of the methanol aqueous solution is more preferably 35 to 40% by weight. When cephalomanine is purified, the mixture containing cephalomanine includes the mixture containing the taxane compound described above.

2) A step of eluting the taxane compound-containing component from the porous material on which the taxane compound-containing component has been adsorbed.
There is no particular limitation as long as it is suitable for elution of the taxane compound-containing component, and examples thereof include a mixture of a water-soluble organic solvent and water. As the water-soluble organic solvent,
Examples include methanol, ethanol, acetonitrile, acetone and the like. The amount of the organic solvent in the mixture is not particularly limited. It is preferable to use a mixture of water and an organic solvent with a different mixing ratio according to the characteristics of the adsorption material.

The amount of the elution solvent used is not particularly limited, but is preferably about 2 to 1000 times, more preferably 5 to 800 times, particularly preferably 5 to 1000 times the weight of the concentrate of the mixture containing the taxane compound. It is 10 to 500 times.

As a method for desorbing the taxane compound-containing component by eluting it from the porous material to which the taxane compound-containing component is adsorbed, the solvent used for elution of the taxane compound-containing component may be brought into contact with the porous material. Specific examples include a mode in which the porous material and the solvent are stirred together for an appropriate time, a mode in which the porous material is filled in a column, and the solvent flows through the column.

In this way, by bringing the above-mentioned elution solvent into contact with the porous material, most of the taxane compound-containing components are eluted and desorbed.

As described above, the purification method of the present invention can effectively separate and purify the taxane compound-containing component effectively by an extremely simple operation as compared with the conventional method.

In the case of an adsorbent using Daiso gel (mean pore diameter: 67 °), which is a mesopore silica gel, after the adsorption, the adsorbent is washed with water and a 35% by weight aqueous methanol solution, whereby cephalomannine and 10- Deacetylbaccatin III is eluted preferentially. Thereafter, the adsorbent can be treated with methanol to elute other taxane compounds. As described above, even when the taxane compounds are used, since the solubility of each compound in the eluate is different, each taxane compound can be effectively separated by utilizing this fact. In addition, there are some differences in the elution state depending on the type of the adsorbent, the size of the pore diameter, and each of the taxane compounds.

According to the method of the present invention, cephalomannine can also be relatively easily separated and purified from taxol and the like by adsorption and washing with the porous silica gel. In addition, the taxane compound adsorbed on the mesopore silica gel and eluted with the methanol eluate has several times the content before adsorption due to the removal of tar components and lignin, etc. It is easily isolated during the purification process.

Further, the porous material from which the taxane compound-containing component has been eluted is washed with a solvent such as methanol or acetone to remove lignin or tar from the porous material.
Then, by washing with a solvent such as dichloromethane or toluene, the obtained porous material after washing can be reused for adsorption and elution of the taxane compound-containing component.

3) Purification Step Further, in the present invention, a purification step of further purifying the taxane compound-containing component purified by the above method to isolate the taxane compound may be provided. By providing such a step, the taxane compound can be purified with higher purity, and each compound can be isolated.
In particular, the taxane compound-containing component obtained by the method of adsorbing and eluting the taxane compound-containing component on a specific porous material as described above makes purification operations by column chromatography, high-performance liquid chromatography (HPLC), etc. difficult. Since such components (lignin, tar, etc.) are effectively removed, such purification operations can be easily combined. Therefore, the taxane compound can be easily isolated and purified to a higher degree.

As a purification means that can be used in the purification step, there can be mentioned a method used in a conventionally known method for purifying an organic compound such as a taxane compound. For example, methods such as high performance liquid chromatography (HPLC), supercritical fluid chromatography, and recrystallization are exemplified. Further, gel filtration chromatography, column chromatography and the like using Sephadex LH-20 (trade name) can also be used. These methods may be performed alone or in combination of two or more. When two or more methods are combined for purification, it is more preferable to carry out another purification method before purifying by HPLC. This can extend the life of the HPLC filler.

By the above-mentioned method, 10-deacetylbaccatin III, baccatin III, 7-β-xylosyltaxol, 10-deacetyltaxol, cephalomannine, taxinin and the like can be isolated and purified.

[0053]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples and the like.

Example 1 Preparation of yew leaf extract 100 kg of yew leaves (including twigs having a diameter of 5 mm or less) was crushed using a grinder: Large 300TC type (manufactured by Taninaka Co., Ltd.) to about 1 mm × 2 mm. Was obtained. This pulverized material was put into a stainless steel container (200 to 400 L capacity, with a valve for extracting the extract from the bottom) with a 100 mesh wire mesh attached to the bottom. Hexane 1
It was immersed in 50 L for 2 days to dissolve the wax content of the leaves, and hexane was removed. Subsequently, 150 L of methanol was added, and the mixture was immersed at 20 to 25 ° C. for 4 days with occasional stirring. The methanol extract was taken out from the bottom of the stainless steel container. A residue obtained by washing the residue remaining in the container with 100 L of methanol was added to the above methanol extract. The methanol extract is concentrated with a large evaporator having a hot water temperature of 35 to 40 ° C,
21 L of a mixture of water and oil was obtained. This mixed solution was put into a 100 L kettle equipped with a stirrer, and further 20 L of methyl acetate and 10 L of saturated sodium sulfate solution were added, followed by stirring for 20 minutes. Thereafter, the kettle was allowed to stand, and the aqueous layer was separated and removed from the kettle. To the remaining oil layer, 10 L of saturated sodium sulfate solution was again added and washed, and then the water layer and the oil layer were separated again to obtain a 15 L oil layer.

The oil layer of 15 L was concentrated to obtain 3050 g of a dark green mixed oil containing a large number of components including a taxane compound, a tar component, lignin and the like. This dark green mixed oil was used as a yew leaf extract.

Analysis and quantification of taxane compounds such as yew leaf extract and extracts obtained in each purification step were performed by high performance liquid chromatography (abbreviated HPLC). The instrument is Shimadzu LC-10A, and the column is Senshu Kagaku Peg.
asil C8 column (4.6 mm in diameter, 25 c in length)
m), UV detector wavelength: 227 nm, eluent: acetonitrile / 10 mM ammonium acetate aqueous solution = 45/55
(Volume ratio) and the flow rate were analyzed at 0.6 mL / min. Prepare a calibration curve of UV absorption intensity using a sample in advance,
The components were quantified using the calibration curve. The content of each component in the yew leaf extract (3050 g) was 2.71 g of 10-deacetylbaccatin III and 0.28 of baccatin III.
g, 7-xylosyltaxol 2.54 g, cephalomannine 0.24 g, 10-deacetyltaxol 0.11
g, taxinin 4.13 g.

Washing of Yew Leaf Extract The yew leaf extract contains carboxylic acid, alkaloid and the like, and it is preferable to remove this in advance. 7 L of 5% sodium hydroxide-half-saturated sodium sulfate solution was added to 15 L of the yew leaf extract, and the mixture was stirred at 200 to 300 rpm for 10 minutes. After standing for 20 minutes, the aqueous layer was removed. This operation was performed once more. Next, 10 L of methyl acetate was added to the yew leaf extract as an oil layer, and 7 L of a 5% hydrochloric acid-5% aqueous methanol solution was further added, followed by washing twice in the same manner as described above. The separated oil layer is 1/10 saturated aqueous sodium bicarbonate-saturated sodium sulfate aqueous solution (10 L).
(Volume ratio), and then the oil layer of the washing liquid was heated to a liquid temperature of 35-4.
The mixture was concentrated at 0 ° C. under reduced pressure to obtain 974 g of a black oil. 2.27 g of 10-deacetylbaccatin III (recovery rate: 83.9)
%), 0.24 g of baccatin III (recovery 86%),
2.34 g of 7-xylosyltaxol (recovery rate 92.
1%) and 0.22 g of cephalomanine (recovery rate 91.1).
%), 10-deacetyltaxol 0.095 g (recovery rate 86.4%), taxinine 3.59 g (recovery rate 8
6.9%) in the black oil.

Synthesis of mesoporous silica (FSM-C16) High purity water glass and 2N aqueous sodium hydroxide solution were mixed with Si
After adjusting so that O ₂ / Na ₂ O = 2.0, the mixture was stirred for 1 day. It was fired at 750 ° C. for 1 hour in an air atmosphere to obtain δ-Na ₂ Si ₂ O ₅ . 50g obtained
Of δ-Na ₂ Si ₂ O ₅ was dispersed in 1 L of water and stirred for 30 minutes to obtain kanemite in a slurry state. The slurry of kanemite was dispersed in a 0.1 mol / L aqueous solution of hexadecyltrimethylammonium chloride, and
Stirring was performed at 0 ° C. for 3 hours. Thereafter, the supernatant was removed by centrifugation, and the precipitate was dispersed in water. Then p with 2N hydrochloric acid
After adjusting to H8.5, the mixture was centrifuged to obtain a precipitate. The precipitate was air-dried and washed with water. The obtained powder was placed in an electric furnace and calcined at 700 ° C. for 3 hours to obtain 45 g of mesoporous silica gel (abbreviated as FSM-C16). Confirmation of the synthesis of mesoporous silica was confirmed by XRD analysis.
The measurement was performed by nitrogen adsorption measurement.

For the XRD analysis, an X-ray generator (source 1.5) was used.
4050 ° (Cu), 3 kW), using a monochromator (tube voltage 40 kV, tube current 30 mA), sampling width 0.01 deg, scanning speed 2 deg / min, divergence slit 1 deg, scattering slit 1 deg, light receiving slit 0.15 mm Was analyzed. Such XRD
The analysis confirmed that the silica gel had a uniform honeycomb structure with a large pore diameter.

Nitrogen adsorption measurement was performed using BER manufactured by Bell Japan Co., Ltd.
A SORP28SA type device was used. Sample is 10 ^-2 T
After drying for 6 hours under the conditions of orr and 40 ° C., the reaction was performed at an equilibrium time of 300 S, an adsorbate N ₂ , an air bath temperature of 40 ° C., a measurement temperature of −196 ° C., and an adsorption cross section of 0.162 nm ² .
When the pore diameter of FSM-C16 was analyzed from a nitrogen adsorption isotherm graph obtained by nitrogen adsorption measurement, it was 27 °. The specific surface area value was 970 m ² / g. The pore volume was 1.26 cm ³ / g.

Adsorption and Desorption of Yew Leaf Extract-Derived Black Oil on Mesopore Silica Gel An industrial purification method was studied using commercially available mesopore silica gel (average pore diameter: 67 °). Dissolve 954 g of the above black oil in 6 L of toluene and add 1 kg of Daiso Gel IR60 (manufactured by Daiso Corporation: particle diameter 63 to 210 μm, average pore diameter 67 °) together with 2 kg.
Stirred at room temperature for 150 hours (150-200 rpm). This mixture was subjected to suction filtration using a large Nutsche to remove toluene. Next, this silica gel was put into a vessel equipped with a stirrer, 12 L of water was added, and the mixture was stirred for 30 minutes (about 200 rpm), and the water washing solution (a) was removed with a large Nutsche. Subsequently, this silica gel was stirred and washed (about 200 rpm) with 1.2 L of 35% by weight aqueous methanol solution for 1 hour (about 200 rpm) and filtered twice to obtain a 35% by weight methanol washing liquid (b). Finally, the mixture was stirred twice with 1.2 L of methanol for 1 hour each time, extracted, concentrated, and then concentrated to remove 218 g of desorbed oil (0.105% of baccatin III: 0.23 g, 1.05% of 7-xylosyl taxol: 2.29 g, 10%). 0.04% of deacetyltaxol: 0.09 g, taxinin 1.61
%: 3.51 g). In addition, operations such as adsorption, washing, and filtration can be smoothly performed by using a simple solid-liquid mixing device with a filter plate.

Using this desorbed oil, the adsorption-desorption operation is performed again in the same manner, and the second desorbed oil 100 g (Baccatin II)
I 0.21%: 0.21 g, 7-xylosyltaxol 2.2%: 2.20 g, 10-deacetyltaxol 0.09%: 0.09 g, taxinin 3.45%: 3.
45 g), a water washing solution (c) and a 35% by weight methanol washing solution (d) were obtained.

In the step of adsorption and desorption to silica gel,
A method of flowing a black oil-toluene mixed solution through Daisogel IR60 packed in a column to adsorb the taxane compound on silica gel, and subsequently eluting the taxane compound using various eluates can also be carried out. On the other hand, in the above-mentioned method in which the black oil-toluene mixture and the silica gel are stirred to perform the adsorption treatment, tar components and the like are evenly attached to the entire adsorbent, so that clogging between silica gel particles during filtration is reduced. The elution operation using various eluates can be performed more easily.

Each of the cleaning solutions (a), (b), (c) and (d)
Were combined and concentrated to remove methanol.
An extraction operation was performed by adding an equal amount of methyl acetate to the concentrate, and 207 g of oil (cephalomannine 21) was obtained from the obtained oil layer.
Contains 8 mg, 2.27 g of 10-deacetylbaccatin III. ) Was recovered. Most of the cephalomannine and 10-deacetylbaccatin III in the yew leaf extract were transferred to this washing liquid recovered oil. Taxol was also mixed because it was slightly soluble in water, but the amount was small. It seems that the solubility of taxol and cephalomanine and 10-deacetylbaccatin III in a solvent dissolved in an organic solvent such as methyl acetate does not change much, but it is adsorbed on silica gel and adsorbed without methyl acetate. It is believed that when the molecule is naked, a difference in the solubility of these compounds in the solvent between the compounds has emerged more clearly, thereby allowing such separation.

By this adsorption-desorption method, it was possible to effectively separate cephalomanine and taxol, which were conventionally difficult to separate. The second desorbed oil contained only trace amounts of cephalomanine, including taxol, baccatin III, 7-xylosyl taxol, 10-deacetyltaxol, taxinin, and the like. It was considered that a part of the other impurities 647 g was dissolved in the washing water, and a part was adhered or adsorbed to the silica gel and did not come out. The silica gel after desorption was found to be usable again when washed with acetone and methanol.

Example 2 Adsorption and Desorption of Black Oil Derived from Yew Leaf Extraction Oil on Mesoporous Silica Gel A purification method was studied using FSM-C16 which is a mesoporous silica gel. 20 g of the black oil of Example 1 was dissolved in 200 mL of toluene, and the mixture was stirred with 20 g of FSM-C16 at room temperature for 2 hours (150 to 200 rpm), and then filtered. The silica gel was washed with water (250 mL) and a 35% by weight aqueous methanol solution (250 mL x 2), and then extracted twice with 250 mL of methanol to obtain 2.99 g of a desorbed oil. Next, to 2.99 g of desorbed oil, toluene and 3 g of FSM-
C16 was added and treated in the same manner, and the second desorbed oil was 1.37.
g was obtained. In this second desorption oil, taxinine was used.
18% (71 mg) was contained.

The taxinin content of the second desorbed oil obtained using FSM-C16 is clearly higher than the detaxed oil content obtained using Daisogel IR60 and that of the second desorbed oil. Met. This is FS
The taxane compound enters the pores of M-C16,
It is considered that the selectivity was higher than in the case of Daisogel by preventing substances having a larger molecular diameter from entering the pores.

Example 3 Purification of 10-deacetylbaccatin III Washing of the silica gel of Example 2 with water and 35% by weight
A washing solution obtained by washing with an aqueous methanol solution was collected. The collected material was concentrated to obtain an oil. OD of 20 g of this oil
It was added to a glass column (diameter 7 cm, filler height 14 cm) packed with 200 g of Sako filler Wakogel LP-60C18 (manufactured by Wako Pure Chemical Industries, Ltd.). Then, acetonitrile / water = 10 / 90-9 as eluent
Column chromatography separation using a solution having a composition of 0/10 (volume ratio) was performed. Eluent: acetonitrile /
The eluted fraction of water = 20 / 80-30 / 70 (volume ratio) was concentrated, 50 mL of methyl acetate was added to a mixture of oil and water obtained by extraction, and the aqueous layer was removed. Next, anhydrous sodium sulfate was added to the obtained oil layer and dried, and then concentrated by an evaporator to obtain 2.3 g of an oil containing 10-deacetylbaccatin III. Eluent: acetonitrile / water =
2. From the eluted fraction of 70/30 to 80/20 (volume ratio)
An oil containing 2 g of cephalomannine was obtained.

2.3 g of an oil containing 10-deacetylbaccatin III was fractionated by liquid chromatography (Shimadzu LC-8).
A, Preparative column YMC-ODS, R-355-15, diameter 5 cm x length 50 cm, particle diameter 10 to 20 m, UV
227 nm, eluent: methanol / acetonitrile / water = 20/60/20 (volume ratio), flow rate 40 mL / mi
n). The fraction containing the peak of 10-deacetylbaccatin III was collected and concentrated by an evaporator (bath temperature 38
C) to give 0.21 g of a white solid. This was recrystallized from diethyl ether to obtain 196 mg of colorless crystals (recovery rate from black oil: 75.0%). This crystal has an optical rotation [α] _D = −41 ° (C =
0.40, methanol, 23 ° C.), mass spectrum, UV spectrum (KBr disk), ¹ H-NM
The R spectrum (CDCl ₃ ) agrees with that of the reagent (SIGMA, USA) and the literature (J. Nat. Prod. Vol.
45,466 (1982) and J. Org. Chem. Vol. 46, 1469 (1981)).
It was confirmed to be III.

Example 4 Purification of Cephalomannine The oil containing 3.2 g of cephalomannine separated in Example 3 was purified by using silica gel (Kieselgel 60, particle size: 6 manufactured by Merk).
The mixture was added to a glass column (diameter 2 cm x packed bed length 32 cm) packed with 50 g (3 to 200 µm), and fractionated with a mixed solution of eluent: hexane / acetone = 2/1 (volume ratio). Each fraction was concentrated, and the concentrate was dissolved in methanol and subjected to HPLC analysis. Then, 265 mg of an oil containing cephalomannine was obtained. This oil was purified using the preparative liquid chromatograph used in Example 3. Acetonitrile / 10 mM ammonium acetate aqueous solution = 50/50 (volume ratio) as eluent, UV detector wavelength 227 n
m, and the flow rate was 40 mL / min. The fractions were concentrated and treated as for 10-deacetylbaccatin III to give 19 mg of a white solid. This is methanol
Recrystallization from an aqueous solution gave 17 mg of colorless crystals. HPL
C was 99.6% pure (recovery 7
3.9%, cephalomanine in black oil). Melting point is 18
At 4 to 186 ° C., the optical rotation [α] _D = −41 ° (C = 0.41).
39, methanol, 23 ° C), mass spectrum,
UV spectrum, ¹ H-NMR spectrum (CDCl
₃ ) was consistent with the value in the literature (J. Org. Chem. Vol. 46, 1469 (1981)), confirming that this crystal was cephalomannine.

Example 5 Next, baccatin III, 7-xylosyltaxol and taxinin were separated. First, 100 g of the second desorbed oil obtained in Example 1 was subjected to the following four steps of purification treatment. (1) Silica gel column chromatography using a hexane / acetone eluent (filler: Wakogel C-300
(Wako Pure Chemical Industries)). (2) Reverse phase column chromatography using acetonitrile / water-based eluate (filler: Wakogel LP-60C18 (manufactured by Wako Pure Chemical Industries)). (3) Column chromatography using methanol as an eluent (the filler was LH-20; components having a molecular weight of about 450 to 1,000 were collected). (4) Silica gel column chromatography using a hexane / acetone eluent (filler: silica gel Kieselgel 60 manufactured by Merk).

By performing these four operations, 46.0 g of pretreated oil was obtained.
(0.52% of baccatin III: 239 mg, 4.6% of 7-xylosyltaxol: 2.13 g, 0.19% of 10-deacetyltaxol: 90 mg, taxinin 7.3
0%: contains 3.36 g. ) Got.

Example 6 15 to 16 g of the pretreated oil obtained in Example 5 was dissolved in a small amount of acetonitrile, and the solution was separated by a preparative liquid chromatograph (Soken Kagaku Hi-Sep LC Prep C-500, ODS packed column, After injection into a particle diameter of 10 to 20 μm, column diameter of 10 cm × length of 50 cm, 2 tubes), operation was performed with an eluent: acetonitrile / water = 68/32 (volume ratio), a flow rate of 160 mL / min, and a UV detector wavelength of 227 nm. A peak having a retention time identical to that of the sample of 7-xylosyltaxol was collected. This fraction was concentrated by an evaporator (bath temperature: 35 to 40 ° C.), and sodium chloride was added to the residue to salt out 7-xylosyltaxol.
Extracted with 50 mL of methyl acetate. The methyl acetate solution was dried over anhydrous sodium sulfate and concentrated. By performing the above operation three times, 3.33 g of a preparative oil (7-xylosyltaxol content: 60%) was obtained.

The fractionated oil was subjected to silica gel column chromatography (silica gel Kieselgel 60, Merk Co., packed bed diameter 3).
fractionation (cm × length 25 cm, hexane / acetone system) yielded 2.7 g of a yellow-white solid. This was filled with silica gel (Kieselgel 60) manufactured by Merck & Co., Ltd. (50
g, packed layer having a diameter of 3 cm × 14 cm) and further fractionated by column chromatography (hexane / acetone = 2/1 (volume ratio)). The fraction containing 7-xylosyl taxol was concentrated and recrystallized from isopropyl alcohol to give 1.96 of colorless crystals of 7-xylosyl taxol.
g was obtained. The recovery from the extracted oil was 77.2%.

The purity was measured by HPLC to be 99.3%. Melting point: 236-238 ° C, Optical rotation [α] _D = −
The data of 23 ° (C = 0.4, pyridine solution, 23 ° C.), mass spectrum, UV spectrum, and ¹ H-NMR spectrum are the analytical values of the standard and the literature (J. Nat. Prod., Vol. 47,
131 (1984)), which confirmed that the obtained crystal was 7-xylosyltaxol.

Example 7 Purification of Taxinin When 7-xylosyltaxol was fractionated by a preparative liquid chromatograph, taxinin was also fractionated. That is, the peak appearing at the same retention time as the taxinin preparation (manufactured by Wako Pure Chemical Industries, Ltd.) was collected to obtain a fraction mainly containing taxinin. This was concentrated by an evaporator, re-extracted with methyl acetate, dried over anhydrous sodium sulfate, and dried by an evaporator to obtain 3.6 g of a colorless solid. This was recrystallized with a mixture of methylene chloride and ethyl acetate to give 3.1.
2 g of a colorless solid were obtained. The recovery of taxinine from the extracted oil was 75.5%. The purity by HPLC analysis was 99.4%. Melting point: 266-269 ° C., optical rotation [α] _D = + 137 ° (C = 0.4, CHCl ₃ , 23
° C), mass spectrum, UV spectrum, ¹ H-
The analysis value of the NMR spectrum (CDCl ₃ ) is the analysis value of the sample and the literature (Shunro Tachibana, Journal of the Mokuzai Gakkaishi, Vol. 40, 1).
009 (1994)), it was confirmed that the obtained crystals were taxinin.

Example 8 Purification of 10-deacetyltaxol When 7-xylosyltaxol was fractionated by a preparative liquid chromatograph, 10-deacetyltaxol was also fractionated. That is, a peak that appeared at the same retention time as that of the sample of 10-deacetyltaxol was collected to obtain a fraction mainly containing 10-deacetyltaxol. This was treated in the same manner as in Example 7 to obtain 81 mg of a colorless solid. This was recrystallized from ethanol / water to obtain 72 mg of colorless crystals (recovery rate: 65.5%). Melting point 194-19
6 ° C., optical rotation [α] _D = −3 ° (C = 0.4, pyridine, 23 ° C.). Mass spectrum, UV, ¹ H-
NMR (CDCl ₃ ) and other spectral data are described in the literature (J.
Nat. Prod., Vol. 44, 312 (1981)), confirming that the obtained crystal was 10-deacetyltaxol.

Example 9 Purification of Baccatin III At the time of fractionation of 7-xylosyltaxol, 0.51 g of a peak component which appeared at the same retention time as that of the specimen of baccatin III (SIGMA) was fractionated. . But H
According to the PLC analysis, other components are also contained, and the preparative liquid chromatography is performed under conditions that are more favorable for the separation of baccatin III, that is, eluent composition: acetonitrile / water / methanol = 20/60/20 (volume ratio). It was to be. The preparative liquid chromatograph was operated at a flow rate of 40 mL / min and a UV wavelength of 227 nm using a small Shimadzu LC-8A. The fraction of the peak having the same retention time as the baccatin III standard was collected. Concentration, extraction with methyl acetate and evaporation to dryness yielded 230 mg of a white solid. This was recrystallized from diethyl ether to give colorless crystals 216.
mg (77.1% recovery). This crystal is HP
LC analysis revealed a purity of 99.4%. The crystal has a melting point of 236 to 238 ° C. (decomposition) and an optical rotation [α] _D = −54.
° (C = 0.41, methanol, 23 ° C.), and the mass spectrum, UV, and ¹ H-NMR spectrum data were obtained from the analytical values of the standard and the literature (J. Org. Chem., Vol. 46, 1469 ( 19
81)). The obtained crystals were found to be baccatin III, because they were consistent with the analytical values described.

Comparative Example 1 Yew leaves were extracted with methanol and then with methyl acetate in the same manner as in Example 1, and 10 g of an alkali- and acid-treated oil (0.22% of 10-deacetylbaccatin III, baccatin III)
0.024%, 7-xylosyltaxol 0.23%,
Contains 0.023% of cephalomanine and 0.37% of taxinine. ) Was dissolved in 50 mL of methylene chloride-50 mL of toluene, and 10 g of mesoporous silica gel having a pore size of 20 mm was used.
(Manufactured by Toyoda Central Research Laboratory) and stirred at 200 rpm for 2 hours using a magnetic stirrer. After filtration, the silica gel after the adsorption treatment is extracted twice with 50 mL of methanol (filtration with stirring for 1 hour with methanol), and the collected methanol solution is concentrated. As a result, only 1.4 g of the obtained desorbed oil is obtained. Met. Analysis of the desorbed oil revealed that there was no taxane compound, and it was found that the taxane compound was hardly adsorbed on the silica gel. As a result, it was found that the pores were small at a pore diameter of 20 °, and the taxane compound was not allowed to enter.

Comparative Example 2 The same purification treatment as in Example 5 was carried out without adsorbing and desorbing 100 g of the same oil as used in Comparative Example 1. As a result, 39 g of the pretreated oil contained (10-deacetylbaccatin III 0.56%, baccatin III 0.06%, 7-
Xylosyltaxol 0.59%, cephalomannine 0.
059%, taxinine 0.94%). Many of the taxane compounds obtained in this example have concentrations of
Was lower than the concentration of the pretreated oil. 15 g of the pretreated oil obtained here was taken, and purified by a preparative liquid chromatograph under the same conditions as in Example 6. As a result, 10-deacetylbaccatin III
And baccatin III overlapped the peaks in the chromatogram and could not be separated.
In addition, taxol and cephalomanine could not be separated because their peaks partially overlapped. 10-deacetylbaccatin II
I and baccatin III can be separated by repurifying with different eluent compositions: acetonitrile / water / methanol = 20/60/20 (volume ratio), but taxol and cephalomannine can be separated by preparative liquid chromatography even if the eluate composition is changed. The separation proved to be difficult in the lithography.

[0081]

According to the purification method of the present invention, a taxane compound-containing component is separated from a taxane compound-containing mixture by a simple method of adsorption and elution, thereby simplifying the purification operation and further purifying the same. This makes it possible to purify the taxane compound while suppressing the loss of the taxane compound at the time, and has an effect that the taxane compound can be efficiently obtained. Further, according to the method of the present invention, cephalomannine can be easily obtained.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4C048 AA06 BB01 BC16 CC01 UU01 XX03 4H006 AA02 AB28 AB84 AD17 BA71 BB11 BB12 BB14 BB16 BB21 BD60

Claims (9)

[Claims] 1. A mixture containing at least one taxane compound selected from the group consisting of 10-deacetylbaccatin III, baccatin III, cephalomannine, 7-β-xylosyltaxol, 10-deacetyltaxol and taxinine. From that the pore size is 25-120 吸着
A method for purifying a taxane compound-containing component, wherein the component containing the taxane compound is adsorbed and eluted using the porous material described in (1). 2. The method according to claim 1, wherein the porous material is silica gel.
Purification method as described. 3. The purification method according to claim 2, wherein the silica gel is mesoporous silica gel or mesopore silica gel. 4. A mesoporous silica gel kanemite the general formula _{(1) C n H 2n +} 1 (CH 3) 3 N + Cl - (1) ( where, n is 12 to 18.) Alkyltrimethylammonium represented by The purification method according to claim 3, which is obtained by reacting ammonium chloride. 5. The purification method according to claim 1, wherein the mixture containing the taxane compound is an extract derived from a plant. 6. The method according to claim 5, wherein the plant is a plant of the Taxus family. 7. The purification method according to claim 1, wherein a mixed solution of a water-soluble organic solvent and water is used as a solvent for eluting the taxane compound-containing component adsorbed on the adsorbent. 8. A method for purifying a taxane compound, comprising a purification step of further purifying the taxane compound-containing component purified by the method according to claim 1 to isolate the taxane compound. 9. A mixture comprising cephalomannine comprising:
A method for purifying cephalomanine, comprising adsorbing cephalomanine using a porous material having a pore diameter of 25 to 120 ° as an adsorbent, and then eluting with a methanol aqueous solution having a methanol concentration of 30 to 40% by weight.