CN1274682C - Process for high efficient separating high pureness taxiol half sythesizing precursor - Google Patents
Process for high efficient separating high pureness taxiol half sythesizing precursor Download PDFInfo
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- CN1274682C CN1274682C CN 200410006973 CN200410006973A CN1274682C CN 1274682 C CN1274682 C CN 1274682C CN 200410006973 CN200410006973 CN 200410006973 CN 200410006973 A CN200410006973 A CN 200410006973A CN 1274682 C CN1274682 C CN 1274682C
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Abstract
The present invention discloses a method for separating and preparing a semisynthetic precursor of taxol with high purity in high efficiency. The mixture of acetonitrile and water is used as a mobile phase; firstly, a C8 column is adopted; then, a C18 column is adopted; extractives of yew leaves are carried out with two times of crude separation; afterwards, the target level separation after the crude separation is carried out is further separated and purified by high-speed countercurrent chromatography; finally, the refining is carried out by a recrystallization method, and 10-DAB of high purity is obtained. The method has the advantages of simple steps, high efficiency and easy repetition, and the purity of the separated and prepared 10-deacetylbaccatin III can reach more than 99%.
Description
Technical field
The present invention relates to the separation method of the semi-synthetic precursor of taxol.
Background technology
Taxol (Taxol) is Americanized scholar Wall and Wani isolated a kind of natural diterpene-kind compound from Pacific yew (yewtree Taxus brevifolia) the bark at first sixties.It is made up of four rings, has 11 chiral centres and a plurality of substituted radical in the molecule, also has the side chain of an alkamine on 13 hydroxyls, and its chemical formula as shown in Equation 1.
Formula 1
Because taxol is a kind of efficient cytotoxin, biological activity with anticancer, and the mechanism of action is different with general cancer therapy drug, the extremely concern in pharmaceutical chemistry field in recent years, and as a kind of cancer therapy drug use clinically evident in efficacy more and more widely.But taxol only depends on from bark of Ramulus et folium taxi cuspidatae and extracts at present, and not only content is lower, only about 0.01%, every extraction 1kg taxol will be cut down 1000～2000 trees, and growth of taxol is slower, resource-constrained, even if all natural resource is felled, also can only satisfy the short-term needs.Therefore, people obtain other approach of taxol for a long time always in research.Summary is got up, and following four kinds of approach is roughly arranged: 1. complete synthesis: the taxol complex structure, 9 chiral centres are arranged in the parent nucleus, and side chain has 2 chiral centres, therefore 2048 non-corresponding isomer should be arranged, and the synthetic of it is a kind of challenge to the organic chemist.Though through the complete synthesis final success that obtained of the effort in more than 20 years, and have very important academic values, because complex synthetic route, step is loaded down with trivial details, and therefore nearly 30 multisteps provide the medicine source unlikely with complete synthesis method at present.2. tissue culture: this method is a promising method.But generally speaking, tissue culture method also is in the research and development stage, realize that the industrial production key is to try every possible means to improve cell yield.3. fungi fermentation: finding taxol growing nonparasitically upon another plant in the fungi of bark of Ramulus et folium taxi cuspidatae, is the impressive progress of taxol research in recent years.With regard to present state of the art, from fungus culture medium, only can obtain the taxol of nanogram level, if will reach commercial production, must reach milligram level level.Therefore, some technical problems solution of still needing.4. semi-synthesis method: the semi-synthetic of taxol mainly is that the Taxan composition in the Chinese yew is carried out structure of modification, obtains the D51-7059 of taxol or biologically active.Comparatively successful promptly is with as shown in Equation 2 10-deacetylbaccatinIII (abbreviating " 10-DAB " again as) is that the taxol that carries out of precursor is semi-synthetic: at first with the C of 10-deacetylbaccatinIII
7The position hydroxyl protection, C
10The position glycoloylization; Again at C
13Introduce protected side chain on the hydroxyl of position; Protecting group and the C on the side chain removed in last hydrolysis
7Protecting group on the position and obtain taxol.
10-deacetylbaccatinIII content in the renewable branches and leaves of multiple Chinese yew is higher, has the basic parent nucleus of Taxan, is fit closely as semi-synthetic precursor.Generally believe that semisynthesis is hopeful to produce in batches in the near future most, thereby alleviate taxol resource anxiety, so that substitute existing plant extraction process the most at last.In addition, utilize semisynthesis, can also carry out chemically modified, make it have better preparation and biological activity taxol and derivative thereof.
As the semi-synthetic precursor of taxol, also extremely scientist's the concern of research of the separation method of relevant 10-deacetylbaccatinIII in recent years.After having reported suitable position as the Margraff R. of French Rhone-Poulenc Rorer etc. with the water treatment Japanese yew, the aqueous solution that obtains is used suitable organic solvent extraction again, selective crystallization obtains the 10-deacetylbaccatinIII of 75%-90%HPLC purity then, or this plant is extracted with methyl alcohol, with a certain proportion of methanol extract is handled then, obtain the 10-deacetylbaccatinIII of 75%-93%HPLC purity at last by selective crystallization, this method is comparatively simple, but the target product purity that obtains is limited.In addition, other report mostly adopts the column chromatography separation method, as silicagel column etc., makes with extra care means with preparation HPLC at last.And the overwhelming majority all is separate object with the taxol, 10-deacetylbaccatinIII is removing taxol in advance after further separate the byproduct that obtains, so the raw materials used Japanese yew bark that is mainly is some condition and be not suitable for from the Japanese yew branches and leaves directly separation and Extraction 10-deacetylbaccatinIII.
Summary of the invention
The purpose of this invention is to provide the method that a kind of high efficiency separation prepares the semi-synthetic precursor of high-purity taxol, can be used for directly separating preparation 10-deacetylbaccatinIII from the extract of Japanese yew branches and leaves.
For achieving the above object, the present invention takes following scheme: the mixture with acetonitrile and water is a moving phase, adopts C earlier
8Post, adopt C then
18Post carries out twice roughing out to the extract of Japanese yew branches and leaves; Adopt high speed adverse current chromatogram further to separate and purifying to the target fraction after the roughing out then; Adopt the method for recrystallization to make with extra care at last, obtain highly purified 10-DAB.
More particularly, please refer to Fig. 1, the present invention takes following scheme:
A. mix sample: with the extract dissolve with methanol of Japanese yew branches and leaves, mix sample, mix sample and volatilize solvent after evenly, mill evenly with Mierocrystalline cellulose;
B. use C
8Column packing is filled the chromatography column chromatography, and with the nitrogengas cylinder pressurization, volume percent is the acetonitrile solution wash-out of 30-50%, collects each cut with run tank;
The fraction that C. will contain 10-DAB merges, and after concentrating, mixes sample once more with the method for steps A;
D. use C
18Column packing is filled the chromatography column chromatography, and with the nitrogengas cylinder pressurization, volume percent is the acetonitrile solution wash-out of 20-30%, collects each cut with run tank;
E. after will containing the fraction merging, concentrating under reduced pressure, vacuum-drying of 10-DAB, separate further,, collect the target fraction according to UV-detector spectrum peak with high speed adverse current chromatogram;
In this step, the solvent system that preferred halohydrocarbon-Fatty Alcohol(C12-C14 and C12-C18)-water is formed carries out the high speed adverse current chromatogram separation; Wherein, halohydrocarbon can be chloroform, methylene dichloride or tetracol phenixin, most preferably is chloroform; Fatty Alcohol(C12-C14 and C12-C18) can be methyl alcohol, ethanol, Virahol, propyl carbinol, most preferably is methyl alcohol.
Can also contain normal hexane in the solvent system in case of necessity.
When carrying out the high speed adverse current chromatogram separation, type of elution is stationary phase on being mutually, is the forward type of elution of moving phase mutually down.
F. behind the target level lease making concentrate drying, can obtain highly purified 10-DAB monomer with acetonitrile or recrystallizing methanol.
In step B and D, the C of preferable particle size between 30-80 μ m
8Post and C
18Post.
Advantage of the present invention is: this method steps is simple, and the efficient height is easy to repetition.Efficient liquid phase chromatographic analysis shows that the 10-deacetylbaccatinIII purity that adopts this method to separate preparation can reach more than 99%.
Description of drawings
Fig. 1 is a process flow sheet of the present invention.
Fig. 2 is through C among the embodiment 1
8The HPLC figure of the 1# sample behind the post.
Fig. 3 is through C among the embodiment 1
18The HPLC figure of the 2# sample behind the post.
Fig. 4 is the paced work curve of 10-DAB standard reference material.
Fig. 5 is the HSCCC separation spectrogram that separates back gained 2# sample among the embodiment 2 through the secondary post.
Fig. 6 is for separating the HPLC analysis of spectra that contains 10-DAB part (3# sample) that obtains through HSCCC among the embodiment 2.
The 10-DAB crystalline HPLC analysis chart that Fig. 7 obtains for recrystallization of 3# sample among the embodiment 3.
Fig. 8 is the HPLC analysis chart of a recrystallization mother liquor of 3# sample among the embodiment 3.
The 10-DAB crystalline HPLC figure that Fig. 9 obtains for secondary recrystallization among the embodiment 3.
Embodiment
Embodiment 1: the secondary column chromatography for separation of Japanese yew branches and leaves alcohol extract
With the extract dissolve with methanol of Japanese yew branches and leaves, mix sample with Mierocrystalline cellulose, mix sample and volatilize solvent after evenly, mill evenly; Because alcohol extract study composition more complicated has adopted two step column separation process, with nitrogengas cylinder pressurization, the first step C
8Post and 45% (v/v) acetonitrile solution wash-out, second step was adopted C
18Post and 25% (v/v) acetonitrile solution wash-out is monitored sepn process with HPLC, collects the target fraction, concentrate drying, and 10-DAB content is wherein carried out quantitative analysis with reference substance.
The 40g study is through first C
8Behind the post, on average can contain the 10-DAB sample by about 10g; Merge four gained altogether about 40g sample (1# sample).Again through second C
18Post on average can contain 10-DAB sample (2# sample) by about 15g after separating.Fig. 2 and Fig. 3 are respectively the HPLC analysis of spectra of 1# sample and 2# sample, and Fig. 4 is the paced work curve of 10-DAB standard reference material.Through quantitative analysis, it is about 0.57% that No. 1 sample contains 10-DAB, and the 2# sample contains 10-DAB about 1.5%.
Embodiment 2: the high speed adverse current chromatogram purifying that contains the 10-DAB fraction after the column chromatography for separation
As shown in Figure 3, still quite complicated through the sample that twice column purification crossed, wherein 10-DAB content also has only about one of percentage.At this moment, can continue purifying with pillar and go down, but consider that its purification efficiency is lower, but C
18Behind the post in the sample 10-DAB content only before crossing post, improve about 2.5 times.Therefore, next adopt the high speed adverse current chromatogram purifying.
At this moment, the solvent for use system is normal hexane-chloroform-methanol-water (15: 35: 17: 8).Fig. 5 is the separation spectrogram of one of them 500mg sample.Fig. 6 has provided the HPLC analysis of spectra that contains 10-DAB part (3# sample) that the adverse current chromatogram purifying obtains.Through quantitative analysis, can on average obtain containing the purifying sample of 10-DAB about 24% through the HSCCC purifying.As can be seen, its purification efficiency is than high many of column chromatography.
Embodiment 3: the final recrystallization that contains the 10-DAB sample
Behind the HSCCC purifying, 10-DAB content is significantly improved.Find that through test the acetonitrile recrystallization can play surprising purification effect, the sample behind the HSCCC purifying can obtain highly purified 10-DAB through recrystallization once or twice.Fig. 7 and Fig. 8 are respectively the 10-DAB crystallization that the 3# sample obtains through recrystallization and the HPLC analysis chart of mother liquor.As can be seen, most chaff interferences have all been stayed in the mother liquor.Fig. 9 is the HPLC dimension analysis chart of the high purity 10-DAB that finally obtains through secondary crystal.Quantitative analysis shows that its purity can reach more than 99%.
In order to confirm that products therefrom is 10-DAB, above-mentioned crystalline powder has been carried out fast atom bombardment mass spectroscopy(FABMS) FAB-MS, the hydrogen spectrum
1HNMR, the carbon spectrum
13CNMR and KBr are infrared, and the IR spectrum is identified.
Mass spectrum identifies that the molecular weight of explanation products therefrom is 544, and is identical with the 10-DAB molecular weight.
Hydrogen spectrum qualification result shows δ (CDCl
3): 8.09 (2H, dd, J=1.8,8, OBz), 7.61 (1H, m, J=1.2,7.3, OBz), 7.48 (2H, t, J=7.6, OBz), 5.64 (1H, d, J=6.7,2-H), 5.25 (1H, s, 10-H), 4.98 (1H, dd, J=1.8,9.8,5-H), 4.89 (1H, t, J=7.3,13-H), 4.32 (1H, d, J=8,20-H), 4.28 (1H, m, 7-H), 4.17 (1H, d, J=8,20-H), 4.01 (1H, d, J=6.7,3-H), 2.62 (1H, m, 6-α H), 2.29 (3H, s, OAc), 2.27 (2H, m, 14-α H, 14-β H), 2.07 (3H, d, J=1.2,18-CH
3), 1.83 (1H, m, 6-β H), 1.74 (3H, s, 19-CH
3), 1.09 (6H, s, 16-CH
3, 17-CH
3).The 10-DAB data basically identical of these data and bibliographical information.δ is that 2.0 climax is the acetonitrile CH of double team in crystallization
3The last proton of CN.
13The CNMR analytical results shows, except δ is that 77.0 and 39.5 peak is respectively deuterated reagent CDCl
3Outside C among the DMSO, δ is that the peak of 211ppm is a ketone group unique in the molecule, and two peaks between 160～170ppm are two ester groups, and nine unsaturated C are represented at seven peaks between 150～130ppm, two two key C wherein, and six phenyl ring C also have a CH
3The three key C of CN.Other 18 is saturated C.Totally two nineteen C conform to 10-DAB.
Infrared spectrum shows, at 3444cm
-1And 1706cm
-1There is strong absorption at two places.
Above structure appraising datum confirms that all resulting product really is 10-deacetylbaccatinIII.
Claims (9)
1. the separation method of the semi-synthetic precursor 10-DAB of taxol, it is characterized in that: the mixture with acetonitrile and water is a moving phase, adopts C earlier
8Post, adopt C then
18Post carries out twice roughing out to the extract of Japanese yew branches and leaves; Adopt high speed adverse current chromatogram further to separate and purifying to the target fraction after the roughing out then; Adopt the method for recrystallization to make with extra care at last, obtain highly purified 10-DAB.
2. the separation method of the semi-synthetic precursor 10-DAB of taxol according to claim 1 is characterized in that, is made up of following steps:
A. mix sample: with the extract dissolve with methanol of Japanese yew branches and leaves, mix sample, mix sample and volatilize solvent after evenly, mill evenly with Mierocrystalline cellulose;
B. use C
8Column packing is filled the chromatography column chromatography, and with the nitrogengas cylinder pressurization, volume percent is the acetonitrile solution wash-out of 30-50%, collects each cut with run tank;
The fraction that C. will contain 10-DAB merges, and after concentrating, mixes sample once more with the method for steps A;
D. use C
18Column packing is filled the chromatography column chromatography, and with the nitrogengas cylinder pressurization, volume percent is the acetonitrile solution wash-out of 30-50%, collects each cut with run tank;
E. after will containing the fraction merging, concentrating under reduced pressure, vacuum-drying of 10-DAB, further separate,, collect the target fraction according to UV-detector spectrum peak with high speed adverse current chromatogram;
F. behind the target level lease making concentrate drying, make with extra care, can obtain highly purified 10-DAB monomer with the method for recrystallization.
3. the separation method of the semi-synthetic precursor 10-DAB of taxol according to claim 2 is characterized in that: described C
8Post and C
18The particle diameter of post is between 30-80 μ m.
4. the separation method of the semi-synthetic precursor 10-DAB of taxol according to claim 2 is characterized in that: in the described step e, the solvent system of forming with halohydrocarbon-Fatty Alcohol(C12-C14 and C12-C18)-water carries out the high speed adverse current chromatogram separation; Wherein, halohydrocarbon is chloroform, methylene dichloride or tetracol phenixin; Fatty Alcohol(C12-C14 and C12-C18) is methyl alcohol, ethanol, Virahol or propyl carbinol.
5. the separation method of the semi-synthetic precursor 10-DAB of taxol according to claim 4 is characterized in that: in the described step e, the solvent system of forming with chloroform-methanol-water carries out the high speed adverse current chromatogram separation.
6. according to the separation method of claim 4 or the semi-synthetic precursor 10-DAB of 5 described taxols, it is characterized in that: also contain normal hexane in the solvent system.
7. the separation method of the semi-synthetic precursor 10-DAB of taxol according to claim 2 is characterized in that: in the described step e, when carrying out the high speed adverse current chromatogram separation, its type of elution is stationary phase on being mutually, is the forward type of elution of moving phase mutually down.
8. the separation method of the semi-synthetic precursor 10-DAB of taxol according to claim 2 is characterized in that: in the described step F, adopt acetonitrile or methyl alcohol to carry out recrystallization.
9. the separation method of the semi-synthetic precursor 10-DAB of taxol according to claim 2 is characterized in that: in the described step F, adopt acetonitrile to carry out recrystallization.
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