JP2007210942A - Method for extracting paclitaxel and 9-dihydro-13-acetylbaccatin iii from taxus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for effectively and environmentally safely isolating analog of taxanes. <P>SOLUTION: The method comprises use of a preparative high-performance liquid chromatography in an industrial scale. The method uses a "Load and Lock Axial Compression Column", wherein the column is packed with any packing material, including small particle size (e.g., about 10 μm). The method is one in which a piston is used to maintain pressure, and the maintaining of the pressure effectively and substantially prevents formation of voids in the bed and the efficiency of the process is effectively increased. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to the isolation and purification of taxane derivatives from naturally derived Taxus species.

  Taxol was first isolated in 1971 by Wani et al. From the yew, Taxus brevifolia. Taxol is one of the taxanes that are diterpenes. Taxanes are diterpene compounds that have found utility in the pharmaceutical field. Taxanes can be found from plant material and have already been isolated. Paclitaxel is a well-known chemotherapeutic agent for the treatment of various metastatic cancers. Approved for the treatment of ovarian and breast cancer.

  Taxol and various taxane derivatives are very cytotoxic and have strong in vivo activity in various leukemia and tumor systems. Taxol is thought to be a particularly useful cancer chemotherapeutic agent.

  The only current source of taxol is some species of yew (Yew, Yew) that grow very slowly. Paclitaxel is a natural product that was first extracted from bark of Taxus brevifolia. T. baccata, tee. T. valiciana, T. T. yunnanensis and T. It has also been discovered from T. canadensis. Current isolation methods are very difficult and yields are low. A method for extracting taxol from yew trees using a methanol solution has been reported. However, since taxanes are present in plant material in relatively small amounts, for example, taxol, will destroy large amounts of the very slow growing yew tree that forms the compound source. Furthermore, a large amount of organic solvent is used in the conventional liquid extraction, which takes time.

  The concentration of paclitaxel in the various raw materials is very low, for example, on the order of about 0.0004 to about 0.01% (w / w) from the bark of Taiyo yew. Due to such a low concentration, it is very difficult to extract and purify the compound from a raw material to a grade that can be used in pharmaceuticals, so far, it has not been put into practical use on a commercial scale. Various normal phase chromatography techniques have been used to date to purify paclitaxel from raw crude extracts.

  The success of low pressure chromatography depends on the nature of the column. The use of silica gel and aluminum trioxide, which are conventional supports in the stationary phase, in distribution systems is associated with various problems. They form a stable stationary phase with most solvent systems, but are highly adsorbed and partly involved in the separation process, thus affecting chromatographic behavior and sample recovery.

  Chromatographic methods have been developed to detect and isolate paclitaxel from various yew species for analytical and preparation reasons. Since these isolation methods are mainly carried out on a small laboratory scale, there are problems of low selectivity and low recovery and high production costs, and paclitaxel, a valuable anticancer compound, is very There is a serious and unrealized need for an economical and practical method of separating from similar cephalomanine and other highly related taxanes.

  The use of cultivatable and renewable plant parts of yew species such as leaves (needle leaves) and twigs should be the most practical and attractive way to increase the supply of paclitaxel. Several needles of the yew species, including Taxus canadensis, have been studied and found to contain an amount of paclitaxel comparable to the bark of Taxus brevifolia.

  Taxus canadensis is an evergreen shrub mainly found in eastern Canada and the northeastern United States. This species is unique in its taxane content. The acicular leaves are paclitaxel (0.009-0,05%), 10-deacetylbaccatin III (10-DABIII, together with 9-dihydro-13-acetylbaccatin III (9-DHABIII, 4), which is the main taxane. 6), baccatin III (5), cephalomanine (3) and a few other taxanes. The concentration of 9-DHABIII in the needles is reported to be 7 to 10 times the concentration of paclitaxel. 9-DHABIII can be an important precursor of a new class of semi-synthetic chemotherapeutic agents with increased water solubility.

  Prior art methods disclose the use of various chromatographic techniques to separate paclitaxel and its related taxanes, which include normal phase chromatography and reverse phase chromatography on silica gel or coupled silica gel columns. including. Prior art methods have been difficult to scale up to larger industrial scale production due to low yields, high production costs, or the need for multiple steps.

  Currently, producing taxol from ornamental yew needles, bark and roots contains an extremely high percentage of unwanted impurities in the extract during extraction (about 40% of the dry plant material). To 50% by weight), which is not economical. Thus, the very high proportion of impurities in the solvent extract in the ornamental yew needle leaves is taxol from this source in addition to the high cost of drying the needle leaves. And the purification of taxanes is expensive and uneconomical.

  Current methods are time consuming and expensive, or are limited to analytical scale in practice. Since paclitaxel is present at low levels in needles and the needles contain a large amount of wax, it is not possible to isolate and purify paclitaxel from needles to clinically acceptable purity. , And even more difficult. The separation of paclitaxel from cephalomanine, an analog closely related to the paclitaxel present in needles and bark, is a difficult task. The two analogs have been separated by selective chemical conversion of cephalomanine in a mixture containing both cephalomanine and paclitaxel. Disadvantages associated with this method include the additional cost of using expensive, sometimes toxic reagents, the additional chromatography required to separate the converted cephalomanine from paclitaxel, cephalomannin during the process, and sometimes paclitaxel. And further chemical transformations necessary for cephalomanine recovery.

  The most relevant prior art is believed to be US Pat. No. 5,969,165 granted on October 19, 1999 to Liu et al., Which provides high yields of taxane analogs from resources containing taxanes. A method to obtain with high purity was proposed. The method uses a polymeric resin adsorbent for separating analogs under low pressure without using complex and expensive isolation / purification steps as currently provided in the prior art. The method of isolating and purifying taxane analogs from a taxane-containing resource included a first step of extracting the taxane source in an organic extract such as dichloromethane. The second step included the step of bringing the extract into contact with the adsorption medium and loading the medium into the column. The column contained an adsorbent such as, for example, aluminum oxide. The next step is to elute at a pressure of about 68.95 to 137.9 kPa (10 to 20 psi) using an organic solvent mixture such as a mixture of hexane and acetone to produce a fraction containing the taxane compound. It included that. The next step included crystallizing the fraction to provide a solid taxane compound and mother liquor and concentrating the mother liquor. The next step involved eluting with a polymeric resin using a polar solvent mixture and a mother liquor to provide at least a second taxane compound.

Thus, the prior art has continued to pursue even more efficient and environmentally safe methods to obtain taxanes with minimal use of plant materials and organic solvents.
While various publications and patents describing the isolation and purification of paclitaxel and taxanes from yew species are on the rise, the currently known procedures for isolating paclitaxel are very complex and difficult, and low Yield. The list of such exemplary patents described below describes a variety of isolation techniques, from normal phase chromatography to reverse phase chromatography. The yield of paclitaxel from various yew species ranges from 0.005 to 0.017%.

US Pat. No. 5,019,504 granted to Christen in 1991;
US Pat. No. 5,380,916 granted to Rao on January 10, 1995;
US Pat. No. 5,380,916 granted to Rao on January 10, 1998;
U.S. Pat. No. 5,407,674, granted to Gabetta et al. On April 18, 1995;
U.S. Pat. No. 5,620,875 granted to Hoffman et al. On April 15, 1997;

U.S. Pat. No. 5,670,673 granted to Rao on September 23, 1997;
US Pat. No. 6,503,396 granted to Kim et al. On January 7, 2003;
Canadian Patent No. 2126698 granted to Nair on November 8, 1993;
Canadian Patent No. 2,157,905 granted to Durand et al. On March 18, 1994; Canadian Patent No. 2213952 granted to G. Caron et al.

  It would be advantageous to provide a more efficient and environmentally safe method for obtaining taxanes with minimal use of plant materials and organic solvents.

  The present invention achieves such desired results and provides a method for isolating commercially important natural products from readily available biomass and for obtaining taxanes from mixtures containing other compounds or materials. Providing an efficient method, providing an isolation process that can be modified to produce paclitaxel and other taxanes on an industrial scale from bark, needles or cell cultures of yew species, plant material, Preferably, taxol and other taxanes are isolated and separated from the raw raw material of ornamental yew in high yield, providing a method that greatly reduces the cost of producing taxane derivatives, and is readily available and renewable A method is provided for providing commercial quantities of the above-mentioned natural products from resources.

  In a first aspect, the present invention is an improvement in a method for isolating and purifying a taxane analog from a resource comprising a taxane analog, the method comprising passing a solution containing the taxane derivative through a chromatography column. Widely suggest. The improvement includes compressing the packing medium in the column to substantially eliminate voids in the column and providing an organic solution comprising a taxane derivative containing impurities. The chromatographic column is maintained under high pressure conditions, for example, from about 172.75 to 2758 kPa (250 to 400 psi), or up to 34475 kPa (5000 psi).

  The present invention provides a second embodiment, wherein the second embodiment includes a preliminary step of passing an organic solution containing a taxane containing impurities through a chromatography column, wherein the packing medium in the column contains voids. Compressed to substantially eliminate and maintained at a high pressure of, for example, about 2068.5 to 2758 kPa (300 to 400 psi), or up to 6895 kPa (1000 psi).

  The present invention provides a third embodiment, the third embodiment comprising passing a paclitaxel syrup extracted in an organic solvent through a chromatography column, wherein the packing medium substantially eliminates voids in the column. And is maintained at a pressure of up to about 6895 kPa (1000 psi), such as about 206.85 to 344.75 kPa (30 to 50 psi).

  The present invention provides a fourth aspect, wherein the fourth aspect is the step of extracting yew biomass with an organic solvent, the step of partitioning the solution with hexane and water, and then the solution with dichloromethane or chloroform. Redistributing with water and recovering a solution of paclitaxel in dichloromethane or chloroform containing impurities.

  One feature of the first aspect of the present invention includes performing a further step of passing an organic solution containing a taxane derivative containing impurities through a chromatography column, wherein the packing medium in the column is a void in the column. And then maintained at a pressure of up to about 2758 kPa (400 psi).

Other features of the first aspect of the invention include the following:
A further step of passing the extracted organic solution containing the taxane derivative containing the impurities through a chromatography column is performed, the packing medium in the column is compressed to remove voids in the column, and then about 2758 kPa (400 psi). ) To provide paclitaxel extracted into an organic solvent, and pass the paclitaxel syrup extracted into the organic solvent through a chromatography column, the packing medium in the column being in the column Compressing and maintaining a pressure of about 344.75 kPa (50 psi);
A preliminary step of extracting yew biomass with an organic solvent, a step of partitioning the solution with hexane and water, a step of redistributing the solution into dichloromethane or chloroform and water, and finally a crude paclitaxel Recovering a dichloromethane or chloroform solution;
Selection of the organic solution as methanol, ethanol, a mixture of methanol and ethanol, a mixture of dichloromethane and methanol, or a mixture of dichloromethane and ethanol;
Selecting diphenyl group-bonded silica gel or C-18-bonded silica gel as the packing medium;
Eluting the column with a gradient solution of acetone / water, methanol / water or acetonitrile / water;
Selecting a flow rate of about 350 ml / min and selecting a pressure maintained between about 4137-5516 kPa (600-800 psi); and recrystallizing and recovering substantially pure paclitaxel.

Features of the second aspect of the invention include:
Selecting acetone / water, ethanol / water or isopropanol / water as the organic solution;
Selecting polystyrene-DVB resin, polymethacrylate resin or polyaromatic resin as packing medium in the column;
Eluting the column with a gradient solution of acetone / water, methanol / water or acetonitrile / water, for example, at a flow rate of about 1 liter / min; and recrystallizing impure paclitaxel from the column eluate Process.

Features of the third aspect of the invention include:
Selecting polystyrene-DVB resin, polymethacrylate resin or polyaromatic resin as the filling medium;
The choice of column elution is to use a step gradient of acetone / water, methanol / water or acetonitrile / water;
Selecting a flow rate of eluate through the column of about 350 ml / min and selecting a pressure of about 4137 to 5516 kPa (600 to 800 psi) or about 6895 kPa (1000 psi); and elution from the column A further step of recrystallizing 9-dihydro-1,3-acetylbaccatin III from a part of the product.

  A feature of the fourth aspect of the invention includes the step of selecting a solution of the taxane analog as the crude crystalline paclitaxel ethanol solution.

  In short, in more general terms, the present invention uses “Load and Lock Axial Compression Column” for preparative high performance liquid chromatography on industrial scale. This suggests a purification technique for extracting an organic solution of paclitaxel.

  Successful use of medium and large diameter columns in preparative high performance liquid chromatography requires appropriate hardware. A “load-lock axial compression column” is one in which a piston is used to fill or unpack the chromatographic bed and to maintain the compression of the bed in use. This effectively and substantially avoids the formation of voids in the floor. The column is packed with any packing material, including small particle size (about 10 μm) media, and a very high plate number is produced. The bed length is adjusted to control the amount of packing material used to prepare the column.

  The use of such a “load-lock axial compression column” in the present invention provides a commercially economical process for extracting paclitaxel from yew species. The process includes a series of procedural steps. The last important step can be carried out in a paclitaxel solution containing impurities prepared by a third step or not prepared. Similarly, the third step can be performed in a paclitaxel solution containing impurities prepared by the second step or not prepared. Additionally, the second step can be performed in a paclitaxel solution containing impurities prepared by the first step or not prepared.

  In a preferred embodiment, the first step comprises extraction from the plant material of the yew species, preferably Taxus canadensis (Canadian yew). As any part of the plant that contains one or more taxanes, for example, bark, roots, leaves or needles, branches, twigs, seeds or whole seedlings can be used. Preferably, the cut off needles and twigs are dried and crushed. The milling of the plant material can be accomplished by means of prior art such as by use of a chipper and / or a mill. Taxanes can be extracted from the whole plant or from parts of the plant such as, for example, steps, roots, leaves (needle leaves), seeds or mixtures thereof. The material to be extracted is either raw or dried. Preferably, needle-shaped leaves are used.

The plant material is preferably first dried and ground to a suitable particle size, usually in the range of about 0.001 to about 10 mm ³ . The plant material is extracted using an organic solvent such as methanol, ethanol, a mixture of methanol and ethanol, a mixture of dichloromethane and methanol, or a mixture of dichloromethane and ethanol. The filtrate from the extract is mixed with water and concentrated to reduce the liquid volume. The concentrate is partitioned between hexane and water to allow a degreasing process. The aqueous liquid is redistributed with water and an organic solvent such as dichloromethane, chloroform or ethyl acetate and concentrated to a concentrated syrup.

  In a preferred embodiment, the second step comprises passing the thick syrup through a column. A preferred solution for this ion exchange is the concentrated syrup prepared in the first step, which is diluted with an organic solvent such as acetone, ethanol or isopropanol. However, any suitable organic solution containing extracted paclitaxel is the starting material in this second step.

  For example, a syrup in dichloromethane / acetone / water is divided into a plurality of fractions by injecting into a medium pressure column containing a polymer adsorbing resin. The polymeric adsorption resin can be, for example, a polystyrene DVB resin, a polymethacrylate resin, or any other suitable polyaromatic resin. The most common type of resin used in polymer adsorbent resins is a polystyrene polymer with crosslinked divinylbenzene. The polymeric adsorption resin should have an appropriate particle size, which makes it appropriate for the column size, specified working pressure and flow rate. One non-limiting example is a particle size of about 5-100 mesh.

  For example, an elution solution with a step gradient of a suitable water-miscible solvent such as acetone / water, methanol / water, ethanol / water or acetonitrile / water is passed through the column. The flow rate is variable and can be changed according to various operating conditions. One non-limiting example is a flow rate of about 2 liters / minute.

The fraction containing up to about 45% acetone / water allows the formation of 9-dihydro-1,3-acetylbaccatin III crystals.
The fraction containing more than about 45% acetone / water is concentrated to remove most of the acetone. They are extracted with a suitable organic solvent such as ethyl acetate, CH ₃ CL or CH ₂ Cl ₂ and concentrated to provide a crude solid residue of paclitaxel.

  The third step involves the purification of crude solid paclitaxel. Preferably, this crude solid paclitaxel is the product of the second step. However, the crude solid paclitaxel produced can be the starting material for this third step.

  The crude solid paclitaxel is dissolved in a suitable water-miscible solvent such as acetone to allow the degreasing process, and the water-miscible organic solvent / water solution such as acetone / water solution is axially compressed. Eluted by column chromatography. The system is a high performance liquid chromatography with means for providing and maintaining a high pressure, such as about 172.75 to 2758 kPa (250 to 400 psi) or about 34475 kPa (5000 psi).

  This column containing the appropriate ion exchange resin as described above is loaded with the paclitaxel solution and eluted, for example, with an acetone / water gradient at the high pressure described above. Such a fraction containing paclitaxel in acetone / water is crystallized and dried to provide semi-pure paclitaxel.

  The final step involves purifying semi-pure paclitaxel. Preferably, semi-pure paclitaxel is the product of the third step. However, the semi-pure paclitaxel produced can be the starting material for this final step.

  The column used in the final step is the same as the column used in the third step. The ion exchange resin or packing medium in the column can be diphenyl group bonded silica gel or C-18 bonded silica gel, or any other reversed phase packing material.

  Semi-pure paclitaxel is dissolved in a suitable water-miscible solvent such as acetonitrile, ethanol or methanol. This solution is loaded into a packed column and used, for example, with a water-miscible organic solvent / aqueous gradient solvent such as acetonitrile / water at a suitable rate such as about 350 ml / min and for example about 4137-5516 kPa ( 600 to 800 psi) or about 6895 kPa (1000 psi) at a suitable pressure maintained high.

  The fraction containing pure paclitaxel was crystallizable and the crystals were recrystallized from acetone / hexane. A pure white powder of paclitaxel was obtained.

Biomass Extract Plant material (Canadian yew, Taxus canadensis) was collected in the fall of 2002 at Prince Edward Island and New Brunswick, Canada, and contained approximately 0.032% paclitaxel. Prior to crushing fresh cuts of Canadian yew, they were dried in a drying kiln at about 60-70 ° C.

  600 kg of ground needle-leaf and twig biomass was placed in a 3000 extractor equipped with a reflective concentrator. Next, 2400 liters of methanol was added. The biomass was refluxed with methanol for about 3 hours, the solvent was filtered, and the first filtrate was recovered. 2000 liters of methanol was added to the biomass residue after filtration, refluxed for about 2 hours, and filtered. The second filtrate was collected and the biomass was discarded. The first and second filtrates were combined and about 10% water was added. The aqueous solution was concentrated with an evaporator under vacuum to about 15% of the initial volume.

  The concentrate was partitioned twice with hexane and water to degrease the concentrate. The aqueous layer was collected and redistributed twice with dichloromethane (or chloroform) and water. The organic layer was collected and concentrated with an evaporator to a thick syrup under vacuum. The hexane layer was collected and the aqueous layer was discarded.

  About 45 kg of syrup was obtained from 600 kg of biomass. The dichloromethane syrup contained about 1-1.3% paclitaxel.

Primary purification of paclitaxel 23 kg of dichloromethane syrup from Example 1 was diluted with 10 liters of acetone in a vessel equipped with a mechanical stirrer, with warming as needed. Agitation was adjusted to 1.5 times per second and 3 liters of deionized water was added slowly over 10 minutes. An aqueous solution of dichloromethane syrup was separated into a plurality of fractions by a stainless steel industrial scale medium pressure column equipped with a medium pressure metering diaphragm pump.

  A slurry made of a methanol solution of 120 kg polystyrene-DVB resin (Rohm-Hass XAD-1600) having a particle size of about 50 to 100 mesh was injected into a column having a diameter of 300 mm and a length of 3000 mm. The column was fixed while pumping the solvent at 30-50 psi (206.85-344.75 kPa), and then methanol was replaced with about 30% acetone water until the column was equilibrated.

  The diluted syrup sample moved to the top of the column. The vessel was rinsed with a few liters of 30% acetone water and the slurry was transferred to the column. The column was then sealed and the elution solution began to flow while pumping into the column. The column was then eluted using a step gradient of 35, 45, 50, 60, 65, 70 and 80% acetone / water. The flow rate was controlled at about 2 liters per minute. About 20 liters of each fraction was collected with the pressure maintained at about 30 to 50 psi (206.85 to 344.75 kPa).

  After first washing with 80% acetone / water, the column is washed with pure acetone and using a mixture of acetone / ethyl acetate (1: 1) until the effluent, which is initially quite dark, becomes colorless. And washed.

After washing, the column was again brought to equilibrium with 30% acetone water, which was regenerated and ready to proceed to the next step.
The collected fractions were monitored by UV absorption at 227 / nm using analytical HPLC and by TLC by spraying with a 10% aqueous ethanol solution. Fractions containing 9-dihydro-13-acetylbaccatin III (mostly in 45% acetone / water) are combined and concentrated to remove most of the acetone and in the hood overnight until crystallization is complete Left alone. The crystals were collected by filtration and recrystallized from methanol to obtain 240 g of pure 9-dihydro-13-acetylbaccatin III as white needle crystals. Yield: 0.08%.

  Fractions containing paclitaxel (mostly in 65% acetone / water) were combined and concentrated to remove most of the acetone. The aqueous solution was extracted with ethyl acetate. The organic layer was collected and concentrated to dryness. The residue was analyzed by analytical HPLC. The residue (crude paclitaxel material) contained about 10-15% paclitaxel. The paclitaxel recovery was about 90-100% from dichloromethane syrup.

Secondary purification of paclitaxel The secondary purification used a preparative high performance liquid chromatography system referred to as “axial compression column chromatography”. The column used was a Varian load-lock axial compression column (250 mm diameter x 1000 mm total length). The chromatography system was a Varian ST-4000 system, equipped with two high-pressure diaphragm pumps, a sample inlet port, a UV detector, and a Varian control software system. The column was equipped with a hydraulic piston and lock adapter for filling and withdrawing the column. After completely filling the column with an ethanol solution of 50 liters of polystyrene-DVB resin (Rohm-HassCG-161m, particle diameter of about 75 μm), the hydraulic piston was operated. The piston was gradually pushed out to compress the column packing media at a pressure of about 3000 psi. While the column was compressed, the lock was switched to its locking position to lock the column. Therefore, the column produced a very high number of plates. The column was fixed while pumping solvent at 172.75 to 2068.5 kPa (250 to 300 psi), and then ethanol was replaced with about 30% acetone water until the column was equilibrated.

  About 1.5 kg of the residue obtained in Example 2 was dissolved in 4 liters of acetone until all solids were dissolved. While stirring, 2 liters of water was gradually added and filtered as necessary. The aqueous solution was then pumped into the column from the sample inlet port.

  The column was then eluted with an acetone / water gradient solvent (starting with 50% aqueous acetone and ending with 100% acetone). The flow rate was controlled at 1 liter per minute. Each approximately 5 liter fraction was collected with the pressure maintained at approximately 2068.5 to 2758 kPa (300 to 400 psi).

  The collected fractions were monitored by UV absorption at 227 / nm using analytical HPLC and by TLC by spraying with a 10% aqueous ethanol solution. Fractions containing paclitaxel (mostly in 70% acetone / water) were combined, concentrated to remove most of the acetone, and left in the hood overnight until crystallization was complete. The crystals were collected by filtration, recrystallized with 70% aqueous methanol, and dried in a vacuum oven to obtain 235 grams of slightly white needle crystals. This semi-pure paclitaxel is about 70-80% pure. Yield: about 90-95% from crude paclitaxel material.

Final purification of paclitaxel Preparative high performance liquid chromatography and axial compression chromatography columns were used for final purification. The column used was a Varian loadlock type axial compression column (diameter 100 mm × total length 1000 mm). The chromatographic system was a Varian SD-2 preparative HPLC system equipped with a sample inlet port, a UV detector, and a Varian control software system. The column was equipped with a hydraulic piston and lock adapter for filling and withdrawing the column. The packing medium used was diphenyl group bonded silica gel or C-18 bonded silica gel.

  After completely filling the column with 6 liters of packing medium in ethanol, the hydraulic piston was activated. The piston was gradually pushed out to compress the column packing media at a pressure of about 3000 psi. While the column was compressed, the lock was switched to its locking position to lock the column. Before using the column in purification, the ethanol was replaced with about 30% acetonitrile water until the column was equilibrated.

  About 50 g of crude paclitaxel obtained in Example 3 was dissolved in 200 ml of acetonitrile until all solids were dissolved. While stirring, 100 ml of water was gradually added and filtered as necessary. The aqueous solution was then pumped into the column from the sample inlet port.

  The column was then eluted with an acetonitrile / water gradient solvent (starting with 30% aqueous acetonitrile and ending with 100% acetonitrile). The flow rate was controlled at 350 ml per minute. Each fraction of about 1 liter was collected with the pressure maintained at about 4137-5516 kPa (600-800 psi).

  The collected fraction was monitored by UV detector at an absorbance of 227 / nm and monitored by TLC by spraying with a 10% aqueous ethanol solution. Fractions containing pure paclitaxel were combined and concentrated to remove most of the acetonitrile and left in the hood overnight until crystallization was complete. The crystals were collected by filtration, recrystallized from acetone: hexane (1: 1) and dried in a vacuum oven to give 36.5 grams of white powder. This pure paclitaxel is more than about 99% pure. Yield: about 85-90% from 70-80% pure crude paclitaxel.

The overall yield was over about 73%.
As mentioned above, the method according to aspects of the present invention is simple. Paclitaxel and other taxanes are provided in high yield and industrial scale purity. Thus, the present invention provides an efficient cost-effective method for mass production of paclitaxel and other related taxanes from plant sources or tissue cultures, particularly Taxus canadensis. The method of the present invention provides for the large-scale production of paclitaxel and related taxanes from yew plants.

  The isolation and purification method according to aspects of the present invention recovers taxane derivatives in high purity from naturally derived yew species. The improvements are reflected in the high taxane recovery and the purity of the isolated taxane.

Claims (13)

A method for isolating and purifying a taxane analog from a resource comprising the taxane analog, comprising:
A step of passing an organic solution containing the taxane derivative through a chromatographic column, and the step of compressing a packing medium in the column so as to substantially eliminate voids in the column, and thereby a taxane derivative containing impurities Providing an organic solution comprising: maintaining the chromatography column under a pressure of up to about 34475 kPa;
A method consisting of: The method according to claim 1, further comprising a step of passing an organic solution containing the taxane derivative containing impurities through a chromatography column,
The method wherein the packing medium in the column is compressed to remove voids in the column and maintained at a pressure up to about 2758 kPa. The method according to claim 1 or 2, wherein
An organic solution containing the extracted taxane derivative containing the impurities is a chromatography column, and the packing medium in the column is compressed to remove voids in the column and maintained at a pressure of up to about 2758 kPa. A further step through the treated column, thereby obtaining paclitaxel extracted in an organic solvent;
The paclitaxel syrup extracted in the organic solvent is a column for chromatography wherein the packing material is compressed to remove voids in the column and maintained at a pressure of up to about 344.75 kPa. Through further processes,
Performing the method. The method according to claim 1, 2 or 3,
Extracting yew biomass with organic solvent, partitioning the solution with hexane and water, redistributing the solution with dichloromethane or chloroform and water, and finally crude paclitaxel dichloromethane or chloroform Recovering the solution, and a method comprising a preliminary step. The method according to any one of claims 1 to 4,
The taxane analog solution comprises a solution comprising crude crystalline paclitaxel in methanol, ethanol, a mixture of methanol and ethanol, a mixture of dichloromethane and methanol, or a mixture of dichloromethane and ethanol. The method according to any one of claims 1 to 5, wherein the filling medium is diphenyl group-bonded silica gel, C-18-bonded silica gel, polystyrene-DVB resin, polymethacrylate resin, or polyaromatic resin. The method according to any one of claims 1 to 6, wherein the column is eluted with a gradient solution of acetone / water, methanol / water or acetonitrile / water. The method according to any one of claims 1 to 7, wherein the flow rate is about 350 ml / min and the pressure is maintained at about 4137 to 5516 kPa. 9. A method according to any one of the preceding claims, wherein the flow rate is from about 1 liter / minute to about 2 liter / minute. The organic solvent in the first step includes dichloromethane, and the elution solution that passes through the column in the third step includes acetone / water, methanol / water, or acetonitrile / water. The method according to item. 11. The organic solvent is dichloromethane, and the elution solution passing through the column is acetone / water, methanol / water, ethanol / water, isopropanol / water, or acetonitrile / water. The method described in 1. 12. A method according to any one of the preceding claims comprising the further step of crystallizing impure paclitaxel from the column eluate. 13. A method according to any one of the preceding claims comprising the further step of crystallizing 9-dihydro-1,3-acetylbaccatin III from a portion of the column eluate.