CN1791590A - Purification of taxanes and taxane mixtures using polyethyleneimine-bonded resins - Google Patents

Purification of taxanes and taxane mixtures using polyethyleneimine-bonded resins Download PDF

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CN1791590A
CN1791590A CN 200480013498 CN200480013498A CN1791590A CN 1791590 A CN1791590 A CN 1791590A CN 200480013498 CN200480013498 CN 200480013498 CN 200480013498 A CN200480013498 A CN 200480013498A CN 1791590 A CN1791590 A CN 1791590A
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taxol
taxols
mixture
described method
extract
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CN100448859C (en
Inventor
詹姆斯·H·约翰逊
T·G·桑班丹
巴里·J·汉德
克里斯多佛·D·豪
罗兰·R·弗兰克斯
布赖恩·A·布赫
约翰·S·尤哈姆
雷克斯·T·加拉格尔
梅尔·A·普兰特
爱德华·M·德西蒙三世
东·S·杨
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Fiat Asia Ltd
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Natural Pharmaceuticals Inc
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Abstract

This invention is directed to a method for isolating and /or purifying one or more taxanes from materials comprising taxanes. In one embodiment, the method comprises the steps of: (a) treating a taxane containing material with an amino containing material attached to a solid matrix, such as a polyethyleneimine-bonded silica chromatographic resin; (b) eluting the desired taxane compound from the resin; and (c) recovering the eluted taxane compound.

Description

Use the method for polyethyleneimine-bonded resin purification of paclitaxel and taxol mixture
Technical field
The present invention relates to a kind of from the material that contains taxol (taxane), the separation or the method for one or more taxol compounds of emanating and composition or the mixture that obtains by this method.The invention still further relates to a kind of purification and comprise the method for the bio-extract of one or more taxols.In one embodiment, this method comprises use attached to the material that contains one or more amino on the matrix (for example solid substrate), and described material is including, but not limited to polyethyleneimine-bonded silicon-dioxide chromatography resin (PBS resin).These resins help high yield and high purity ground purification of paclitaxel.In one embodiment, described chromatography resin comprises non-functionalized and/or non-deutero-polymine (" the PEI ") polymkeric substance on the silicon-dioxide that is bonded to various particle diameters or aperture.In another embodiment, polymine is methylated and forms bonding the silicon-dioxide of diethylamino methyl (" DEAM ").In an alternative embodiment of the present invention, DEAM and PEI can be used in the normal-phase chromatography under the acid pH.
Background technology
The present invention relates to use the amino material that contains on solid substrate of PBS resin even load for example from the material that contains one or more taxols, to emanate or isolation of taxol A (paclitaxel) or other taxol.The present invention also relates to purify and contain the bio-extract of taxol.
As everyone knows, for example various taxols such as taxol A can use chromatographic technique to purify.In the document relevant, reported that bondings such as silicon-dioxide, aluminum oxide, for example C18 and C8 for example have the silica resin and the polystyrene divinylbenzene resin isochromatic spectrum medium of alkyl with this purpose.But because the characteristic of method of purification itself, these media all have limitation.
Have been found that with being used for emanating and compare, attached to containing amino material (for example PBS resin) more excellent selectivity and resolving power can be provided on the matrix with other common chromatography resin of purification of paclitaxel.Therefore, in taxol purification field, can utilize attached to the amino material (for example PBS resin) that contains on the matrix to come more effectively to separate than other resin.For example, according to document description, the separation of taxol A and Cephalomannine (cephalommanine, taxol B) is difficult.As described herein, use one or more PBS resins can promote the separation of these compounds greatly.In addition, compare with reversed-phase resins such as for example C18, C8 and polystyrene divinylbenzenes, because can be with an organic solvent when using the PBS resin, thus can this resin of easier filling, and the loadings of sample is higher.
The PBS resin mainly is used in the ion-exchange chromatography purposes.Taxol seldom contains for example ionogenic group such as carboxylic acid or amino, and therefore interactional mode is not traditional ion-exchange.Surprisingly, when segregation or purification of paclitaxel, use the PBS resin that excellent selectivity and resolving power can be provided.In the present invention, when using a spot of acid modification agent or salt in moving phase, the PBS resin will play a role best.
In addition, we find that the amino of PEI polymkeric substance can be through alkylation, arylation or acidylate and functionalized.We find that also the preparation that can use the PBS resin to carry out taxol compound separates with analytical.The separation of preparation property can by intermittently, semicontinuous or continuous mode carries out.Semicontinuous mode can be simulation moving-bed (SMB) stratographic form.
Summary of the invention
The present invention relates to use and adhere to (for example bonding) amino material that contains on solid substrate and from the material that contains one or more taxols, emanate or isolation of taxol A (paclitaxel) or other taxol, described attached to containing amino material including, but not limited to emanating or the polymine matrix of isolation of taxol on the solid substrate, for example be bonded to the polymine on the silicon-dioxide.Other is suitable for containing amino material and can comprise United States Patent (USP) the 5th, 085 on the matrix (for example solid substrate), and No. 779 and the 5th, 092, those disclosed material in No. 992.This paper introduces this two pieces of patents by reference in full.
In an alternative embodiment, the present invention is intended to use the PEIS resin to assist to separate from the composition that contains taxol or purify and comprises the various taxols of taxol A.It is believed that compared with prior art, the purposes that is used for these resins of this purpose is novel and excellent.Preferred resin comprises PEI and DEAM.
In an alternative embodiment, the present invention aims to provide a kind of method of emanate one or more taxols and analogue thereof from the taxol mixture.The taxol mixture can comprise bio-extract, and perhaps the taxol mixture can be obtained by semi-synthetic or total synthesis method.Described isolation process may further comprise the steps:
(a) use attached to amino material (for example PBS resin) the processing taxol mixture that contains on the matrix;
(b) described one or more taxols of wash-out and analogue thereof from the chromatography resin; With
(c) described one or more taxols and its analogue in the fraction of the one or more elutants of recovery.
In another alternative embodiment, the present invention aim to provide a kind of from the material that contains taxol purification of paclitaxel and/or increase contain the method for the taxol concentration in the material of taxol, Ramulus et folium taxi cuspidatae (Taxus) extract is for example arranged the described material that contains taxol or by taxol mixture semi-synthetic or that total synthesis method obtains, the described material that contains taxol comes from the vegetable material in the one group of plant that is selected from Japanese yew by name (Yew).Said method comprising the steps of:
(a) use attached to the material that amino material (for example PBS resin) processing comprises taxol and its natural analog that contains on the matrix;
(b) wash-out taxol and analogue thereof from the chromatography resin; With
(c) taxol and the analogue thereof in the fraction of the one or more elutants of recovery.
Can further understand the present invention and advantage thereof with reference to following detailed description and accompanying drawing.
Description of drawings
Fig. 1 represents to use the exemplary chemical reaction synoptic diagram of chromatography resin of the present invention.
Fig. 2 represents to use the exemplary chemical reaction synoptic diagram of chromatography resin of the present invention.
Fig. 3 represents to use PEI resin isolation taxol A of the present invention and taxol B, C and other impurity.
Fig. 4 represents to use DEAM resin of the present invention with semi-synthetic taxol A and other taxol separation of by-products.
Fig. 5 represents the taxol molecule of indefiniteness.
Fig. 6 represents the exemplary compound (" elementary taxol ") of indefiniteness.
The exemplary taxol molecule of Fig. 7～9 expression indefinitenesses.
Figure 10 represents the comparison of the retention time of taxol standard substance on various media.
Embodiment
" alkoxyl group " used herein is meant straight chain, side chain or the cyclic saturated hydrocarbon that is connected on the Sauerstoffatom.Preferably, alkoxyl group has 1～6 carbon atom.Alkoxyl group also can refer to the alkoxyl group that replaces, and this alkoxyl group can comprise following substituting group: for example alkanoyloxy, thiazolinyl, alkyl, alkyl silyl, alkyl sulphonyl, alkyl sulphur oxygen base (alkylsulfoxy), alkylthio; Alkynyl; For example an alkylamino, dialkyl amido and amino such as an arylamino, ammonia diaryl base; Amide group, aryl, arylalkyl, carboxyl, carboxyl alkoxyl group, formamido-, carboxylic acid group, haloalkyl, halogen, hydroxyl, itrile group, nitro, phosphate, siloxy-, sulfate, sulfoamido, sulfonyloxy; And their combination.Wherein, the preferred example of alkoxyl group comprises methoxyl group, oxyethyl group, propoxy-, ring propoxy-, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert.-butoxy, cyclobutoxy group, pentyloxy, isopentyloxy, neopentyl oxygen, cyclopentyloxy, hexyloxy and cyclohexyloxy.
" alkyl " used herein is meant straight chain, side chain or cyclic saturated hydrocarbon.Preferably, alkyl has 1～6 carbon atom.Alkyl also can refer to the alkyl that replaces, and it can comprise following substituting group: for example alkanoyloxy, thiazolinyl, alkyl, alkyl silyl, alkyl sulphonyl, alkyl sulphur oxygen base, alkylthio; Alkynyl; For example an alkylamino, dialkyl amido and amino such as an arylamino, ammonia diaryl base; Amide group, aryl, arylalkyl, carboxyl, carboxyl alkoxyl group, formamido-, carboxylic acid group, haloalkyl, halogen, hydroxyl, itrile group, nitro, phosphate, siloxy-, sulfate, sulfoamido, sulfonyloxy; And their combination.Preferred substituted is amino, the group that contains carboxylic acid, haloalkyl, halogen, hydroxyl, itrile group, nitro and sulfate groups such as alkoxyl group, for example dialkyl amido, ammonia diaryl base.The example of preferred alkyl is including, but not limited to methyl, ethyl, propyl group, sec.-propyl, cyclopropyl, normal-butyl, isobutyl-, sec-butyl, the tertiary butyl, cyclobutyl, amyl group, 1-ethyl propyl, cyclopentyl, hexyl and cyclohexyl.
" aryl " used herein is meant phenyl or naphthyl replacement or unsubstituted.Substituent example on the aryl is including, but not limited to alkanoyloxy, thiazolinyl, alkoxyl group, alkyl silyl, alkyl sulphonyl, alkyl sulphur oxygen base, alkylthio; Alkynyl; For example an alkylamino, dialkyl amido and amino such as an arylamino, ammonia diaryl base; Amide group, aryl, arylalkyl, carboxyl, carboxyl alkoxyl group, carboxy and amide groups, carboxylic acid group, haloalkyl, halogen, hydroxyl, itrile group, nitro, phosphate, siloxy-, sulfate, sulfoamido, sulfonyloxy; And their combination.Preferred substituted is amino, the group that contains carboxylic acid, haloalkyl, halogen, hydroxyl, itrile group, nitro and sulfonic acid groups such as alkoxyl group, alkyl, for example dialkyl amido and ammonia diaryl base.
" arylalkyl " used herein is meant the alkyl that is connected on the aryl.The example of arylalkyl is a phenmethyl.
" alkalescence crust card fourth III (basic baccatin III) structure " used herein is meant the compound with structural formula shown in Figure 6, wherein R 1, R 2, R 4, R 7, R 10And R 13Be hydrogen, alkyl, acyl group, aryl, arylalkyl, vinyl, ether, ester group, glycosyl, oxygen base or hydroxy-protective group independently of one another.Crust card fourth III with structural formula shown in Figure 7 is also included within the alkalescence crust with the 10-deacetylation crust card fourth III with structural formula shown in Figure 8 and blocks in the definition of fourth III, and Ac is ethanoyl (CH in Fig. 8 3C (O)-), Bz is benzoyl (PhC (O)-or C 6H 5C (O)-).
" ester group " used herein be meant have ester functional group promptly-straight chain, side chain or the cyclic substituents of C (O)-OR.The example of ester group comprises acyl groups such as the ethanoyl that for example is bonded on the hydroxyl and benzoyl.
" ether " used herein be meant have ether functional group promptly-straight chain, side chain or the cyclic substituents of C-O-C-.The example of ether is including, but not limited to HOCH 2CH 2OC (CH 2OH) H-.
" glycosyl " used herein or " glycosyl " are meant any in the multiple sugar derivatives, and this sugar derivatives contains the non-glycosyl group that is bonded on oxygen or the nitrogen-atoms, and generates for example sugar such as glucose when hydrolysis.The example of preferred glycosyl is an xylosyl.
" halogen " used herein is meant fluorine, chlorine, bromine and/or iodine.
" heterocyclic group " used herein is to contain at least one for example saturated, unsaturated or aromatic ring-shaped compound of non-carbon atoms such as oxygen, nitrogen or sulphur in the finger ring.The example of heterocyclic group comprises for example furyl, morpholinyl, piperidino-(1-position only) (piperadino), piperazinyl, N methyl piperazine base, pyrrolinyl (pyrrollyl), pyridyl and thienyl such as 2-furyl.
" oxo base " used herein is meant the substituting group by the glycosyl oxidization deriving, and described glycosyl is for the xyloside described in No. the 5th, 356,928, the United States Patent (USP) for example etc.
" taxol or taxol molecule " used herein blocks the molecule of fourth III structure including, but not limited to containing the alkalescence crust, and wherein (2R, 3S)-C 6H 5CH (Rx) CH (OH) C (O)-group forms ester with hydroxyl on the C-13 position that is positioned at alkalescence crust card fourth III structure.The group that Rx represents can be that amino, amino salt (for example ammonium salt), the amino of protecting with the amido protecting group maybe can be transformed into amino substituting group.Alkalescence crust card fourth III structure and (2R, 3S)-C 6H 5Various isomer, homologue and the analogue of CH (Rx) CH (OH) C (O)-group are also included within the definition of taxol molecule.10-deacetylation crust card fourth III structure is also thought in the scope of taxol molecule.Be included in material in the definition of taxol or taxol molecule including, but not limited to for example elementary taxols such as taxol A, taxol B (Cephalomannine), taxol C, taxol D, taxol E, taxol F and taxol G.In addition, the definition of taxol or taxol molecule comprises Docetaxel (docetaxel) (TAXOTERE ).(for example seeing Fig. 5～6).
" vinyl " used herein is meant the straight or branched substituting group with carbon-to-carbon double bond.The example of vinyl is including, but not limited to 1-methyl isophthalic acid-propenyl (CH 3CH=C (CH 3)-) and 2-methyl isophthalic acid-propenyl ((CH 3) 2C=CH-).
Japanese yew is the general name of multiple Ramulus et folium taxi cuspidatae seeds; Taxus is the common genus in Ramulus et folium taxi cuspidatae (Taxaceae) section.Since 1962, taxol the earliest from the bark of the yewtree (Taxus brevifolia) that picks up from the State of Washington segregation come out, in 1971, report that two other Ramulus et folium taxi cuspidatae seeds are also to contain taxol in european yew (Taxus baccata) and the taxus chinensis in northeast (Taxuscuspidate) subsequently.Through further investigation, also report in other a large amount of Ramulus et folium taxi cuspidatae kinds and the cultivated variety and also contain taxol.These seeds are including, but not limited to Mexico red bean China fir (Taxus globosa), Florida Ramulus et folium taxi cuspidatae (Taxus floridana), T. canadensis (Taxuscanadensis), Xizang Taxus chinensis (Taxus wallichiana), taxusyunnanensis (Taxusyunnanensis), China Ramulus et folium taxi cuspidatae (Taxus chinensis), and a large amount of ornamental cross-fertilize seed, for example Taxus x media (Taxus media) cultivated variety and Ka Bi tower taxus chinensis in northeast (T.cuspidata " Capitata "), the Taxus x media cultivated variety has for example Deng Sifumi Taxus x media (T.media " Densiformis "), west this Taxus x media (T.media " Hichsii "), Brownie Taxus x media (T.media " Brownii "), general dark green Taxus x media (T.media " Dark Green Spreader "), blue handsome Taxus x media (T.media " Runyan "), Sarasvati Taxus x media (T.media " Wardii ") and Dao Tuoni Taxus x media (T.media " Tautonii ") etc.In the present invention, can be from including, but not limited to any Ramulus et folium taxi cuspidatae seeds of above-mentioned kind and cultivated variety, obtaining extract or semi-synthetic reaction mixture.Pointed out to be used for other Ramulus et folium taxi cuspidatae seeds: Chadwick of the present invention in the following document, L.C. and Keen, R.A., in May, 1986, " A study of the Genus Taxus " (research of Taxus), Res.Bull. (Research Bulletin), 1086, Ohio Agricultural Research and Development Center (Ohio farming research and centre of development); Appendino, G., nineteen ninety-five, " The Phytochemistry of theYew Tree " (vegetable chemistry of yew tree): Phytochemistry, Natural Products Reports (vegetable chemistry, natural products report) 12 (4): 349-360; Convention On InternationalTrade in Endangered Species of Wild Fauna and Flora:Eleventh mooting ofthe Plants Committee (endangered wild fauna and flora international trade pact: the 11st meeting of the plant council), LangKawi (Malaysia), 3-7 day September calendar year 2001, Document PCllDOC.22-p.1, the U.S.; And Greer, Schutzki, R.E., Fernandez, A. and Hancock, T.F., 1993.10 the moon/December, " Electrophoretic Characterization of Taxus Cultivars " (electrophoresis of Yew planting mutation characterizes): HortTechnology, 3 (4): 430-433.These reference are here all introduced by reference in full.
Find surprisingly now, adopt the positive liquid-phase chromatographic column of filling the PBS resin, can emanate and the purification of paclitaxel compound from the Ramulus et folium taxi cuspidatae seeds with high yield, this taxol compound comprises taxol and paclitaxel analogs and its homologue.Use described method, can be from the natural biological extract with by a large amount of paclitaxel analogs of emanating out semi-synthetic or the material that total synthesis method obtains.
Raw material of the present invention can be the vegetable material that is selected from the class plant that is commonly referred to yew tree.Only plant is the Ramulus et folium taxi cuspidatae seeds in such plant.In the Ramulus et folium taxi cuspidatae seeds, preferred especially Taxus x media cultivated variety.For example, preferred cultivated variety is including, but not limited to this Taxus x media of west or general dark green Taxus x media.Although using some part of yew tree in the present invention is easily, can be from whole strain plant or for example extract taxol and natural analog thereof single part such as timber, trunk, tree root, leaf (needle), seed or their arbitrary combination.The material that extracts can be fresh or exsiccant.Preferred bark or the needle of using.In addition, use method of the present invention can or use purification of paclitaxel the culture supernatants that the isolated culture technology obtains from the vegetable cell that grows up to.In addition, described method is applicable to from the mixture of the mixture handled by chromatographic technique or these technical finesses of no use and separates and purification of paclitaxel.This method also is applicable to separation and the purification by semi-synthetic or the taxol that total synthesis method obtains.
In one aspect of the invention, use separates from the mixture that contains taxol or one or more taxol compounds of emanating attached to the amino material that contains on the matrix (for example silicon-dioxide).Separated or the material of purifying of this class is including, but not limited to taxol A, B, C, D, E, F, G, Docetaxel and Nuo Na taxol (Nonataxel).This class is attached to containing amino examples of material including, but not limited to the PBS resin on the matrix.These resins are used in the ion-exchange purposes usually, therefore can not be used to separate the neutral molecules such as taxol that for example generally do not contain ionogen.By using for example PBS resin, selectivity and resolving power when having improved the isolation of taxol mixture surprisingly.For example, reversed-phase resins such as PBS resin ratio such as C18, C8 and polystyrene dialkylene benzene etc. are easier is loaded into higher level.Also can advantageously use for example PBS resin with organic solvent.
The PBS resin can be obtained by the polyethyleneimine polymers on the silicon-dioxide that is bonded to various apertures and/or particle diameter.This silicon-dioxide has the mean pore size in 60～300 scopes.Preferably, silicon-dioxide has the mean pore size of about 100～300 , more preferably from about 120 .In addition, silicon-dioxide has about 0.25 median size to about 500 micrometer ranges.Preferably, silicon-dioxide has about 10～120 microns median size, and more preferably from about 20～60 microns, most preferably from about 40 microns.
Suitable PBS resin of the present invention can comprise DEAM.This resin can be bought from J.T.Baker, CAS number of registration product code: 7317,7471,7472,7473, and name is called " BAKERBOND  DEAM Chromatography Packing ".Another kind of suitable PBS resin can comprise PEI.This resin also can be bought from J.T.Baker, CAS number of registration: 126850-07-5, and product code: 7134,7180,7264,7368,7476 and 8179, name is called " Polyethyleneimine Bonded Silica Gel ".
In an alternative embodiment of the present invention, can be non-derivative, for example PEI attached to the material (for example PBS resin) that contains amino on the matrix.Chromatography resin of the present invention can be including, but not limited to the PBS resin of derivatize, in this PBS resin, and the primary amino of polyethyleneimine: amine moiety and/or secondary amino group and reactive partial reaction.PBS resin of the present invention can be used following general formula:
The general structure of PBS resin
Figure A20048001349800151
Silica sphere
PEI：R＝H
DEAM：R＝CH 3
Said structure only is for purpose illustrative, indefiniteness.The structure of PBS resin can have many other branch's forms, or comprises one or more attached to the position on the silica particles.And the R group can be including, but not limited to H-, methyl, acyl group, alkyl, aryl, arylalkyl, alkylsulfonyl or their any combination.
The amino of PEI polymkeric substance can be functionalized by alkylation, arylation, acylations or other method.Make aminofunctional will make resin have selectivity, resolving power or other performance that needs of increase.For example, DEAM is the object lesson of functionalized PEI.
Can use resin of the present invention to emanate or purification of paclitaxel from the taxol mixture, described taxol mixture is including, but not limited to bio-extracts such as for example Ramulus et folium taxi cuspidatae Extracts.In an alternative embodiment, can use the present invention from the taxol mixture of producing by solvent distribution, centrifugation, filtration, precipitation or their arbitrary combination (biological example extract), to emanate or purification of paclitaxel.Can use resin of the present invention to emanate and/or purification of paclitaxel from the mixture that contains natural deutero-taxol, wherein taxol is not formed by synthetic or semisynthesis yet.In addition, can use resin of the present invention from the mixture that comprises the taxol that obtains by semi-synthetic or complete synthesis process, to emanate or purification of paclitaxel.
As everyone knows, for example various forms of chromatograms such as silicon-dioxide, aluminum oxide, C8, C18, polystyrene divinylbenzene can be used for purification of paclitaxel A and other taxol from Ramulus et folium taxi cuspidatae Extract.Can with other method that skilled in the art will recognize that for example solvent distributes and crystallization as chromatographic additional.Can be used in combination other technology of this class and PBS resin, thereby make that purification of paclitaxel A obtains simplifying with other taxol from Ramulus et folium taxi cuspidatae Extract.For example, we find that the PBS resin is effective especially in that taxol A is separated with taxol B, N-methyl taxol, taxol laurate etc.Also can use other taxol that exists in taxol that the PBS resin makes correlation types such as for example elementary taxol (see figure 6) and the Ramulus et folium taxi cuspidatae Extract to separate.Can use the PBS resin that taxol is separated with non-target component, thereby increase the taxol concentration in the mixture.
In an alternative embodiment, raw material can comprise the vegetable material that is selected from the class plant that is commonly referred to Japanese yew.Only plant is the Ramulus et folium taxi cuspidatae seeds in such plant.The raw material that uses among the present invention can including, but not limited to: (1) is by any raw material that comprises one or more taxols of the method except that semi-synthetic or total synthesis method preparation; (2) any raw material that comprises one or more taxols for preparing by chromatography; (3) not any raw material that comprises one or more taxols for preparing by chromatography; (4) by the prepared any raw material that comprises one or more taxols of solvent distribution, centrifugation, filtration, precipitation or its any combination; (5) from any raw material that comprises one or more taxols of Chinese yew; Or (6) from any raw material that comprises one or more taxols of one or more Chinese yews, and wherein taxol is not only derived from yewtree.Raw material can comprise by the prepared any raw material that comprises one or more taxols of the arbitrary combination of the parameter that lists above.The method that preparation contains the raw material (the semi-synthetic or complete synthesis reaction mixture of biological example extract) of taxol is known in this area.
In another alternative embodiment, the present invention aims to provide a kind of method of one or more taxols of emanating from the mixture that contains taxol, and this method may further comprise the steps: (a) with the described mixture of PBS plastic resin treatment; Wherein said one or more taxols derive from one or more Chinese yews, and wherein said one or more taxols not only derive from yewtree; (b) with described one or more taxols of elutriant wash-out from the PBS resin; (c) one or more taxols of recovery institute wash-out.
In another alternative embodiment, the present invention aims to provide a kind of method of one or more taxols of emanating from the mixture that contains taxol, said method comprising the steps of: (a) with the described mixture of PBS plastic resin treatment; Wherein said mixture comprises and is lower than 25 weight % or greater than the elementary taxol of 40 weight %; (b) described one or more taxols of wash-out from the PBS resin; (c) one or more taxols of recovery institute wash-out.
In another embodiment, the present invention aims to provide a kind of method of one or more taxols of emanating, and this method may further comprise the steps: the mixture that (a) contains taxol with the PBS plastic resin treatment; Wherein said mixture comprises the elementary taxol of about 25 weight %～40 weight %; Wherein said one or more taxols not only derive from yewtree; (b) described one or more taxols of wash-out from the PBS resin; (c) one or more taxols of recovery institute wash-out.
In an alternative embodiment of the present invention, raw material can comprise and is lower than about 25 weight % or greater than the elementary taxol of about 40 weight %, described elementary taxol is including, but not limited to taxol A, B, C, D, E, F or G.All the other materials in the extract can comprise other compound, and these other compounds are including, but not limited to impurity.Other appropriate level of elementary taxol can comprise about 0.5%～1%, 1%～10%, 10%～15%, 15%～20%, 20%～25%, 25%～30%, 30%～35% or 35%～40% in the raw material.Other content also can comprise about 40%～50%, 50%～60%, 60%～70%, 70%～80%, 80%～90% or 90%～100%.
In another alternative embodiment, bio-extract derives from Chinese yew.In another alternative embodiment, bio-extract derives from any Chinese yew, but does not comprise yewtree.In another alternative embodiment, bio-extract derives from any Chinese yew, but do not comprise yewtree, and comprise the elementary taxol of about 25 weight % to about 40 weight %, this elementary taxol is including, but not limited to taxol A, B, C, D, E, F or G.
In another embodiment, the present invention aims to provide a kind of method of one or more taxol compounds of purifying from bio-extract, and wherein bio-extract derives from one or more Chinese yews.In one embodiment, bio-extract is from one or more Chinese yews, but do not comprise yewtree.In another embodiment, bio-extract comprises and is lower than 25 weight % or greater than the elementary taxol of 40 weight %.In another embodiment, bio-extract comprises about 25% to about 40% elementary taxol, and wherein bio-extract derives from one or more Chinese yews, but does not comprise yewtree.
In addition, in an alternative embodiment, bio-extract can comprise isopropylcarbinol, the content of this isopropylcarbinol is lower than about 50%, 40%, 30% or 20%, preferably be lower than 10%,, most preferably be lower than 3%, 2%, 1%, 0.5% or 0.25% more preferably less than 5%.
Method of the present invention can make the purity of bio-extract increase about 10%～20%, 20%～30%, 30%～40%, 40%～50%, 50%～60%, 60%～70%, 70%～80%, 80%～90%, 90%～100%, 100%～110%, 110%～120%, 120%～130%, 130%～140%, 140%～150%, 150%～200%, 200%～250%, 250%～300%, 300%～350%, 350%～400%, 400%～450%, 450%～500%, 500%～550%, 550%～600%, 600%～650%, 650%～700%, 700%～750%, 750%～800%, 800%～850%, 850%～900%, 900%～950%, 950%～1000%.Term used herein " purity " is meant the weight percent that is present in one or more taxols in exsiccant raw material or the bio-extract.
Can use resin of the present invention one or more taxols of purifying from the taxol mixture, described taxol mixture is obtained by semi-synthetic or total synthesis method whole or in part.In an alternative embodiment, can use resin of the present invention from crude product mixture, purify semi-synthetic taxol A or other semi-synthetic taxol.Because most of building-up reactionss all produce by product or have unreacted raw material, they are structurally near target product, so the method for these products of purifying is very important in many cases.For example, have been found that the PBS resin can improve and promote to be made it and many related impuritieses and the compound separation that structurally is associated for example shown in Figure 10 by the purification of the semi-synthetic taxol A of method production described herein.
In an alternative embodiment, the present invention relates to one or more taxol compounds of from material, purifying by semi-synthetic or total synthesis method preparation.Fig. 1 and 2 has shown a kind of exemplary nonrestrictive semisynthesis that adopts resin of the present invention.The name that these methods are described in a PCT/US03/10556 number application that is called " Conversion of Taxane Molecules " and submission on August 4th, 2003 is called in the PCT/US03/24666 number application of " Methods and Compositions for Converting TaxaneAmides to Paclitaxel or Other Taxanes ".Above-mentioned each application all is incorporated herein by reference in full.
In an alternative embodiment, these materials comprise the elementary taxol of C-2 ' benzoyl that is lower than about 10 weight %, preferably are lower than approximately 5%, more preferably less than about 3%, most preferably are lower than about 1%.In another alternative embodiment, the material that contains taxol comprises and is lower than about 9%, 8%, 7%, 6%, 4%, 2%, 1%, 0.5%, 0.1% or 0.01%.Taxol impurity is including but not limited to the C-2 ' benzoic ether of taxol A, B, C, D, E, F or G.Taxol impurity also comprises the C-2 ' benzoic ether that is lower than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.5% taxol B, C, D, E, F or G.Term weight percent used herein is meant the per-cent of one or more compounds in these materials that are present in the drying solid form.
In an alternative embodiment, the present invention aims to provide a kind of from the method by one or more taxols of segregation semi-synthetic or the material that comprises taxol compound that total synthesis method obtains, and described isolation process may further comprise the steps: (a) with the described material of PBS plastic resin treatment; The molecule that wherein is used as reactant in semi-synthetic or total synthesis method not only derives from yewtree; (b) described one or more taxols of wash-out from the PBS resin; (c) one or more taxols of recovery institute wash-out.
In another embodiment, the present invention aims to provide a kind of from the method by one or more taxols of segregation semi-synthetic or the material that comprises taxol compound that total synthesis method obtains, and described isolation process may further comprise the steps: (a) with the described material of PBS plastic resin treatment; Wherein said material comprises the 2 ' benzoic ether that total amount is lower than taxol A, B, C, D, E, F or the G of about 8 weight %～3 weight %; (b) described one or more taxols of wash-out; (c) one or more taxols of recovery institute wash-out.In another embodiment, the present invention aims to provide a kind of method of one or more taxols of emanating from the material that comprises taxol compound, and the method that obtains described material does not comprise that C-2 ' the hydroxyl to the taxol molecule carries out benzoylated step.
In another embodiment, the present invention aims to provide a kind of from the method by one or more taxols of segregation semi-synthetic or the material that comprises taxol compound that total synthesis method obtains, and described isolation process may further comprise the steps: (a) with the described material of PBS plastic resin treatment; Wherein said material comprises the 2 ' benzoic ether that total amount is lower than taxol B, C, D, E, F or the G of 0.5 weight %; (b) described one or more taxols of wash-out; (c) one or more taxols of recovery institute wash-out.
In another alternative embodiment, the present invention aims to provide a kind of method for preparing the taxol mixture.This method may further comprise the steps: with the PBS plastic resin treatment by the material that comprises one or more taxols semi-synthetic or that total synthesis method obtains.Employed taxol or other compound derive from one or more Chinese yews in the semisynthesis, perhaps derive from one or more Chinese yews that do not comprise yewtree.In another embodiment, the material that contains taxol comprises the 2 ' benzoic ether of taxol A, the B, C, D, E, F or the G that are lower than 3 weight %.In another embodiment, the material that contains taxol comprises the C-2 ' benzoic ether of taxol A, the B, C, D, E, F or the G that are lower than 0.5 weight %.
In another alternative embodiment, the handled material of the present invention can comprise benzoyl oxide, phenylformic acid and Benzoyl chloride, and the amount of benzoyl oxide, phenylformic acid and Benzoyl chloride preferably is lower than 10%, preferably is lower than 5%, more preferably less than 1%, most preferably be lower than 0.3%.
About by material semi-synthetic or that total synthesis method obtains, when handling these materials with the present invention, the purity of target product therefrom is at least 70%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
In another alternative embodiment, the material that the present invention handles can comprise molecular weight and be about the taxol etc. that 1104 taxol impurity and/or molecular weight are about 871 butylene oxide ring ring open loop.
Method of the present invention can make that the purity of specific taxol increases about 10%～20% in the raw material, 20%～30%, 30%～40%, 40%～50%, 50%～60%, 60%～70%, 70%～80%, 80%～90%, 90%～100%, 100%～110%, 110%～120%, 120%～130%, 130%～140%, 140%～150%, 150%～200%, 200%～250%, 250%～300%, 300%～350%, 350%～400%, 400%～450%, 450%～500%, 500%～550%, 550%～600%, 600%～650%, 650%～700%, 700%～750%, 750%～800%, 800%～850%, 850%～900%, 900%～950%, 950%～1000%.Term purity used herein is meant the weight percent that is present in one or more taxols in exsiccant raw material or the bio-extract.
A scheme of the present invention relates to uses the PBS resin to separate the taxol compound that comprises taxol A with preparative-scale.The separation of preparation property can with intermittently, semicontinuous or continuous mode carries out.Semicontinuous and continuous mode can be simulation moving-bed (SMB) stratographic form.
In another embodiment, the present invention includes use PBS resin separates by liquid phase chromatography.In such embodiment, in the isolating moving phase of liquid chromatography, use a spot of acid or salt properties-correcting agent.Acid of using in the moving phase or salt properties-correcting agent can be including, but not limited to acetate, formic acid, ammonium acetate or ammonium formiates.
The PBS resin can be with positive mode work, and taxol is shown unique selectivity.For the standard type chromatogram, on C-18 and silicon-dioxide, other taxol all can be before taxol A and wash-out afterwards.In the normal-phase chromatography on the PBS resin, the taxol of other similar of great majority is wash-out before taxol A all, and this makes can simplify the separation of preparation property.In most of common taxols, 10-deacetylation taxol wash-out after taxol A on the PBS resin.Fortunately, the retention time of 10-deacetylation taxol prolongs widely, makes separate easily in preparation property system.This is very important, because 10-deacetylation taxol is the important by-products in the semi-synthetic preparation, and is present in usually in the natural extract of Ramulus et folium taxi cuspidatae seeds.
Figure 10 represents the comparison of the retention time of taxol standard substance on various media.In Figure 10, footnote 1 expression Phenomenex TMSynergi TMHydro-RP, 4mm (250 * 4.6mm) HPLC posts: acetonitrile/water gradient eluent (40% to 60% acetonitrile, 45 minutes), 1.5ml/min (ml/min).Footnote 2 expression Amicon Si-100-10sp (250 * 4.6mm) HPLC posts: Isocratic elutriant, 60% ethyl acetate/40% hexane, 1.0ml/min.Footnote 3 expression J.T.Baker  Wide-Pore PEI, 5 microns (250 * 4.6mm) HPLC posts: Isocratic elutriant, 80% ethyl acetate/20% hexane (containing 0.5% acetate), 1.0ml/min.Footnote 4 expression J.T.Baker  Wide-Pore DEAM, 5 microns (250 * 4.6mm) HPLC posts: Isocratic elutriant, ethyl acetate (containing 0.5% acetate), 1.0ml/min.Footnote 5 expressions are by the resulting retention time of mixture of independent injection taxol standard substance.Footnote 6 expressions are with respect to the retention time of taxol A.The mean time that the independent injection of footnote 7 expressions taxol standard substance is three times.
Bonding has the resin of PET that taxol is shown similar selectivity, but near some taxols wash-out (for example 10-deacetylation-7-Epitaxol is seen Figure 10) taxol A still.For example the PEI of chemically modified form such as DEAM has kept the selectivity of same way as, and has shown bigger with the isolating ability of taxol A wash-out taxol immediate with it.
The PBS resin has high avidity to taxol.See that as Figure 10 the mixture of about 60% ethyl acetate and about 40% hexane is enough to from the silicon-dioxide with rational time wash-out taxol A.In some cases, PEI needs stronger mixed solvent, for example about 79.75% ethyl acetate, 19.75% hexane and 0.5% acetate.DEAM is stronger than PEI to the avidity of taxol A, and the mixture that needs about 99.5% ethyl acetate and about 0.5% acetate is as moving phase.The employed stronger solvent system of PEI and DEAM can dissolve taxol to a greater degree, thereby allows higher loadings.
In some variation patterns, use polyethyleneimine-bonded silica resin from the semi-synthetic or complete synthesis taxol reaction mixture of mixture of taxols such as for example Ramulus et folium taxi cuspidatae Extract, to separate the various taxols that comprise taxol A. production comprises paclitaxel and is described in No. 60/401191 application (being called for short ' 191 application) of submitting on August 4th, 2002 at appropriate method and the composition of interior semi-synthetic taxol mixture, the U. S. application of submitting in the PCT/US03/10557 that submitted on April 5th, 2003 and on February 4th, 2003 number is that 60/444847 name is called the provisional application of " Method and Compositions for Preparing a Pharmaceutical Compound (e.g., Paclitaxel or other Taxanes) Using a Benzoylating Agent EssentiallyFree of Ring Chlorination ". These apply for that each all integrally is incorporated herein by reference.
In an alternative embodiment, method of the present invention comprises following one or more step: (i) fill pillar with the PBS resin of appropriate amount; (ii) use organic solvent balance pillar, described organic solvent preferably contains the acetone of acetate; (iii) the semi-synthetic or complete synthesis reaction mixture of Ramulus et folium taxi cuspidatae Extract mixture is loaded on the pillar; (iv) use the described mixture of organic solvent wash-out, the preferred acetone/acetate of described organic solvent; (v) elutriant is collected as one or more fractions; Determine that (iv) the target taxol appears in one or more fractions; (v) by crystallization and purification target taxol.
According to an alternative embodiment, be included in crude extract in order to purify or the extract of partly purifying in taxol and other taxanes substance, the raw material that contains taxol is carried out positive liquid chromatography (" NPLC ") handles.Usually the several variablees of research realize separating and purifying with liquid chromatography, and these variablees comprise composition, column dimension and the eluent flow rate of column filling (for example stationary phase or sorbent material), elutriant (for example moving phase).These variablees are known to those skilled in the art or do not need a large amount of tests just can easily determine.
Column size and temperature, flow velocity and chromatographic separation time are not crucial to implementing the present invention, and mainly based on the requirement of high efficiency chromatography, these requirements are known or do not need a large amount of tests just can easily determine those skilled in the art for they.
In an alternative embodiment, use the ethyl acetate that contains 0.5% acetate to make moving phase, on DEAM resin (ball of 20 μ m, 100 ), carry out the chromatogram of semi-synthetic taxol A and purify.At suitable absorbing wavelength place, this wavelength is preferably 254nm and 280nm, monitors isolating progress with uv-absorbing.Taxol A peak is collected in the fraction.The rising part (from the baseline to the summit) at peak can be collected in a plurality of fractions, is generally 2 or 3 fractions.The earliest that fraction contains most of 7-Epitaxol and 10-deacetylation-7-Epitaxol impurity in these fractions.The rest part at peak is collected in one or more fractions.Can use stagewise gradient wash-out 10-deacetylation taxol from this pillar of methyl alcohol and 50: 50 mixtures of the ethyl acetate that contains 0.5% acetate.
In an alternative embodiment, for brief and easy handling, preferably do not use linear gradient, but adopt stagewise gradient to come remaining impurity of wash-out.Use linear gradient to need more complex apparatus.Use the pre-mixing solvent system to realize the stagewise gradient wash-out by changing the single valve that moving phase is supplied in the pump.In another alternative embodiment, preferably methyl alcohol is postponed to be incorporated in the moving phase system, because a spot of methyl alcohol can quicken the wash-out of the slower impurity of wash-out, this impurity takes off the benzoyl taxol for for example 2-.
In an alternative embodiment, preferably use liquid chromatographic system of the present invention with preparation sexual norm (greater than the amount of 100mg).Preparation property pillar general diameter is 7～300mm, and length is 10～100cm.The technician of chromatographic field can easily select to be fit to by the pillar of the bed size of purification material quantity.Various factorss such as resolving power according to the particle diameter that comprises column dimension, resin and aperture and target peak are regulated the flow velocity of moving phase.The typical flow of preparation property pillar can be in the scope of 10ml/min to 41/min.
The time that chromatographic run needs arrived about 30 hours scope at about 10 minutes.Although can use high slightly temperature, the temperature of chromatographic separation is generally room temperature.
When according to the invention process chromatographic separation, pillar can be generally 10～500p.s.i.g work down in low pressure (LPLC) to the pattern of middle pressure (MPLC).Also can under high pressure (HPLC) pattern, move, be generally 500～2000p.s.i.g.
In another scheme, the semi-synthetic or complete synthesis reaction mixture of Ramulus et folium taxi cuspidatae Extract mixture can be loaded on the pillar after the dissolving.For example with an organic solvent or other method that those skilled in the art will know that dissolve described mixture.The preferred organic mixture comprises acetone/acetate.Then institute's dissolved mixture is loaded on the pillar that is filled with an amount of PBS resin.
Can select to be used for solvent of the present invention (eluent) with reference to the stratographic standard operating procedure.The organic solvent that can use middle polarity such as acetone, ethyl acetate, tetrahydrofuran (THF) or acetonitrile for example usually is as eluent.Also can use other ketone, ether and the ester that contain 1～5 carbon atom.If mixture contains higher polar taxol, then the properties-correcting agent of eluent can comprise higher polar solvent, for example lower alcohol, acetate and water; And if mixture contains lower polar taxol, then the properties-correcting agent of eluent comprises lower polar organic solvent, for example alkane and halohydrocarbon.According to by the character of purifying mixture, the per-cent of properties-correcting agent can be 0%～100%.Those skilled in the art can easily determine this per-cent.
In another embodiment, Ramulus et folium taxi cuspidatae Extract mixture or crude product mixture can be dissolved in the organic solvent that comprises ethyl acetate/THF (tetrahydrofuran (THF)) and form paclitaxel solution.Stir this paclitaxel solution under mild heat, impurity and fragment are removed in vacuum filtration then.
Thick paclitaxel solution also can be diluted in the organic solvent described organic solvent ethyl acetate/THF solution.With this injection of solution and/or be loaded on the pillar of the PBS resin that contains appropriate amount.This pillar can carry out balance in advance with organic solvent, described organic solvent ethyl acetate/acetate.After paclitaxel solution being injected/be loaded on the pillar, ethyl acetate can be expelled in the pillar.The material of institute's wash-out can be collected in a plurality of fractions.Can use the stagewise gradient that comprises the methanol/ethyl acetate mixed solvent.The taxol that wash-out needs from pillar is collected in one or more fractions then.Can use washing step.
Sometimes, it is favourable using the gradient solvent system of stagewise gradient or continuous gradient.The concentration extreme value of gradient depends on: (1) concentration of the needed organic solvent of wash-out taxol from the sorbent material; (2) organic solvent dissolves each other fully and is present in single requirement in mutually under the needed concentration of wash-out taxol.For example, bring into use 100% ethyl acetate, rapid or continuous gradient mode switches to 1%～10% methyl alcohol with one step, multistep then.This system is essential for separating the very big taxol of various polarity difference, and those skilled in the art determines this system easily.
The analytical technology of using this area to know, for example thin-layer chromatography (TLC), infrared spectra (IR), nucleus magnetic resonance (NMR) spectrum, high performance liquid chromatography (HPLC), RPLC and mass spectrum (MS) can detect the existence of target taxol in one or more fractions.
After pillar is collected the target taxol,, can use other chromatographic process or crystallization and/or one or many recrystallization that it is further purified according to the required purity of final product taxol molecule or taxol mixture.Can use binary or ternary solvent system (being at least a solubilising solvent and at least a contrary solvent (anti-solvent)) to carry out crystallization and recrystallization.Wherein, the example of solubilising solvent comprises acetone, methyl tertiary butyl ether, methylene dichloride, THF, methyl alcohol, ethanol, Virahol and acetonitrile.The example of contrary solvent comprises for example varsol such as hexane and heptane and water.In most applications, solubilising solvent and contrary solvent dissolve each other with used ratio.Wherein, the example to the useful solvent system of taxol molecule comprises acetone/hexane and methanol.
Fig. 1 and 2 represents the chemical reaction synoptic diagram of use PBS resin of the present invention (for example DEAM).Particularly, its expression is converted into taxol A or other taxol with the taxol acid amides and uses the semisynthesis of PBS resin purification of paclitaxel from reaction product.The method representation that the taxol acid amides is converted into taxol A or other taxol is in the aforesaid patent application of this paper.
More than described being suitable for concrete chromatographic technique of the present invention and condition, will describe preferred embodiment below according to segregation of the present invention, separation and purification of paclitaxel derivative.
Embodiment
The following example that is provided only is for illustration purpose, rather than for example limits the scope of the present invention to specific step or composition.
Embodiment 1
Present embodiment has shown use PEI resin purification of paclitaxel A (paclitaxel) and taxol B (Cephalomannine) from the half Deng Sifumi Taxus x media extract of purifying.Here, give the J.T.Baker PEI resin (40 micron grain sizes, 275 dust apertures, J.T.Baker Item #7264) of 75 liters pillar filling 24.5kg.This resin flow velocity is that the acetone that contains 0.5% acetate of 2 times of column volumes (promptly 150 liters) of 3.2L/min carries out balance.Then the half Ramulus et folium taxi cuspidatae Extract sample introduction of purifying is loaded on the pillar, the concentration of this extract in acetone is about 250mg/mL, and its flow velocity is 0.5L/min.The concentration of taxol is 2.5 weight %, or is every 100g PEI resin 2.5g sample introduction solid.The weight percent of taxol A and taxol B is 10.8 weight % in the sample introduction solid.This sample introduction with the acetone that contains 0.5% acetate with 3.2L/min (the flow velocity wash-out of superfacial velocity=4.4cm/min).Collect 10 fractions altogether.Fraction 1 and fraction 10 are respectively 75L (1 times of pillar volumes), and fraction 2～9 is respectively 19L (1/4 times of pillar volume).Taxol A and B total amount that product fraction 2～5 contains account for 87.96% of sample introduction, and the weight percent of combined prod fraction is 58.59%.This product is adapted to pass through crystallization and further purifies.
Embodiment 2
Present embodiment has shown that the taxol A (paclitaxel), the taxol B (Cephalomannine) that exist in the yewtree extract that uses the PEI resin partly to purify separate with taxol C.Here, the solid sampling with the half yewtree extract of purifying is dissolved in the acetone that contains 0.6% acetate.This solution is loaded on the J.T.Baker PEI resin (40 micron grain sizes, 275 dust apertures, J.T.Baker Item#7264).The loading capacity of sample introduction is 3%, or is every 100g PEI resin 3g sample introduction solid.Eluting solvent is the acetone that contains 0.6% acetate.When wash-out sample introduction component from pillar, approximately per 1/20 column volume just is collected as a sample.Analyze these fractions with HPLC, draw with data, as shown in Figure 3.Current operation shows taxol A, taxol B is separated with impurity with taxol C.
Embodiment 3
Present embodiment has shown the use DEAM resin thick semi-synthetic taxol A (paclitaxel) that purifies.Here, the molecule that is used as reactant in semi-synthetic or total synthesis method derives from the Chinese yew except that yewtree.In the present embodiment, the equipment that uses is NovaPrep 200 Preparative High Performance Liquid Chromatography System (NovaPrep 200 preparation property highly effective liquid phase chromatographic systems), and this equipment and wavelength set are that Hitachi's L-7400 ultraviolet rays detector of 254nm is used in combination.These two equipment all are directly connected to operation 2.11.V version LC ReSponder control software (R﹠amp; S Technology is on PC interface Inc.).
Described pillar is Load ﹠amp; Lock 2 " Preparative LC column (R﹠amp; STechnologies, Inc./Varian), its size is as follows: internal diameter=5cm, length=25cm, volume=490.87cm 3Fill the silica dioxide gel that following bonding has diethylamino methyl (DEAM) for this pillar: sphere, 20 microns particle diameter, the mean pore size of 120 dusts.Fill the resin that 270g is compressed to 800psi for this pillar.
Be called the primary amine of describing among the U.S. Patent application PCT/US03/10557 of " Conversion of Taxane Molecules " (conversion of taxol molecule) by name and transform chemical process, prepare the thick taxol A of 7.1g altogether.Use this thick taxol of about 35mL dissolution with solvents, this solvent comprises 90 volume % ethyl acetate/10 volume % tetrahydrofuran (THF)s.Mild heat (about 35～40 ℃) stirred this solution 30 minutes down.Before injection, the vacuum filtration clear soln is to remove O-fiber or particle.After the filtration, solution is transferred in the graduated cylinder, the ethyl acetate/tetrahydrofuran solution that used 90: 10 is diluted to final volume and is 42.5mL (diluting 6 times).Again mix before the sampling to guarantee homogeneity.The loading capacity of this operation is 2.625%, or is every 100g DEAM resin 2.625g sample introduction solid.Flow velocity is set at 90mL/min.Use standard flow to balance each other 20 minutes on this pillar by ethyl acetate+0.5 volume % acetate is formed.20 minutes the moment, the previously prepared sample solution of whole volumes is expelled on the pillar, inject cleaning procedure with the 20mL ethyl acetate immediately.Collect fraction (listed institute all is from injection point if having time---the time 0 begins to calculate) in the following manner.
Table 2
Fraction Beginning Finish Total time Filling area % Filling quality (mg)
1 6:15 15:00 8:45 10.73 12.2
2 15:00 15:15 0:15 74.95 96.4
3 15:15 16:30 1:15 95.43 995.0
4 16:30 29:00 12:30 99.25 4284.6
5 29:00 35:15 6:15 100.00 555.3
Summation 5943.4mg
When 29 minutes (promptly from injection point---time 0), begin 50% stagewise gradient, form by 50: 50 methanol/ethyl acetate cleaning solvent moving phase.Operation moves 20 minutes when finishing altogether.This washing step allows to be eluted in quickly the taxol A 10-deacetylation taxol A compound of wash-out afterwards.In case ultraviolet rays detector detects specific absorption and increases sharply, this indicates the wash-out of 10-deacetylation taxol A, closes last fraction immediately and collects valve (being fraction 5).In 10 minutes, from pillar, flush out whole 10-deacetylation taxol A under these conditions.But in order to guarantee to clean out all residual substances from pillar, as regular practice, washing step always carried out whole 20 minutes.Therefore, beginning to all processes of finishing the pillar washing step from injection is 49 minutes.The chromatogram trajectory table of the bleeding point of expression fraction 1～5 is shown among Fig. 4.
Fraction 3,4 and 5 is incorporated in transmits in the pond, so that carry out subsequently crystallization work.The fraction analysis (HPLC) of the fraction of being transmitted provides 5.835g taxol A altogether.Divided by the total mass of the taxol in all fractions, the rate of recovery is 98.18% as calculated.The taxol purity that enters the coarse raw materials in the pillar is 87.9 weight %, and after pillar was purified, the taxol A purity of the merging fraction of being transmitted was 98.71 area % (HPLC).
Embodiment 4
Give the J.T.Baker PEI resin (40 micron grain sizes, 275 dust apertures) of 44 liters pillar filling 10kg.This resin is with the 50 volume % ethyl acetate that the contain 0.5% acetate/50 volume % heptane of 3 times of column volumes (promptly 135 liters) the flow velocity balance with 1.0L/min.The flow velocity of all the other fractions is 1.0L/min.Half sample introduction (deriving from blue handsome Taxus x media) of purifying is loaded on the pillar, and the concentration of this sample introduction in ethyl acetate is 250mg/ml.The purity of elementary taxol is 57 weight % in the sample introduction.At first, first fraction is the 50 volume % ethyl acetate that contain 0.5% acetate/50 volume % heptane of 170 liters.The second, collect the 50 volume % ethyl acetate that contain 0.5% acetate/50 volume % heptane of three 10 liters.The 3rd, the product fraction is 90 volume % ethyl acetate/10 volume % methyl alcohol of 90 liters.At last, this pillar washs with the ethyl acetate/methanol of 45 liter of 90 volume %/10 volume %.The elementary taxol of 93.5 weight % is contained in the product pond.The recovery per-cent of taxol A and B is 95%.
In whole specification sheets, when the present invention being described as having, comprising or comprising specific components, perhaps, when method being described as having, comprising or comprising the particular procedure step, the present invention also refers to be made up of described component or treatment step basically, or is made up of described component or treatment step.In addition, should be appreciated that as long as the present invention still can implement, the order of step or the order of some action of carrying out are not crucial.And, as long as the present invention still can implement, can carry out two or more steps simultaneously.In addition,, one or more step of the present invention or key element can be omitted, perhaps when this paper does not have clear and definite disclosed any composition or step not to exist, also invention described herein can be implemented suitably as long as the present invention still can implement.
In addition, under the situation that does not break away from its spirit or essential feature, the present invention can show as other particular form.Therefore, will be understood that previous embodiments is illustrative, rather than limit invention described herein.
This paper is incorporated herein the every piece of patent being mentioned and the full content of non-patent literature by reference clearly.

Claims (48)

  1. One kind from the mixture that contains taxol the segregation one or more taxols method, said method comprising the steps of:
    (a) with the described mixture of PBS plastic resin treatment; Wherein said one or more taxols derive from one or more Chinese yews, and wherein said one or more taxols not only derive from yewtree;
    (b) with elutriant described one or more taxols of wash-out from the PBS resin; With
    (c) one or more taxols of recovery institute wash-out.
  2. One kind from the mixture that contains taxol the segregation one or more taxols method, said method comprising the steps of:
    (a) with the described mixture of PBS plastic resin treatment; Wherein said mixture comprises and is lower than 25 weight % or greater than the elementary taxol of 40 weight %;
    (b) with elutriant described one or more taxols of wash-out from the PBS resin; With
    (c) one or more taxols of recovery institute wash-out.
  3. One kind the segregation one or more taxols method, this method may further comprise the steps:
    (a) contain the mixture of taxol with the PBS plastic resin treatment; Wherein said mixture comprises the elementary taxol of about 25 weight %～40 weight %; Wherein said one or more taxols not only derive from yewtree;
    (b) described one or more taxols of wash-out from the PBS resin; With
    (c) one or more taxols of recovery institute wash-out.
  4. 4. one kind from the method by one or more taxols of segregation semi-synthetic or the material that comprises taxol compound that total synthesis method obtains, and described isolation process may further comprise the steps:
    (a) with the described material of PBS plastic resin treatment; The molecule that wherein is used as reactant in semi-synthetic or total synthesis method not only derives from yewtree;
    (b) described one or more taxols of wash-out from the PBS resin; With
    (c) one or more taxols of recovery institute wash-out.
  5. 5. one kind from the method by one or more taxols of segregation semi-synthetic or the material that comprises taxol compound that total synthesis method obtains, and described isolation process may further comprise the steps:
    (a) with the described material of PBS plastic resin treatment; Wherein said material comprises the C-2 ' benzoic ether that total content is lower than taxol A, B, C, D, E, F or the G of about 8 weight %;
    (b) described one or more taxols of wash-out; With
    (c) one or more taxols of recovery institute wash-out.
  6. 6. one kind from the method by one or more taxols of segregation semi-synthetic or the material that comprises taxol compound that total synthesis method obtains, said method comprising the steps of:
    (a) with the described material of PBS plastic resin treatment; Wherein said material comprises the C-2 ' benzoic ether that total content is lower than taxol B, C, D, E, F or the G of 1.0 weight %;
    (b) described one or more taxols of wash-out; With
    (c) one or more taxols of recovery institute wash-out.
  7. 7. the method for a segregation taxol A from the mixture that contains natural deutero-taxol said method comprising the steps of:
    (a) with the described taxol mixture of PBS plastic resin treatment;
    (b) wash-out taxol A from the PBS resin; With
    (c) the taxol A of recovery institute wash-out.
  8. 8. method of one or more taxols of purifying from bio-extract said method comprising the steps of:
    (a) prepare bio-extract with the method except that chromatography;
    (b) with the described bio-extract of PBS plastic resin treatment;
    (c) described one or more taxols of wash-out from the PBS resin; With
    (d) one or more taxols of recovery institute wash-out.
  9. 9. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from Taxus x media cultivation mutation.
  10. 10. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from this Taxus x media of west.
  11. 11. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from general dark green Taxus x media.
  12. 12. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from European yew.
  13. 13. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from taxus chinensis in northeast.
  14. 14. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from the Florida Ramulus et folium taxi cuspidatae.
  15. 15. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from T. canadensis.
  16. 16. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from Xizang Taxus chinensis.
  17. 17. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from taxusyunnanensis.
  18. 18. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from Chinese Ramulus et folium taxi cuspidatae.
  19. 19. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from the Deng Sifumi Taxus x media.
  20. 20. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from the Brownie Taxus x media.
  21. 21. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from this Taxus x media of west.
  22. 22. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from blue handsome Taxus x media.
  23. 23. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from the Sarasvati Taxus x media.
  24. 24. as each described method of claim 1～3, wherein, the described mixture that contains taxol comprises the bio-extract that derives from the Dao Tuoni Taxus x media.
  25. 25., wherein, be taxol A by isolating described taxol as each described method of claim 4～6.
  26. 26., wherein, be taxol B by isolating described taxol as each described method of claim 4～6.
  27. 27., wherein, be taxol C by isolating described taxol as each described method of claim 4～6.
  28. 28., wherein, be taxol D by isolating described taxol as each described method of claim 4～6.
  29. 29., wherein, be taxol E by isolating described taxol as each described method of claim 4～6.
  30. 30., wherein, be taxol F by isolating described taxol as each described method of claim 4～6.
  31. 31., wherein, be taxol G by isolating described taxol as each described method of claim 4～6.
  32. 32., wherein, be Docetaxel by isolating described taxol as each described method of claim 4～6.
  33. 33. as each described method of claim 1～8, wherein said PBS resin has about 60 mean pore sizes to about 300 scopes.
  34. 34. as each described method of claim 1～8, wherein said PBS resin has about 100 mean pore sizes to about 200 scopes.
  35. 35. as each described method of claim 1～8, wherein said PBS resin has the mean pore size of about 120 .
  36. 36. as each described method of claim 1～8, wherein said PBS resin has about 0.25 median size to about 500 mu m ranges.
  37. 37. as each described method of claim 1～8, wherein said PBS resin has the median size in 1～100 mu m range.
  38. 38. as each described method of claim 1～8, wherein said PBS resin has about 10 median sizes to about 120 mu m ranges.
  39. 39. as each described method of claim 1～8, wherein said PBS resin has about 20 median sizes to about 60 μ m.
  40. 40. as each described method of claim 1～8, wherein said PBS resin has the median size of about 40 μ m.
  41. 41. as each described method of claim 1～13, wherein said PBS resin is DEAM.
  42. 42. as each described method of claim 1～8, wherein said PBS resin is PEI.
  43. 43. as each described method of claim 1～8, wherein said PBS resin has about 60 mean pore sizes to about 800 scopes.
  44. 44. as each described method of claim 1～8, wherein said PBS resin has primary amino or secondary amino group on the polyethyleneimine: amine moiety.
  45. 45. as each described method of claim 1～8, the amino of wherein said PEI polymkeric substance functionalised.
  46. 46. as each described method of claim 4～6, wherein described one or more taxols of institute's wash-out have the purity at least about 70%.
  47. 47. as each described method of claim 4～6, wherein described one or more taxols of institute's wash-out have the purity at least about 80%.
  48. 48. as each described method of claim 4～6, wherein described one or more taxols of institute's wash-out have the purity at least about 90%.
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Cited By (3)

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CN102219764A (en) * 2011-05-12 2011-10-19 河南省科学院化学研究所有限公司 Method for separating and purifying paclitaxel industrially
CN106943985A (en) * 2017-04-11 2017-07-14 南昌大学 Preparation method for the amino modified mesoporous silicon oxide of separating and purifying taxol
CN110531000A (en) * 2019-09-20 2019-12-03 杭州师范大学 It is a kind of for distinguishing the metabolic markers and its detection method of pseudotaxus chienii and taxusyunnanensis

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CN108101869A (en) * 2017-12-20 2018-06-01 上海金和生物制药有限公司 A kind of extracting method of natural Japanese yew alcohol

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US5475120A (en) * 1990-11-02 1995-12-12 University Of Florida Method for the isolation and purification of taxol and its natural analogues
EP1051505B1 (en) * 1998-01-26 2003-05-28 Man Woo Han Process of extracting taxol from taxus cuspidata
US5969165A (en) * 1999-01-07 1999-10-19 508037 (Nb) Inc. Isolation and purification of paclitaxel and other related taxanes by industrial preparative low pressure chromatography on a polymeric resin column

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102219764A (en) * 2011-05-12 2011-10-19 河南省科学院化学研究所有限公司 Method for separating and purifying paclitaxel industrially
CN102219764B (en) * 2011-05-12 2013-06-26 河南省科学院化学研究所有限公司 Method for separating and purifying paclitaxel industrially
CN106943985A (en) * 2017-04-11 2017-07-14 南昌大学 Preparation method for the amino modified mesoporous silicon oxide of separating and purifying taxol
CN110531000A (en) * 2019-09-20 2019-12-03 杭州师范大学 It is a kind of for distinguishing the metabolic markers and its detection method of pseudotaxus chienii and taxusyunnanensis
CN110531000B (en) * 2019-09-20 2022-07-12 杭州师范大学 Metabolism marker for distinguishing white bean Chinese yew and taxus yunnanensis and detection method thereof

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