Method for separation and purification of 13-dehydroxybaccatin III and 10-deacetylpaclitaxel from taxans-containing materials
This invention is directed to a method for efficiently separating and purifying 13-dehydroxybaccatin III and 10-deacetylpaclitaxel with a high purity of 90% or higher purity, preferably 99.5% or higher purity, from taxane-containing materials such as Taxus species, and the thusly obtained highly pure 13-dehydroxybaccatin III and 10-deacetylpaclitaxel can be used as semi-synthetic precursors of paclitaxel and/or docetaxel that are anticancer agents.
1. A method for separating and purifying 13-dehydroxybaccatin III and 10-deacetylpaclitaxel simultaneously from a taxane-containing material, the method comprising the steps of:
(a) extracting the taxane-containing material with at least one organic solvent selected from the group consisting of low-molecular alcohols of C1 to C4 and halogenated alkanes (C1-C2), and concentrating the solvent including the extracted taxane-containing material;
(b) partitioning the concentrated solution obtained from step (a) by adding at least one organic solvent selected from the group consisting of halogenated alkanes (C1-C2), ethers, ethyl acetate and butanol, that is not mixable with water, and liquid/liquid extracting the partitioned solution to separate an organic solvent layer of the at least one organic solvent selected from the group consisting of halogenated alkanes (C1-C2), ethers, ethyl acetate and butanol;
(c) concentrating the extracted solution obtained from step (b) under reduced pressure, dissolving the concentrated solution in 0.5%(v/v) to 10%(v/v) methanol/dichloromethane mixture solution, and performing normal phase low-pressure liquid chromatography to obtain paclitaxel, 13-dehydroxybaccatin III and 10-deacetylpaclitaxel active fractions at the same time; and
(d) as a step of separating and purifying 13-dehydroxybaccatin III and 10-deacetylpaclitaxel from the 13-dehydroxybaccatin III and 10-deacetylpaclitaxel active fractions obtained from the normal phase low-pressure liquid chromatography in step (c),
(d-1) dissolving the 13-dehydroxybaccatin III active fraction obtained from the normal phase low-pressure liquid chromatography in step (c) in at least one organic solvent selected from the group consisting of alcohols of C1 to C4, acetonitrile, and purified water, and performing reverse phase low-pressure liquid chromatography to obtain 13-dehydroxybaccatin III active fraction; and
(d-2) dissolving the 10-deacetylpaclitaxel active fraction obtained from the normal phase low-pressure liquid chromatography in step (c) in at least one organic solvent selected from the group consisting of alcohols of C1 to C4, acetonitrile, and purified water, and performing reverse phase low-pressure liquid chromatography to obtain 10-deacetylpaclitaxel active fraction.
2. The method according to claim 1 further comprising, after the performing the reverse phase low-pressure liquid chromatography of step (d-1), at least one step of:
precipitating the 13-dehydroxybaccatin III active fraction obtained from the reverse phase low-pressure liquid chromatography using an organic solvent, purified water or mixture thereof to obtain 13-dehydroxybaccatin III-containing precipitates; and
performing reverse phase high performance liquid chromatography.
3. The method according to claim 2 , wherein the step of obtaining the precipitates comprises:
dissolving the 13-dehydroxybaccatin III active fraction by reverse phase chromatography using an organic solvent selected from the group consisting of tetrahydrofuran, alcohols of C1 to C4, halogenated alkanes of C1 to C2, ketones, and mixtures thereof; and
adding to the obtained solution a substance selected from the group consisting of alkanes of C4 to C8, purified water and mixtures thereof in a volume of 1 to 20 times of the volume of the solvent used for dissolving the 13-dehydroxybaccatin III active fraction to form precipitation.
4. The method according to claim 1 further comprising, after performing the reverse phase low-pressure liquid chromatography of step (d- 2 ), the step of performing reverse phase high performance liquid chromatography of the obtained 10-deacetylpaclitaxel active fraction.
5. The method according to claim 1 , wherein the taxane-containing material is Taxus species plant body selected from the group consisting of Taxus brevifolia, Taxus canadensis, Taxus cuspidate, Taxus baccata, Taxus globosa, Taxus floridana, Taxus wallichiana, Taxus media and Taxus chinensis , or a cell, tissue or cell culture derived from the Taxus species plant body.