CN1069641C - Taxad alcohol cleaning and purifying method - Google Patents
Taxad alcohol cleaning and purifying method Download PDFInfo
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- CN1069641C CN1069641C CN 98121946 CN98121946A CN1069641C CN 1069641 C CN1069641 C CN 1069641C CN 98121946 CN98121946 CN 98121946 CN 98121946 A CN98121946 A CN 98121946A CN 1069641 C CN1069641 C CN 1069641C
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- taxol
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Abstract
The present invention relates to a method for purifying taxol coarse products and cleanly preparing refined products, which uses a filling chromatographic column to elute coarse products containing 10 to 48% of taxol by gradient at normal pressure. The prepared chromatographic column is filled by silica gel of 50 to 300 meshes. Nontoxic normal alkane (n-pentane to n-octane) and acetic ester (methyl acetate, ethyl acetate and propyl acetate) are used as an eluting agent. A solution after eluted can recover a solvent by rotary evaporation, and 99.5% to 99.9% of taxol is obtained by vacuum freeze drying segment products after the solution is concentrated. The method has the advantages of simple manufacturing process and low cost, and has no pollution in the process of purification. The solvent and the eluting agent can be recovered. The present invention is convenient for industrialized production.
Description
The present invention is a kind of method of taxad alcohol cleaning and purifying.
Taxol is a kind of active natural organic-compound of broad spectrum anticancer that has.Wani (JAmer.Chem.Soc. in 1971,1971,93,2325) etc. reported first is separated from India's yewtree (Taxuobrevifolia) and has been obtained taxol, and confirm that taxol has strong widely lethal effect to lung cancer, liver cancer, mastocarcinoma cell and other tumour cell, side effect is very little.Its molecular formula is C
47H
21NO
14, molecular structure is as follows:
1979 (Nature, 1979,277,665) such as Schiff confirm taxol pharmacological action mechanism uniqueness, can promote the polymerization of tubulin, again can the unlikely depolymerization of stabilize microtubules.Nineteen eighty-three U.S. food and Drugs Directorate (FDA) approval to enter the I phase clinical, entered the third stage clinical trial at present, be considered to the most promising anticarcinogen of finding over nearly 20 years.
The source of taxol is very limited.It is to extract and purifying the about 40～160mg/kg of content in the bark from the bark of Taxaceae trees.The growth of this seeds is very slow, and about 6 inches of the trunk diameter of a common century-old veteran generally can only strip the bark of 2.5kg on every tree.The pure taxol that extracts 25kg provides 10,000 2 thousand routine cancer patients's clinical trials, is equivalent to the bark of 30,000 yew trees of needs.There is the yew tree natural resources in countries such as the U.S., Canada, India, all contain taxol in bark, trunk and the China fir leaf of China's taxus chinensis in northeast (T.cuspidata), taxusyunnanensis (T.yunnanensis) and beautiful Ramulus et folium taxi cuspidatae (T.chinensis var.mairei), but content is lower.In order to obtain the source of taxol, protect rare seeds, carried out short strongization and the tissue culture method of Chinese yew abroad, also have the work of semi-synthetic and complete synthesis taxol.Yet.Because culture method only obtains the amount of nanogram level every day, complete synthesis yield is lower, and cost is huge, is difficult to commercialization.Therefore, extracting taxol from tree, particularly as far as possible all extract taxol, is present unique, business-like method.
Separation commonly used and purification step remove and desolvate for using methyl alcohol or 95% ethanol lixiviate bark, the medicinal extract water dissolution, after the degreasing with methylene dichloride or chloroform extraction; After extracted component concentrates, obtain the monomer or the mixture of bearing taxanes through chromatography.It is more or less freely to use extraction to extract the taxol crude product from bark, and it is bigger still 10～48% thick product direct purification to be obtained 99.5～99.9% smart product technology difficulty.In addition, solvent or eluent cause serious environmental to pollute without reclaiming directly discharging.
Senilh (J.Natural Products, 1984,47,131) etc. extract taxol from European China fir (Taxul bacata) and use following steps:
(1) uses the alcohol extraction bark, concentrate;
(2) in water and methylene dichloride, distribute;
(3) " filtration " chromatography is separated;
(4) silica gel column chromatography separates;
(5) aluminum oxide chromatographic separation;
(6) the silica gel medium pressure column chromatography is separated;
(7) preparation HPLC separates.
Polysciences Inc. is isolation of taxol from the T.brevifolis bark:
(1) methyl alcohol or alcohol extraction bark concentrate behind the hybrid extraction and remove most of alcohol;
(2) concentrated solution dichloromethane extraction, extraction matter concentrate drying is to powder;
(3) powder dissolves with propyl alcohol and ligroin (1: 1), and indissolvable component removes by filter;
(4) filtrate of containing taxol concentrates, and is dissolved in 30% propyl alcohol filtration naphtha, through the Florisil post
Filter;
(5) effusive taxol component crystallization purifying secondary from post;
(6) the crystalline taxol further separates in silicagel column, in this step, and the most similar product
Cephalomannine separates from taxol;
(7) effusive taxol recrystallize secondary from post;
(8) unsegregated mixture and other solute, circular treatment obtains pure taxol.
Miller (J.Org.Chem., 1981,46,1469) adopts the following step:
(1) with methanol extraction Taxus wallichiana tree root, trunk, leaf, concentrated extract is to solid;
(2) in water and hexane, distribute;
(3) use chloroform extraction, concentrate then;
(4) silica gel column chromatography;
(5) secondary silica gel column chromatography;
(6) counter-current distribution method;
(7) second adverse current apportion design;
(8) preparation H-PLC separation and purification.
Use the normal phase silica gel column chromatography method from Ornamental yew (Taxus Xmedia Hicksii), to extract (usp.5,279,949):
(1) with the bright leaf of 70% alcohol extraction;
(2) activated carbon decolorizing after-filtration in the extract;
(3) extraction matter concentrates and removes most of organic solvent;
(4) water soluble ingredient is centrifugal separates with the precipitated solid that contains taxol;
(5) solid enters the normal phase silica gel column chromatography separation:
(6) the secondary column chromatography is separated, and the low pressure silica gel column chromatography separates thick taxol component;
(7) reverse-phase chromatography final purification.
Caster etc. (usp.5,440,055) use supercritical methanol technology, use N simultaneously
2O, CO
2, C
3H
8, Freon-22 or CHClF
2Deng as supercritical gas, extract taxol.
Rao etc. (usp.5,380,916,1992) use the taxol after ozonize is separated to get pure taxol (simultaneously referring to WO92/07842).
In March, 1998 Durand etc. (usp.5,723,635) has invented the method for utilizing centrifugal partition chromatograph method separating and purifying taxol.This method is used chromatographic column separating and purifying taxol from thick material well, and the primary treatment amount is big:
(1) under the preparative column scale, handles taxol bulk processing thing with reverse-phase chromatography;
(2) wash-out taxol and other product from the sorbent material;
(3) in the pillar of wash-out, reclaim taxol and resemblance;
(4) use ozone purifying final product.
Above-mentioned many separation purification method comprise crystallization process, the high pressure normal phase chromatography, the high pressure reverse-phase chromatography, centrifugal partition chromatograph method and supercritical extraction etc. all need to carry out under high pressure, vacuum or the supercritical state, and product purity is only 99%, major impurity is cephalomannine, is difficult to separate; Prepare highly purified taxol, also need with processing such as ozone or four cesium chlorides; Solvent, leacheate do not reclaim, and cause environmental pollution.
The objective of the invention is to set up that a kind of processing method is simple, pollution-free in the purge process, leacheate or the recyclable method that re-uses, is convenient to industrialization and can obtain high-purity taxol of solvent.
The present invention uses commercially available 10～48% paclitaxel prodrugs to be raw material, and normal paraffin, acetic ester, analysis are analytical pure with reagent such as ethanol, acetonitriles, uses after distilling.Analyzing with methyl alcohol is that high performance liquid chromatography is pure, and water is the secondary redistilled water, and silica gel, activated carbon fiber are analytical pure.The high performance liquid chromatograph that raw material and product analysis are used is produced as U.S. Beckman company, 110B double pump sample introduction.128 diodes shake row detector or 126 UV-detector, 4.6 * 250mmC
18Chromatographic column, flow velocity are 1.0ml/min, and sample size is 3 μ l.The operation of Pentium II MMX266 computer control efficient liquid phase chromatographic analysis.Sbsz-I type Rotary Evaporators, KLG-II type freeze drier, the super digital display of CS501-SP school temperature device, the automatic polarimeter of WZZ-2A, WKS-1A numeral fusing point instrument.Mass spectroscopy is at Mei GuoCE ﹠amp; Carry out on the TOF-2000MIP time of flight secondary ion massspectrometry instrument that A company produces, electron energy 15kev, specific charge m/e is in 0～1000 scope interscan.Liquid-matter connects spectrum analysis and carry out Zorbax2.1 * 100mmC on the HP1100-MS of U.S. Hewlett-Packard
18Chromatographic column, 5 μ m, flow velocity are 0.3ml/min, sample size is 5 μ l, the APES source, transmission voltage is 70 volts, sweep limit is that m/z is between 200～1200.
The present invention needed from packing before purification of paclitaxel.People's frock post beats pillar when adding silica filler evenly packs it into, and filler should not have tangible tomography or crackle in the pillar post after installing.After the taxol crude product is dissolved in acetic ester, pour chromatographic column into, add normal paraffin, acetic ester and composition thereof gradient elution, each gradient solvent and mixed solvent ratio are: normal paraffin (100%), normal paraffin: acetic ester (4: 1～2), normal paraffin: acetic ester (1: 1～4), acetic ester (100%).
The thick product purity of commercially available taxol is 10～48%, and it is mixed with the acetate solution usefulness to be purified that taxol concentration is 2.5～10% (wt%), and the too rare purification efficiency of concentration is not high, and the too big purification effect of concentration is bad.
Gradient elution is Skellysolve A, normal hexane, normal heptane, octane with the solvent normal paraffin, and acetic ester is methyl acetate, ethyl acetate, propyl acetate.
Be to improve the purifying purification efficiency, purifying is with chromatogram column length 30-80cm, and column diameter 2.0～8.0cm is for well.For product after making purifying is not with variegated, the activated carbon fiber of 25～35% silica gel volumes is equipped with in the present invention with the chromatographic column upper end, silica gel adds from the post upper port during dress post, there is teflon piston the lower end of post, the piston lower end is the 4# core layer of pad one deck fiberglass, after silica gel adorned, adorn activated carbon fiber at last, silica gel and activated carbon fiber size are respectively 50～300 orders and 100～200 orders.
Through 90～120 ℃ of oven dry activation 1-3 days, make its thorough drying, before silica gel and the activated carbon fiber dress post to remove the excess water of absorption.
The taxol crude product detects primary product through the efficient liquid phase chromatographic analysis (as Fig. 1) of LC-MS respectively at retention time 39.838,59.844,61.636,65.884min place, and the 0.976min place is the sample introduction peak.The mass spectroscopy of LC-MS on the throne shows (as Fig. 2), and the product of 61.636min goes out to obtain mass signal at 61.800min after flowing into mass spectrograph.Use the API-ES positive ion source, mass-spectrometric data is 854.3, illustrates that quality is 853.3.The high performance liquid chromatograph and the 4.6 * 250mmC that use Beckman to produce
18Chromatographic column is analyzed taxol crude product (as Fig. 3), respectively 18.00,29.10,34.39, the 36.05min place measures primary product, the 2.24min place is the sample introduction peak.Contrast with standard taxol appearance time, show that the 36.05min place is taxol.It is 48.10% that usable floor area normalization method and marker method record its content.
Collect each section leacheate, I～IV concentrates through rotary evaporation, can use repeatedly after distilled solvent or the leacheate rectifying, and avoiding directly, discharging pollutes.Concentrated solution separates obtaining all cpds in leacheate I～VI through-40 ℃ of vacuum lyophilizations.Each component of efficient liquid phase chromatographic analysis and crude product contrasts, and does not contain taxol in I～II, contains the taxol of about l% in the III, contain 99.5～99.9% taxol (the efficient liquid phase chromatographic analysis result as shown in Figure 4) in the IV, mass spectroscopy to the product IV shows that its quality is 853.4, sees Fig. 5.Separating the taxol that obtains is the white crystals sprills.Fusing point is 215.4～215.6 ℃, and specific rotation is-49 ° (in methanol solutions).
Separate behind the product, silica gel that takes out from post and activated carbon fiber can be used repeatedly through 250～350 roastings 1 to 3 day, have reduced preparation cost.
The method major equipment of taxad alcohol cleaning and purifying crude product of the present invention and reagent are packing and leacheate.After gradient elution contained the thick product of 10～48% taxols, through reclaiming leacheate and solvent, lyophilize can obtain 99.5～99.5% taxol under the normal pressure.Technology is simple, and leacheate does not have mould easy recovery, compares with the purification process of existing taxol, and it is low to have a cost, and purge process is pollution-free, and solvent and leacheate can reclaim, and are convenient to plurality of advantages such as suitability for industrialized production.
The TOF-SIMS mass spectrum of high-efficient liquid phase chromatogram Fig. 5 component VI of the Beckman instrument of high-efficient liquid phase chromatogram Fig. 4 component VI of the Beckman instrument of LC-MS mass spectrum Fig. 3 coarse raw materials of LC-MS high-efficient liquid phase chromatogram Fig. 2 coarse raw materials of Fig. 1 coarse raw materials
Embodiment 1:
Take by weighing 100～200 order silica gel, 225 grams and 50～100 order activated carbon fiber, 10 grams, after 110 ℃ of oven dry activate 48 hours in muffle furnace, hand-stuff in the pillar of 60cm length, post φ 2cm.There is teflon piston the pillar lower end, and the piston upper end is the 4# core layer of pad one deck 0.5 gram glass fibre.Take by weighing 2 grams, 48% taxol crude product, be dissolved in the 40ml methyl acetate, pour the chromatography chromatographic column into.Add 300ml hexane (100%), 200ml hexane successively: methyl acetate (4: 1), 600ml hexane: methyl acetate (1: 1), 600ml methyl acetate (100%) obtain cut I, II, III and IV respectively.Difference rotary evaporation components I-IV under 77 ℃, the concentrated component that obtains is through-40 ℃, 10
-2τ dry 40 minutes down.Component IV after vacuum drying treatment is a white powder, is 99.9% taxol through efficient liquid phase chromatographic analysis, and fusing point is 215.6 ℃, and specific rotation is-49 ° (in methanol solutions).
Embodiment 2:
Take by weighing 100～200 order silica gel, 280 grams and 50～100 order activated carbon fiber, 10 grams, after 110 ℃ of oven dry activate 48 hours in muffle furnace, hand-stuff in the pillar of 80cm length, post φ 3cm.There is teflon piston the pillar lower end, and the piston upper end is for filling the 4# core layer of 0.5 gram glass fibre.Take by weighing 3.0 grams, 10% taxol crude product, be dissolved in the 35ml ethyl acetate, pour the chromatography chromatographic column into.Add 600ml heptane (100%), 400ml heptane successively: ethyl acetate (2: 1), 400ml heptane: ethyl acetate (1: 1), 300ml ethyl acetate (100%) obtain cut V, VI, VII and VIII respectively.87 ℃ are rotated evaporated components V-VIII down, and the concentrated component that obtains is through-40 ℃, 10
-2τ dry 40 minutes down.Component VIII after vacuum drying treatment is a white powder, is 99.5% taxol through efficient liquid phase chromatographic analysis, and fusing point is 215.4 ℃, and specific rotation is-49 ° (in methanol solutions).
Embodiment 3:
Rotary evaporation is collected among the embodiment 1 and 2 solvent and leacheate reclaim hexane, methyl acetate, heptane, ethyl acetate respectively through 10,000 stage number rectifying, and the solvent after this rectifying can use repeatedly.Silica gel after the use and activated carbon fiber take out separate collection from post, dried 5 hours down for 110 ℃ in muffle furnace, are warming up to 300 ℃ of roastings 48 hours then.Silica gel and activated carbon fiber that this method is handled can use repeatedly.
Claims (4)
1, a kind of method of purification of paclitaxel, the chromatogram that adopts silica gel to fill, carry out gradient elution, the Fractional Collections elutriant, evaporation, concentrate and drying, it is characterized in that: crude product to be purified is dissolved in acetic ester, being mixed with taxol concentration is the solution dress post of 2.5～10wt%, the addition sequence of eluting solvent is a normal paraffin, 4: 1～2 normal paraffin: acetic ester, 1: 1～4 normal paraffin: acetic ester, acetate fat, described normal paraffin is a Skellysolve A, normal hexane, normal heptane or octane, acetic ester are methyl acetates, ethyl acetate or propyl acetate.
2, the method for purification of paclitaxel according to claim 1, it is characterized in that: chromatogram column length 30～80cm, column diameter 2.0～8.0cm, in the chromatographic column except filling gel, its upper end is filled with the activated carbon fiber of 2～5% volumes, silica gel is 50～300 orders, and activated carbon fiber is 100～200 orders.
3, the method for purification of paclitaxel according to claim 1 is characterized in that chromatographic column is with the oven dry activation 1～3 day under 90～120 ℃ of temperature before the dress post of filling gel and activated carbon fiber.
4, the method for purification of paclitaxel according to claim 1 is characterized in that in the chromatographic column that used silica gel and activated carbon fiber are in 250～350 ℃ of following heat-activated 1～3 day, renewable use.
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|---|---|---|---|
| CN 98121946 CN1069641C (en) | 1998-10-10 | 1998-10-10 | Taxad alcohol cleaning and purifying method |
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|---|---|---|---|
| CN 98121946 CN1069641C (en) | 1998-10-10 | 1998-10-10 | Taxad alcohol cleaning and purifying method |
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| CN101880264B (en) * | 2010-05-26 | 2012-03-21 | 徐丹 | Method of preparing taxol by separating 10-deacetyl taxol acylate |
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