MXPA97006180A - Isolation and purification of paclitaxel and cefaloman - Google Patents

Abstract

A new process for the extraction, isolation and separation of taxanes, particularly paclitaxel, from natural sources such as bark, aciculas and sprouts of Taxus species, tissue cultures and fungi, in which the taxanes are separated from the extracts is described. crude by spreading between polar and non-polar solvents, precipitation in non-polar solutions, reacting the mixture by halogenation of unsaturated taxane derivatives, followed by chromatographically separating and crystallizing taxanes from a mixture of polar and non-polar solvents, more particularly, The process of halogenation of unsaturated side chain taxane derivatives, particularly cephalomannin in the presence of paclitaxel, in which bromine is preferably added to the double bond of unsaturated taxanes leaving paclitaxel unchanged, paclitaxel is easily separated from the mixture including less polar halogenated taxane derivatives

Description

PATIENT AND PURIFICATION APPEARANCE, PFTCLlTaXRI ... CÍÍL 'ÍOMN N NA The invention relates to the isolation and purification of paclitaxel from any source that contains paclitaxel, including organic matter such as this material. vegetable, crops and fungi, and in particle! of T. irevi fo-l i a, the bark of the Pacific yew T. bacca t d, T. yuuua -nensi, T. Walichiana. More particularly, this invention provides for the separation and purification of pallaxol from mixtures of taxanes containing various concentrations of paclitaxel and other taxanes, including closely related laxanes having unsaturated side chains, such as cephalomapin. & ieat = £. da.JLa_ipsrepci! 5D Paclitaxel is a well-known chemotherapeutic drug for the treatment of various metastatic cancers. It has been approved by the Food and Drug Administration (FDA) for the treatment of ovarian and breast cancers and is currently undergoing clinical trials for the treatment of lung and colon cancers. The compound is a natural product extracted mainly from the bark of the Pacific yew, Taxus brevifolía, and is also found in T. bacca ta,. wal ichiana and T. yunnaneneis and other biomass extracts from plant materials, including T. hicksii, T. densiformis, T. gem, T. wardii, T. cuspidata, T. capi tata, T. brownii, and T. dark green spreader, which contain a mixture of taxon-like molecules. Paclitaxel is also available from cells of cultivated plants and fungi. The compound is commercially available in quality for reagents, for example, from the Aldrich Chemical Co., product No. 41-701-7, Sigma Chemical Company, products No. 7402 and T 1912, depending on the source, derived from Fluka. Chemie AG. product N ° 86346 and ICN Bio edicals product N "193532.

The concentration of paclitaxel in various raw materials is typically low, for example, in the order of between 0.0004-0.08% (w / w) in the Pacific yew bark. Such low concentrations make it very difficult to extract and purify the compound for clinical standards from raw materials, and therefore impractical at a commercial level. At present, several processes are known for the extraction and purification of paclitaxe. Wani et al., J. Am. Chem. Soc. 93.9: 2325-2327 (1971), describes the extraction of trunk bark from T. brevifol ia with ethanol, which is then concentrated and extracted with chloroform and water. , and where paclitaxel is in the chlorophoric phase. Paclitaxel is further purified by column chromatography on columns of florisil, sephadex and silica gel. A method of the National Cancer Institute (NC?) (1983) is based on the extraction of trunk bark of T. brevifolia with methanol followed by extraction with methylene chloride. The chloride extract is dried and then dissolved in acetone followed by the precipitation of impurities with n-hexane. The soluble fraction is further purified by column chromatography. None of the Wani et al. And NCi procedures, however, are very effective or commercially practical, since they result in very low yields of the order of about 0.02% or less. This is due to the presence of other taxanes, such as the narrow analog of paclitaxel cephalomannin, which have similar structures and physical properties very close to those of paclitaxel. See FIG. 1 which illustrates the chemical structures of paclitaxel and cephalomannin. In a process developed by Potier and others,. Nat .. Prod. 47.1: 131-137 (1984), the stage of precipitation in the NCI process is replaced by a step that employs a method of extraction with a pair of solvents, that is, using successive extractions with solvents progressively more polar. After the addition of chromatography on alumina and silica columns, paclitaxel is concentrated as a mixture of paclitaxel and cephalomannine. Paclitaxel is then separated from the cephalomannin by HPLC, with a yield of pacíit.a-xel considerably higher than that obtained by any of the Wani et al. Or NCl methods. However, the method of Potier et al., Similar to the methods of Wani et al. And NCi, has the main disadvantage of requiring the separation of multiple taxanes with similar separation parameters in a final concentrate by the use of chromatographic separations. multiple conventional graphs to obtain a purified paclitaxel product. A large-scale commercial processing of paclitaxel employing multiple conventional chromatographic separations to provide clinically acceptable pure paclitaxel would be needed by these methods, such are impractical commercially due to the high expense associated with such multiple chromatographic separations. Multiple separations are needed in large part because of the similarity in structure and properties of paclitaxel and cephalomannin. As shown in the PIG. 1, the only difference in their structures is that the amino group of the side chain of paclitaxel is acylated with benzoic acid, and in the cephalomannine the amino group of the side chain is acylated with tíglico acid that contains a double bond. Methods other than the chromatographic separation of paclitaxel from cephalomannin are known, such as chemical modifications of the double bond of the side chain in cephalomannin. For example, Kingston et al., J. Nat. Prod., 55: No. 2, 259-261 (1992) describes the catalytic oxidation of the double bond of the side chain of cephalomannin in the presence of 0sO 4 to obtain a diol, since then it is separated from paclitaxel by chromatographic procedures and crystallizations. There are problems with this method in the use of mixtures of unpurified taxanes since the oxidation catalyzed by OsO ^ is not susceptible to crude extracts due to the low selectivity for the double bond of the side chain of the cephalomannin, which if it could be used , would significantly reduce the cost of the extraction and purification process. Additionally, using OsO ^ in the manufacture of pharmaceutical products is undesirable due to the severe toxicity of the compound. In U.S. Patents No. 5,334,732 and. . 36,684 of Murray et al., Oxidation of the side chain of cephalomannin by ozone is described. These methods are also undesirable since the use of ozone in an oxidation process with crude extracts produces many unwanted reactions with paclitaxel; oxidation by ozonolysis is strong and non-selective in compounds with many functional groups such as paclitaxel. and cephalomannine, and may cause unwanted oxidations of other functional groups, such as aldehydes, ketones, amines, etc., in the paclitaxel molecule, or of the double bond found within the taxane ring of paclitaxel or cephalomannin. There is also the expensive requirement of an ozone generator. Thus, the isolation and purification of paclitaxel from a crude biomass containing a complex mixture of taxanes or, at the other extreme, from more purified mixtures containing mainly paclitaxel and cephalomannine is currently limited to non-economic chromatographic separation techniques. and / or to non-selective oxidation methods, thereby presenting a serious and unfulfilled need for an economically practicable method for separating the valuable anti-moral compound paclitaxel from its close analogue cephalomannoma, as well as from other closely related taxanes. Therefore, an objective of this invention is to provide a simpler and more cost-effective method than currently available methods for the economical isolation and purification of the important paclitaxel chemotherapeutic compound.

SUMMARY OF THE INVENTION In order to fulfill the aforementioned objective, the present invention now provides a new and unique process for the isolation and purification of paclitaxel from crude biomass extracts containing a complex mixture of taxane-type compounds, including paclitaxel, and especially of the crude bark of T. brevifolia, T. baccata, T. yunnanenaie and T. wal ichiana, as well as of plant material such as aciculture and twigs of various taxa species and which also includes purification downstream of paclitaxel produced from sources such as cell cultures of taxa and fungi species that produce paclitaxel. Thus, in one aspect, the present invention provides a process for the isolation and purification of paclitaxel from organic material containing, inter alia, a mixture of taxanes, which comprises solvent extraction of the organic material to form a composition comprising paclitaxel. , and then chromatographically separate paclitaxel and other taxanes from the composition with an organic solvent. This is preferably followed by a "flash" chromatographic separation step of paclitaxel and other taxanes in a normal phase chromatographic column containing silica gel as an absorbent to form a refined mixture comprising paclitaxel, cephalomannine and other taxanes. The resulting mixture is then reacted with a halogen, preferably bromine, under conditions effective for selective halogenation of the unsaturated side chain residue of cephalomannin to produce a diastereomeric mixture of dihalocepharomannins; paclitaxel is then easily and conveniently separated from the mixture in high yield. In another aspect of the invention, any step in the process for the isolation and purification of paclitaxel from raw organic material can be carried out by the stage of selective halogenation of cephalomannine, before or after extraction and purification by techniques conventional chromatography, for example, either the mixture of a crude extract or a more refined mixture essentially only of paclitaxel, cephalomannin and other taxanes. In still another aspect of the invention, there is provided a highly efficient and economical method for separating paclitaxel with a virtually quantitative yield from its narrow-cephalicomannin analogue by a new chemical modification of the cephalomannin. The present invention will be more fully understood by reference to the following detailed description of the preferred embodiments of the invention, the examples and the drawings.

BRIEF DESCRIPTION PE THE DRAWING? FIG. 1 illustrates a generalized representation of the structures of paclitaxel and cephalomannin. The PIG. 2 illustrates a generalized representation of the structures of various unsaturated taxanes and functional groups contained therein with various unsaturated side chains, which can be halogenated according to this invention. The PIG. 3 is a process diagram of a preferred embodiment of an aspect of the invention in the isolation and purification of paclitaxel from T. brevifolia. The PIG. 4 illustrates a preferred reaction scheme for the selective bromination of cephalomannin. FIG. 5 is a UV spectrum of paclitaxel obtained from this invention. FIG. 6 is an IR spectrum of paclitaxel. obtained from this invention. FIG. 7a is a proton NMR spectrum of paclitaxel obtained from this invention. FIG. 7b is a carbon 13 NMR spectrum of paclitaxel obtained from this invention. FIGS. 8a and 8b are paclitaxel EI-MS obtained from this invention. FIG. 9 is a paclitaxel DCI-MS obtained from this invention. FIG. 10 is a FAB-MS (positive ion mode) of paclitaxel obtained from this invention. The PIG. ll is a FAB-MS (negative ion mode) of paclitaxel obtained from this invention. FIG. 12 is an HPLC analysis of paclitaxel obtained from this invention. FIG 13 is a TGA spectrum of paclitaxel obtained from this invention. FIG. 14 is a DSC spectrum of paclitaxel obtained from this invention.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES In the present process of the invention, a simple and inexpensive method is provided for extracting purified paclitaxel from various biomass sources, such as plant material containing a mixture of taxanes, for example, from the T-bark. brevifolia (Pacific yew), T. yannanensis, from needles and branches of other Taxus species and from other sources of paclitaxel such as those produced from cell cultures of Taxus species and fungi that produce paclitaxel. The process comprises the selective halogenation, preferably bromination, of unpurified, partially purified or purified mixtures of paclitaxel, cephalomannine and other taxane derivatives containing unsaturated side chains, to selectively transform certain taxanes without destroying paclitaxel. In one embodiment of this process, biomass containing a complex mixture of various taxanes, if such is the case, is preferably processed to a high surface-to-volume ratio to increase mass transfer of paclitaxel during an extraction phase. initial, preferably with a lower alcohol, such as methanol, ethanol, etc. For example, using the bark of T. brevifolia as a source in which the concentration of paclitaxel is typically less than 0.1% w / w, the bark is ground to a fine mixture and then extracted with methanol for a time considered sufficient, so that substantially all the paclitaxel in the sample is extracted. The methanol extract is then concentrated, for example, by means of a rotary evaporator, to preferably concentrate approximately 20 times less in volume than the original extract. Additional extraction can be carried out, for example, by spreading the methanol concentrate between a suitable solvent, such as methylene chloride, chloroform or ethylene dichloride, and water, preferably in a 1: 1 v / v ratio, in the water-soluble components are extracted in the aqueous phase. Such components may be, for example, paclitaxel glycosides soluble in water and other more polar compounds in the mixture that may be reserved for further processing of potentially valuable components. In this embodiment, the organic solvent phase comprising paclitaxel is evaporated to a residue comprising paclitaxel, and can be further purified by sedimenting the impurities. For example, by dissolving the residue in acetone, the non-polar bituminous impurities are separated by precipitation with the addition of an equal volume of hexanes, and can then be separated by filtration. The acetone-hexanes-soluble filtrate is then concentrated and a residue precipitated in fresh hexanes. The precipitate obtained is then dried under high vacuum (1 mm to 2 mm) at 40 ° C.

In this embodiment it is preferred that this residue is subsequently dissolved in a minimum amount of chromatographic solvent, such as methylene chloride or ethylene dichloride, and then subjected to flash chromatography on a column of silica gel. The mobile phase can be, for example, a mixture of acetone and methylene chloride or ethylene dichloride in a ratio of 1: 9 to 3: 7 v / v. A few first fractions of the column contain slightly polar compounds, followed by fractions containing varying concentrations of paclitaxel and cephalomannin. After the fractions have been eluted from the column, the silica gel is washed, for example, with acetone and methanol, and the eluent is discarded. Fractions containing paclitaxel and cephalomannine are subsequently combined and evaporated to re-form a residue comprising paclitaxel. This residue, subsequently, is preferably dissolved in a chlorinated solvent, such as carbon tetrachloride, methylene chloride, chloroform or ethylene dichloride., to form a solution in which various unsaturated taxanes are halogenated under conditions effective for the selective halogenation of the double bond of the unsaturated side chain in the cephalomannin. { and other taxanes containing unsaturation in the side chain) to produce a diastereomeric mixture of dihalocefalomanins in solution with paclitaxel and other taxane compounds halogenated in the side chain. Although all halogens are contemplated for use in this invention, bromine is preferred because of its high efficiency and low cost. Preferably, all of the present cephalomannine is substantially completely converted to the diastereomeric mixture of dihalocepharomannins to provide an easy separation of paclitaxel from the mixture. The taxanes in the chlorinated solvent are also brominated preferably under dark conditions, with vigorous mixing, and at a temperature close to 0 ° C. The reaction rate is desirably slow so that the rate of production of hydrobromic acid is limited and there is little or no hydrolysis of the paclitaxel residues. After chromatographic analysis, for example HPLC, to determine whether all, or indeed all, of the cephalomannin present has reacted completely, the addition of bromine is terminated, and the brominated solvent solution containing, inter alia, paclitaxel and isomers of dibromocefalomanin is preferably washed, for example, first with a dilute solution of sodium sulphite followed by sodium bicarbonate, stopped removing and neutralizing any bromine or hydrobromic acid formed during the reaction. The organic layer is further washed with water and dried with anhydrous sodium sulfate and then evaporated to dryness and the solid residue is dissolved in a solvent, for example, methylene chloride, and the fractions containing Dibromocephalomanin isomers can be separated chromatographically from fractions containing paciitaxel, for example, by column chromatography on a column of silica gel, preferably with a mixture of acetone and ethylene dichloride of 1: 9 v / v. Containing paclitaxel based on TLC and HPLC analysis are combined, and then evaporated to a dry solid residue, from which purified paclitaxel can be recovered by dissolving the solid in acetone and crystallizing the paclitaxel with hexanes.The crystals are filtered, washed and They dry up to a final product.The process is simple and easy to perform.Preferably, the analysis of product streams The products and by-products for paclitaxel and cephalomannin are made by HPLC and TLC, so that any loss of product can be avoided. As shown, the halogenation, particularly bromination, of cephalomannin is a new and convenient way to increase the selectivity of paclitaxel during the chromatographic separation of mixtures containing both paclitaxel and cephalomannin, and unlike various chemical modifications presented by the taxanes, the conditions for bromination can be controlled so that there is no signifi cant loss of paclitaxel during the process. FIG. 3 illustrates a preferred process scheme for the isolation and purification of paclitaxel from T. brevifolia, and FIG. 4 illustrates a preferred reaction scheme for the selective broadening of cephalomannin. According to this invention, the separation process does not depend on the concentration of paclitaxel present, nor on the composition of the complex mixtures which are the starting points for the isolation and purification of paclitaxel. Therefore, the present method of the invention can be conveniently applied to the isolation and downstream purification of paclitaxel from other sources of paclitaxel such as cultured plant cells and fungi that produce paclitaxel, with the stage of selective halogenation contemplated for use at any stage of the procedure to facilitate the isolation and purification of paclitaxel. For mixtures containing cephalomannine and amounts of about 1% to about 99, 9% paclitaxel, the halogenation process is similar to that described above. The mixture is preferably dissolved in a large amount of chlorinated solvent, for example CC1 * or CHC1, and after cooling to about 0 ° C with stirring, a stoichiometric amount of bromine (1.2 molar equivalents relative to cephalomannin) is added. diluted with CC14 or CHCI3 until the cephalomanin is completely joke. The total reaction should be carried out in the dark at temperatures that should not exceed 20G'C, preferably 5? C, and controlled, for example, by HPLC analysis. After the bromination ends, the reaction mixture is washed to remove the excess bromine. In all cases, the cephalomannine and unsaturated taxanes are brominated with a high recovery of paclitaxel. The resulting mixtures containing paclitaxel and brominated compounds are separated and purified using a variety of methods such as chromatography and crystallization. The transformation of the cephalomannin to the less polar dibromo derivative gives possibilities for an easier separation of the paclitaxel from the mixture. The number of molar equivalents of bromine added to the mixture depends mainly on the concentration of cephalomannine and the other unsaturated compounds. Generally, a less pure mixture (which contains a high amount of unsaturated taxanes in relation to cephalomannine) requires more molar equivalents of bromine to completely brominate the unsaturated taxanes that a purer mixture would require. FIG. 2 illustrates the structures of various unsaturated taxanes. If the mixture has a high content of unsaturated compounds, such as taxi ci, ta-icine, taxinin and / or brevifolol, the molar equivalents will be higher because they will absorb more than i molar equivalent of bromine. The solvents that can be used for the halogenation process can be inert to the halogen employed, especially the preferred bromine. Useful and preferred solvents according to this invention are chlorinated solvents such as CC14, CHC13, CH ^ Cl; C ^ H ^ Cl ^, being CC1 «, the most preferred. The halogenation process is more effective within the temperature range from about -20 ° C to about +20, = * C, preferably from about -5 ° C to about S, JC, with bromine. A preferred reagent for the above reactions is 0.0M to 0.1M bromine in carbon tetrachloride, or chloroform, which is commercially available. The conventional approach would suggest that by using a halogen such as bromine to brominate taxane compounds containing various functional groups that are sensitive to bromine or other halogens, various undesired reactions would occur with paclitaxel or the other compounds listed in FIG. 2. However, on the other hand, it has been found that the selectivity for the double bond of the side chain of cephalomannin is unexpectedly very high, as well as for the selective bromination of other taxanes containing exocyclic double bonds. In the process of this invention, paclitaxel is not significantly degraded or brominated during the reaction. Paclitaxel, however, can degrade to several unidentified compounds if the reaction is exposed to a large amount of light, or if a large excess of halogen is used. Any degradation of paclitaxel during the halogenation reaction (bromination) can be easily avoided by periodically monitoring the reaction by HPLC. li = BQßlOS The following examples relate to the purification of paclitaxel by a preferred embodiment of the process of the invention. All chemicals were used as received from the manufacturer / supplier. The Pac! The ith-xel and the cephalo-anin resulting from all product streams and by-products were controlled by thin layer chromatography using Merck No. 5554 silica gel plates F2S4 and by HPLC. The HPLC system consisted of a Waters 510 pump, a 660E system control, a 7J2 WISP or 71 OE WISP autoinjector, a Waters 490 programmed multiple wavelength detector, a Waters 990 photodiode array detector and a NEC computer ACIV and a Watere LamhdaMax model 481 metro spectrofot and a data module. The HPLC columns used include a Waters μbondapak reversed phenyl phase of 3.9 mm x 300 mm and a phenyl reserve column. The silica gel for the "flash" chromagraph was of a mesh size 32-63 supplied by ICN Biomedicals. It should be understood, however, that the following examples are for illustrative purposes only, and are not intended to limit the scope or spirit of this invention or the claims in any way.

EJBMPLfl i Purification of Paclitaxel of Biomass in Crude Stage 1 Cortex of the Pacific yew, Taxus brevifol ia, crumbles to between 2 and 4 mm in size. The concentration of paclitaxel in the cortex is between 0.03-0.1% w / w. 45 kg of the bark are fed to a stainless steel tank. This is extracted three times with 150 liters of methanol.

Each extraction is performed over a period of 5 days with frequent recirculation of the extract to promote mixing. The extract is concentrated by rotary evaporation to a concentrate between 10-15 liters of volume. The temperature of the extract did not exceed 40 ° C. Cas 99% of paclitaxel is extracted in the methanol phase by this method.

Step 2 The methanol concentrate of otap 1 is subjected to partition, for example, between methylene chloride or ethylene dichloride and the like, and water. To 15 liter of the methanal extract equal volumes of methylene chloride and water are added. The mixture is stirred slowly for 1 'minutes and allowed to stand for a period of 2 hours. The two phases are separated. The full metal chloride phase is further processed for the isolation of the pacl axel. If paclitaxel remains in the aqueous phase during the analysis, it is subsequently reextracted with methylene chloride and the methylene chloride fraction is combined with the same fraction as the first extraction. Methanol (0.5-1 liter) is added to the mixture if emulsions are formed during mixing and required to break. The methylene chloride extract is evaporated and dried until dry. The solid residue is between 0.9-1.1 kg containing 1.2-2.2% w / w paclitaxel. The temperature of the product during processing is not allowed to exceed 40C, C.

Step 3 The aqueous fraction of step 2 contains paclitaxel glycosides, 10-desacetyl-baccatin III, baccatin III and other polar compounds. To 35 liters of the aqueous fraction is added 5 liters of brine solution. This is extracted with 20 liters of ethyl acetate. The two phases are separated. The upper ethyl acetate layer contains paclitaxel glycosides, 10-deacetyl-1-baccalane III, baccatin III and other polar compounds. The lower aqueous phase is reextracted with ethyl acetate and the traction of ethyl acetate is combined with the same fraction of the previous extraction. The fraction of collected ethyl acetate, which is between 30-35 liters, is concentrated to CO? I nuación by rotary evaporation to a viscous dark brown solution of between 2.8-3.2 liters. This is stored for further processing to isolate 03 ql cs.

Stage ^. The solid residue of methylene chloride from step 2 is dissolved in 2 liters of acetone. An equal volume of hexane is added under conditions of intensive agitation. Polar impurities are separated by precipitation under these conditions. They are allowed to settle and the supernatant is decanted for further processing. The precipitate is washed with acetone / hexane (1/1 v / v) and the filtrate is combined with the previous supernatant. The supernatant is evaporated to one third (1/3) of the volume by rotary evaporation. The residue v scoso is between 0.5 1 / 3.0 1 and is yellowish brown.

Step 5: The acetone / hexanes residue of step 4 is added dropwise to 10-15 of hexanes, while stirring vigorously. A light yellow material begins to precipitate. After about 8 hours, this material is filtered and dried under high vacuum (1 mm to 2 mm) at 40 ° C. C to give approximately 0.5-0.6 kg of material.

Stage 6 The solid residue from step h is subsequently dissolved in 0.5 liter of an acetonide-methylene chloride mixture 1: 9 v / v, and subjected to flash chromatography on a gel column. silica using the my solvent but the mobile phase. The amount of silica gel used is between 3.5-4 kg. The fractions were collected in 1 liter volumes. Each sample was analyzed by TLC and HPLC. The cephalomanin was coeluted with paclitaxel. The fractions containing paclitaxel and cephalomanin ee are combined and subjected to rotary evaporation to dryness. The solid residue is a crude mixture of paclitaxel and cephalomanin of between 55 g and 70 g containing 4b-b5% w / w of paclitaxel, or between 36 g-40 g of paclit.axel.

Step 7 A crude mixture of paclitaxel and cephalomannine from step 6 found containing 28.8% w / w of cephalomannin and 51.2% w / w of paclitaxel after the analysis is subsequently modified chemically to remove paclitaxel from the cephalomanin. 10 g of the crude product are dissolved in 1 liter of a chlorinated solvent, such as, for example, carbon tetrachloride, chloroform, methylene chloride or ethylene dichloride. In this preferred embodiment, a 0.01M solution of bromine on carbon tetrachloride is reacted very slowly with the crude solution under dark conditions and at a temperature of 0 ° C with vigorous mixing. The progress of the reaction is monitored by HPLC. The bromination reaction is terminated when the cephalomannin has been fully reacted. The trace amounts of bromine are removed by washing with an aqueous solution of its sodium phytate. The brorahydric acid formed during the reaction is removed by washing with a dilute solution of sodium bicarbonate (0.5% w / w). The resulting organic extract is then dried with anhydrous sodium sulfate and concentrated in a rotary evaporator to a solid residue having 13.2 g of weight.

Step 8 The brominated residue from step 7 is dissolved in a mixture of acetone / methylene chloride, 1: 9 v / v, and separated atographically on a column of silica. The collected fractions were analyzed by TLC and HP C Fractions containing paclitaxel were pooled together and evaporated to dryness by rotary evaporation. The solid residue is white and has a mass of 6.1 g.

Etáp¿L2. The solid residue from step 8 was then dissolved in acetone and crystallized with an equal volume of n-hexane or hexanes. The crystals were washed with cold acetone / hexane solution, l / 1 v / v, and then dried under vacuum at 40 ° C. The solid crystals weighed 4.84 g and contained > 97% w / w paclitaxel as measured by HPLC. gJEWPI.9.? Bromination of Partially Purified Cefalomanin A solution of 0.63 g of 91.5% cefalo anin (0.0007 mol) containing approximately 6-7% of paclitaxel dissolved in 150 ml of carbon tetraclaride was added to a flask of round bottom of three mouths, 500 ml, equipped with a separating funnel of 250 me. The flask was immersed in an ice-salt bath. When the temperature reached -5 ° C, a solution of bromine (0.1221 g) in carbon tetrachloride (76.31 ml, 0.01M) was slowly added with stirring at such a rate that the reaction temperature did not exceed C. The ratio of cephalomanin to bromine was 1: 1.1 moles. The addition required approximately 3 hours and the resulting solution was light brown and cloudy.

The brochure was monitored by HPLC analysis every hour. The reaction is complete when all the cephaloma-nine present becomes the 2", 3-bromobromide, which, based on HPLC, required approximately 8 hours. The reaction mixture was light yellow to colorless, due to the bromine consumption. The reaction mixture was subsequently transferred to a one liter separatory funnel and first voided with 0.5% aqueous sodium sulfite (300 ml), 0.5% aqueous sodium bicarbonate (300 ml) and then twice. with deionized water (200 ml each) until a final pH of 6.5. The combined aqueous layer was extracted once with CH ^Cls and the CH ^Cl ^ layer was mixed with the previous organic extract. It was then dried with aaS04, filtered and evaporated to dryness. The yield was 0.76 g of a light cream colored solid having a yield of about 100% based on the raw material. The cream solid material was chromatographed on a column of silica gel (50 g, ICN Silitech, 32-63 D, 60 A) using acetone / CH2Cl2 (10:90) as the eluent. Fractions of fifty ml were collected and monitored by TLC (Silicagel 60 Fas, Merck No. 5554, developed with acetone / CH2Cl- (20/80), and detected using vanillin-sulfuric acid in methanol spray reagent.) . The fractions with a single spot in R £ - 0.64 (Nß 26 - N ° 38 fractions) were mixed, concentrated to dryness to give 0.485 g of a clear to light white crystalline solid, mp sd ^ C, identified as 2", 3" -dibromocefalomanin. The yield was estimated to be 70% based on the starting cephalomannin.

EXAMPLE 3 Bromination of a Crude Mixture Containing Cefalsanine, Paclitaxel and other Taxane Type Compounds Using an apparatus similar to that used in Example 2, a sample of crude paclitaxel (2.0 g) of a mixture of paclitaxel at 51 , 2%, 28.8% cephalomannine and approximately 20% of other taxanoe or non-taxanic impurities, based on HPLC, was dissolved in 150 ml of carbon tetrachloride and 150 ml of CH2Cla, to give a clear light yellow solution. The flask was immersed in an ice-salt bath and stirred. When the temperature reached -5 < : 2C, a solution of 0.1332 g of 100% bromine in 83.13 ml (0.01 M) of carbon tetrachloride (cephalomannan i M: 1.2 M bromine) was added to the solution at such a rate that the temperature of the reaction mixture did not exceed b ^. The addition required approximately three hours and resulted in a brownish, turbid yellow solution. After the addition of bromine was completed, the reaction was allowed to continue under the same conditions for an additional 8 hours, with HPLC analysis of paclitaxel and cephalomannin made every hour. The reaction is complete when the solution is colorless or light yellow and all the cephalomannine has been converted to the brominated derivative. If, after the additional 8 hours, the solution still contains more than 1-2% of cephalin, maintaining the initial conditions, 10 ml of 0.01 M bromine are added dropwise in carbon tetrachloride and left to react for 1 hour before analyzing again with HPLC. The excess bromine in the reaction mixture was removed by washing with 0.5% aqueous Na2SO3 (300 mL), 0.5% aqueous NaRCO * (200 mL), and deionized water (2 x 200 mL). The reaction mixture was dried using anhydrous NaSO4 and concentrated to dryness under high vacuum to give 2.35 g of dry powder of light cream to white color. The dried material was then purified on a silica gel column using the conditions listed in Example 2. The ratio between the mixture to be separated and the silica gel was 1:60, thus 120 g of gel was used of silica. Each fraction was controlled by TLC and every third fraction by HPLC. Fractions with the same K £ in TLC and the same retention time in HPLC were mixed to provide two combined fractions. The fractions (N "25 - N ° 39) that showed a single spot of TLC with Rr 0.64 represented dibromocepharomycin and the fractions (N" 41 - N "81) that showed a single spot of TLC with Rc 0.49 represented The fractions No. 25 - No. 39, during concentration to dryness at about 40 ° C under high vacuum, gave a solid from white to light yellow, 0.460 g (66.6% theoretical yield). of the dibromocefa omanin obtained is as follows: mp 158-160 ° C (chromatographic purity 96.19%) Rg = 0.64 (single spot) on Silica gel 60 FZ _? Piat (Merck, No. 5554) Solvents: acetone: CH ^ l ^ (20:80) Spraying Reagent: Vanillin / Sulfuric acid in methanol Mass Spectrum [FAB] *: [M + H = 990, 992, 994 [M + Na] = 1014 [M + KJ * = 1030 The concentration of the second combined fractions (No. 41 - No. 81) gave 1.16 g (> 100% theoretical yield) of paclitaxel, which was recrystallized using 50:50 acetone / hexane, filtered, washed with the same ratio of cooled solvent and dried under high vacuum at 40 C, C for 24 hours. The yield was 0.902 g (45.11% based on the raw material and 88.1% based on the HPLC analysis of paclitaxel in the raw material) of a white crystalline material. The analysis of the separated and purified paclitaxel is as follows: p.f. of 214 ° C-216 ° C Rt = 0.49 in the presence of authentic sample on silica gel plate F s _ [Merck Nu b554] Solvent System: acetone / CH ^ Cl (20:80) Spraying Reagent : Vanillin / Sulfuric acid in methanol Methanol UV spectrum in CH30H: 228.4 (297146.8) ^ in nm, U) 206.6 (26540.1) IR spectrum in KBr (crtr3 :) 3500, 1105, 1070, ( Tertiary and secondary OH), 3430, 1650, 1580 (-CONH-), 3070, 1610, 1520, 780, 710 (mono-substituted aromatic rings), 2950, 2910, 1480, 1450, 1370, (CH?, CH2, CH), 3020, 1315, 980 (double bond) 1730, 1270 (aromatic esters) 1715, 1240 (> C = 0), 1730, 1180 (acetates), 850 (epoxy ring) Both UV and IR spectra are adapted to those of pure paclitaxel.

EXAMPLE 4 Separation and Purification of Paclitaxel from a Raw Mix of Taxans, and Analysis of the Same. A solution of 10.00 g of crude paclitaxel (on the basis of HPLC analysis the ex content of 28.8% of cephalomannin, 51.2% of paclitaxel and about 20% of other taxane or non-taxanic impurities) dissolved in 1.5 1 carbon tetrachloride in a two-necked, 2 1 flask, equipped with a 500 ml separatory funnel, a reflux condenser, a thermometer and a magnetic stirrer-2.3, and immersed in a bath of ice-salt. The reaction mixture was stirred until the temperature reached -5 ° C and then 41.2 ml of 0.1 M bromine (0.655 g of bromine) in carbon tetrachloride was added dropwise over about 3 hours. The molar ratio in re cephalomannine and bromine was 1: 1.2. The temperature did not exceed 5 ° C. After the addition of bromine was complete, stirring continued while maintaining the temperature from -1 ° C to 5 ° C. The reaction was monitored by HPLC every hour until all the cephalomadne had been converted to the dibrominated derivative (approximately 8 hours). The final color of 1500 1600 ml of solution was light yellow or cream, depending on the color of the starting mixture and the possible presence of a small excess of bromine. To remove any trace of bromine, the reaction mixture was washed with aqueous NaaSO3 at 0.5% (500 ml), 0.5% aqueous NaHCO3 (500 ml), and deionized water (2 x 500 ml). The reaction mixture was then dried with anhydrous Na 3 SO 4 and concentrated to dryness under vacuum to give 13,20 g of a light cream to white solid material. This material was separated chromatographically in a column. of silica gel under the conditions listed above in Examples 2 and 3. A 100 x 5 cm glass column was prepared by the suspension method with 600 g of silica gel (ratio 1:50). The column was eluted with acetone / CH2Cl2 (10:90). 1 1 aceto-na / CH2Cla (25:75) was used as a final wash of the column. Each fraction was analyzed by TLC and every third fraction by HPLC. The fractions (Nß ll - N ° 22) had a single spot at Rf = 0.64 and after combination, concentration and drying in a Buchi rotary evaporator (40"C, high vacuum), they gave 3.25 g (95%) ) of 2"," -dibrs ocefalomanin as a solid from white to light yellow.

The analysis of this compound is as follows: p.f .: 158-160 ° C Re -0.64 (a single spot) on Silica gel 60 F2a4 plate [Merck Nü 5554] Solvent system: Acetone / CH ^ Cl ^. (20:80) Spraying Reagent: Vanillin / Sulfuric Acid in Methanol.

Elemental Composition and Molecular Weight (based on HR FAB-) C4SHs < lNO 4'7í, Br2 [M + H] ~: Calculated: 990.191000 Found: 990.191103 (? m = 0.3 ppm) C4sH54N014'79Brβ Br [M + H] *: Calculated: 992.181000 Found: 992.189057 (? M = 8.1 ppm) C4s_HS4N0X4ß Br2 [M + H] *: Calculated: 994.175000 Found: 994.187011 (? M = 12.1 ppm) C4BH33NOa.4Na ':, 9Brβ:? Br [M + Na] *: Calculated: 1014.161000 Found: 1014.171002 (? M = 9.9 ppm) C4BHB3NOa.4K'7S, BrßlBr [M + K] *: Calculated: 1030.097000 Found: 1030.144940 (? M = 46.5 ppm) UV spectrum in CH3OH (? M? X nm, (e)]: 274.2 (1550.8); 227.1 (18619.4); 221.8 (18325.1) IR spectrum in KBr (cm ") ) 3500, 1105, 1070, (tertiary and secondary OH), 3420, 1670, .1580 (-CONH-), 3110, 3060, 1605, 1505, 770, 710, (onosubstituted aromatic compounds), 3060, 2960, 2915, 2870, 1465, 1370, l-CH3, -CHa-, = CH-), 3020, 1670, 1310, 980 (double bond), 1730, 1270 (aromatic esters), 1715, 1240 (> C = 0), 1730, 1180 (acetates), 855 (epoxy rings), 520 (brominated compounds) NMR of nH in CDC13 1.94 (d, 3H, -C0C (Br) γg-b ") (300 MHz): 1.98 (d, 3H), -HC (Br) £ H ^ -4") (ppm, chain protons 4.63 (qt, 1H, 1H, £ g3 (Br) 3 ') lateral only) 3 C NMR (300 MHz) 170.21 and 170.25 (Cl *) (in ppm, C of the 172.26 and 172.32 (C-1") side chain only) 72.76 and 72.90 ( C-2 *) 69.71 and 69.88 (C-2") 55.34 and 54.52 (C-3) 30.39 and 30.77 (C-4") 27.21 and 27.62 (C-5") EI-MS: [M] * 568, 551, 509, 491, 449, 431, (m / z) (fragments 405, 391, 186, 329, 326, 308, major) 278, 264, 24b, 217, 200, 188, 159, 149, 122, 10b, 91, 83, 77, 55, 43.

DCIMS: [M + H3 * 569, 552, 510, 492, 474, 450, (m / z) (fragments 432, 424, 392, 387, 370, 329, major) 327, 309, 279, 265, 264 , 246, 218, 200, 188, 167, 149, 125, 124, 106, 101, 100, 91, 83, 69 FAB * MS: 1030 [M + KK; 1014 [M + N?? P; 992 (m / z) [M + H] ~ (See Anal. Blem.); 974 [M-H20K; 932 [M-AcOH] "; 914 [M-AcOH-H20] *; 912 IM-HBr] *; 870 [M-BzOH] *; 854 l870-HaO-2HJ; 832 [M-2HBr] *; 705 [M-243 -Ac] *; 569 [T] ~ 551 [T-H20], • 509 [T-AcOH] *; 491 [T-AcOH-H30] ~; 448 [T-Bz-OH] ~; 429; 424 fSH2] *; 413; 405 [S-H20] ~; 391 [S-0-H201 *; 387 [T-ACOH-BZOHJ *; 376; 347 [SO-CO-HCHO] *; 338; 327 [387-T-Ac-OH]; 315; 284 [327-Ac] *; 279; 264 [832-T] * O [424-2HBr] *; 246 [264-HaO] ~; 231; 218 [ 264-HCO-OH] *; 188; 167 [SO, Hß0NBr2] *; 149 [167-H20] *; 133; 122 [Bz-OH] ~; 113; 105 [Bz] *; 91 [C ". H] ~; 83; 77 [CßHe] *; 76; 57; 55; (T = taxane ring in the compound; chain (side) of S-acid in the compound) HPLC: Condition 1 CN Column 10 μ (250x4.6 mmn) Solvent System (40:60) Flow rate 1 ml / min Detector Waters 490 a 227 n Injection volume 20 μl RT -di roeuctt .. oar. 26.06 min Condition 2 Column Curosil G 6 μi (250 x 3.2 m) Solvent system CHaCN: II; .0 (4b: 55) Flow rate 0.75 ml / min Waters detector 490 at 227 nm Injection volume 20 μl "• i '.J'- ihcs-oc.tuoMplm * diastereoisomeric IOrtials: RTX - 23.53 RTZI = 24.50 Thermogabic Analysis 28.04"C, (100.0%), 100.00 ° C (TGA): (99.64%), 150.00 ° C (98.88%), Temperature 175.00 ° C, (95.35%), 180.00 ° C, (% Stability) (86.74%), 200.00 C, (60.38%), 25.00 ° C (45.03%).

Differential Scanning Calorimetry (DSC): 173.76 ° C, 187.73 ° C.

Fractions from No. 26 to No. 28 that had a single spot on TLC (Re 0.49, the same as the authentic paclitaxel sample) and a single peak on the HPLC were combined, concentrated and dried on a Buchi rotary evaporator. (40 ° C, high vacuum mm at 2 rpm), to give 6.10 g of a white solid. This material was crystallized from 60 ml of a mixture of acetone / hexane (50:50), filtered, washed with the same ratio of cooled solvents and dried under high vacuum at 40 ° C (24 hours) to obtain 4, 84 g (92%) of a white crystalline solid identified by comparison with an authentic sample of paclitaxel. The analysis of purified paclitaxel is as follows: p.f. : 214-216OC TLC: R_: 0.49 (in the presence of the authentic sample) Pas4 plate of silica gel 60 (Merck N'5 5554) Solvent system: (20:80) Spraying reagent: ain i 1 lina / Sulfuric acid in methanol.

Elementary analysis ^ • 47 ^ 51 ^ 1"•% C% H% N Calculated 66.11 6.02 1.64 Found 65.97 5.89 1.63 FIG. 5 UV spectrum in CH30H; ("« ««: In nm, (e) 227.2 (29824.1) 208.0 (26256.3) FIG. 6 IR spectrum (KBr) 3500, 1105, 1070 (tertiary OH and (cm-1) secondary), 3430, 1650, 1580 (-C0NH-), 1610, 1520, 780, 710 (monosubstituted aromatic rings), 2950 , 2910, 1480, 1450, 1370, (-CH3, -CH2-, > CH-groups) 3020, 1315, 980 (double bond) 1725, 1270 (aromatic esters) 1710, 1240 (> C = 0) 850 (epoxy rings) FIG. 7a XH NMR spectrum: 1.88 (S, 10H, C-1); 5.66 (d, 1H, - (300 MHz, CDCl 3) C-2); 3.82 (dd, 1H, C-3); 2.38 (S, 3H, CH3COO to C-4); 4.94 (dd, 1H, C-5); 1.88 (ddd, 1H, C-6); 2.48 (ddd, lH, C-6); 2.53 (d, 10H, C-7); 4.38 (dd, 1H, C-7); 6.27 (S, 1H, C-10); 2.23 (S, 3H, CH3C00 to C-10); 6.20 (qt, 1H, C-13); 2.27 (ddd, lH.C-14); 233. { dd, 1H.C-14); 1.13 (S, 3H, C-19); 1.23 (S, 3H, C-18); 1.78 (S, 3H, C-18); 1.68 (S, 3H, C-19); 4.20 (dd, 1H, C-20); 4.30 (S, 1H, C-20); 3.77 (S, 1H, C-2 '); 4.78 (ddd, 1H, C-2 '); 5.20 (ddd, 1H, C-3 '); 7.10 (d, 1H, N-1); 7.30 + 7.53 (m, 10H, protons p and m in aromatic rings Aj., B & Cx); 7.64 (t, lH, A-p); 7.72 (dd, 2H, Cx-o); 8 (ll (, 2H, A, -o).

FIG. 7b 13 C NMR spectrum 79.1 (C-1); 75.1 (C-2); 4b, 8 (300 MHz, CDC13) (C-3); 81.2 (C-4); 84.4 (C-5); (ppm) 35.6 (C-6); 72.1 (C-7); 56.7 (C-8); 203.6 (C-9); 75.6 (C-10); 133.3 (C-11); 141.9 (C-12); 72.3 (C-13); 35.7 (C-14); 4.3.2 (C-15); 21.8 (C-16); 26.9 (C-17); 14.7 (C-18); 9.5 (C-19); 76.5 (C-20); 73.3 (C-2 '); 55.1 (C-3 '); 20.7 (£ H-, CO to C-10); 22.6 (CjJ3C0 to C-4); 170.3 (3C0 to C-10); 171.1 (CJg3CO to C-4); 167.0 (ArCO-Aa; 167.0 (ArC-Ca); 172.7 (PhlSCO-); 129.3 (aC-A; 133.8 (aC-B ^); 138.1 (aC-C; , 3 (? -C, A?), 127.0 (? -C, Ba), 127.0 (oC, C_), 128.7 (m-CA, 128.6 (m- C ^ a.); 129.0 (m-CC i 133.6 (p-CA-; 131.9 (p-CB; 128.3 (pC, C).

FIGS. 8a and 8b EIMS: [M] * = 853 568 [T] *; 550 [T-HaO; 508 [T- (m / z, the fragments AC? H] ~ 490 [T-AcOH-H ^ O] *; 448 major) [T-2ACOH] * O [T-BzOH] "; 386 [T- AC? H-BzOH] ~; 326 [T-BzOH-2AC-OH] ~; 308 [326-H20] ~; 286 [MT] "O [S]"; 280; 268 [SO] -; 240 [S - 0-CO] ~; 210 [SO-CO-HCOH] *; 122 [BzOH] *; 105 [Bzl *; 91 [C7H7] ^; 77 [CeHs] - \ - 51; 43 [Ac] *.

FIG. 9 DC-MS: [M + H] * = 854 569; 551; 509; 492; 449; 387 (m / z; fragments 327; 311; 287; 269; 240; 224 major) 222; 210; 165; 149; 123; 105 92; 71 FIG. 10 FAB 'MS: 892 [M + K] *; 876 [M + Na] -854 (m / z; the fragments [M + Hj *; 569; 551; 523; 509; 495; major) 369; 327; 286; 240; 210; 177; 155; 149; 119; 105; 85; 69 FIG. 11 FAB * MS 852 - [M + HJ * FIG. 12 HPLC: Column μBondapak Phenyl Solvent System CH ^ CN: CH30H: H ^ 0-13: 20:48 Flow rate 1 ml / min Waters detector 490uv at 227 nm Injection volume 20 μl FIG. 13 TGA: Temperature (stability) 50.00ac (100.0% 205.00ßC (99.86%), 215.00 ° C (99.10%), 220.00aC (92.19%), 250.00 ° C (56.66%), 275.00 ° C (45.92%).

FIG. 14 DSC: 210.85ßC Water content (% H20): 0.90% (Karl Fischer) HO.VEPAP PK LA I VEN IOM Having described the present invention, it is considered as novelty, and therefore, the content of the following claims is claimed as property :

Claims (34)

1. RfílYímilCAClQ? ÜS l. A method for the isolation and purification of paclitaxel from organic material comprising a mixture of taxanes; wherein said process comprises; (1) extracting a composition comprising a taxazo compound of said organic material; (2) chromatographically separating a mixture comprising paclitaxel, cephalomannine and other taxanes of said composition; and then (3) reacting said mixture with a halogen under conditions effective for the selective conversion of cephalomannin into a diastereomeric mixture of dihalocepharomannins; and then (4) separating said paclitaxel from said mixture.
2. 2. The method according to claim 1, wherein the organic material is extracted with a first extraction solvent which is then evaporated to form a first residue comprising paclitaxel, then extracting said first residue with a second solvent of extraction which is then evaporated to form a second residue comprising paclitaxel, and then further purifying said second residue by crystallization. The method according to claim 2, wherein said first extraction solvent is methanol and wherein said first residue comprising paclitaxel is partitioned between water and a solvent selected from the group consisting of methylene chloride, ethylene dichloride and chloroform, and said residue is extracted into said solvent and dried to said second residue, and said second residue comprising paclitaxel is dissolved in acetone and the non-polar impurities are separated by precipitation with hexanes, wherein the solution of acetone-hexanes is then evaporated to approximately one third (1/3) of the volume to form a third viscous residue comprising paclitaxel. 4. The method according to claim 4, wherein said solvent and water are present in a ratio of about 1: 1 v / v. The method according to claim 3, wherein said third viscous residue is precipitated by the addition of about ten times the volume of hexanes to form a light yellow precipitate to form a fourth solid residue comprising paclitaxel. 6. The method according to claim 4, further comprising dissolving said fourth solid residue in acetone with methylene chloride and / or ethylene dichloride, which is then subjected to flash chromatography on a gel column. silica in a chromatographic solvent to obtain eluent fractions containing mixtures of paclitaxel and cephalomannine which are combined and dried to form a fifth residue comprising paclitaxel. The method according to claim 6, wherein said chromatographic solvent is a mixture of acetone and methylene chloride or ethylene dichloride in a ratio of about 1: 9 to about 3: 7 v / v. 8. The method according to claim 6, wherein said fifth residue is dissolved in a chlorinated solvent selected from the group consisting of CC14, CHC13, C2H4C12 and CH2C12 and reacted with a halogen. 9. The method according to claim 8, wherein said halogen is bromine. The method according to claim 9, wherein said bromine is present in a solution concentration of about 0.01M to about 0.1M in halogenated solvents. 11. The method according to claim 10, wherein the reaction with bromine is carried out at a temperature ranging from about -20 ° C to about 20 ° in the dark 12. The method of agreement with claim 11, wherein substantially all of the cephalomanin present in said chlorinated solvent solution is converted to a stereoisomer mixture of dibromocephal ornanes, and the paclitaxel is then separated from said reaction mixture. according to claim 12, wherein said organic matter comprising paciitaxel is selected from the group consisting of Taxus brev ifolia root, a plant material of the taxus species, a cell culture of the taxus species and a fungus that produces paclitaxel. The method according to claim 12, wherein said paclitaxel is separated from said reaction mixture by silica gel chromatography with a chromatographic solvent comprising a mixture of acetone and methylene chloride or ethylene dichloride present in a ratio that varies from approximately 1: 9 to approximately 3: 7 in volume. 15. The method according to claim 12, wherein said separated paclitaxel is crystallized with a mixture of acetone and hexanes. 16. A method for separating paclitaxel from a mixture comprising paclitaxel and cephalomannin, comprising the steps of a) reacting said mixture with a halogen at a temperature and for a time sufficient to halogenate substantially all of the cephalomannine, and b) separating the paclitaxel. of the halogenated cephalomannin. 17. A method according to claim 16, wherein the halogen is selected from the group consisting of fluorine, chlorine, bromine and iodine. 18. The method according to claim 17, wherein the halogen is bromine. 19. The method according to claim 18, wherein the bromine is present in a concentration ranging from about 0.01M to about 0.1M. The method according to claim 16, wherein the reaction of a) is carried out at a temperature ranging from about -20 ° C to about 20 ° C, C. 21. The method according to claim 19, in bromine is present in a chlorinated solvent selected from the group consisting of CC14, CHC1, and CH2C12. 22. The method according to claim 21, in the chlorinated solvent is CC14 or CHC13. 23. The method according to claim 16, wherein the reaction of a) is carried out in the dark. 24. The method according to claim 16, wherein the separation of b) is carried out by chromatography with silica gel in a suitable solvent. 25. The method according to claim 24, wherein the solvent is a mixture of acetone and methylene chloride, 1,2-dichloroethane in a volume ratio of about]: 9 to about 3: 7. 26. The method according to claim 25, wherein the fractions containing paclitaxel are evaporated to a solid residue. 27. The method according to claim 26, further comprising purifying the pacl Ltaxel by crystallization. 28. The method according to claim 27, wherein the solid residue is dissolved in acetone. 29. The method according to claim 28, wherein the paclitaxel is separated by crystallization with hexanes. 30. The method according to claim 25, wherein the paclitaxel is separated from a diastereomeric mixture of 2", 3" -dibromocefalomanin. 31. The method according to claim 16, wherein the reaction of a) is controlled by high performance liquid chromatography. 32. The method according to claim 16, wherein the mixture is derived from a source containing paclitaxel. The method according to claim 32, wherein the source containing paclitaxel is selected from the group consisting of the bark of Taxus brevifolia, plant material of a Taxus species, a cell culture of Taxus species, and a fungus that produces paclitaxel. 34. A method for separating pac itaxel from a mixture of paclitaxel and cephalomannine, comprising the steps of a) reacting said mixture with bromine at a temperature and for a time sufficient to brominate suetanially all of the cephalomannine, and b) separating paclitaxel from the brominated cephalomanin.