Purification paclitaxel

We have developed a new photoreactive analogue of paclitaxel, 3 -N-BzDC-3 -N-debenzoylpaclitaxel (109) and its ditritiated derivative ([3H]-109) has been evaluated for its ability to photolabel tubulin and P-glycoprotein.66 Radiolabeled photoreactive analogue [3H]-109 was synthesized by N-acylation of 3 -N-deben-zoyl-2, 7-bis(0-TES)paclitaxel (108) with N-(2,3-ditritio-3-(4-benzoyl-phenyl)propanoyloxy)succinimide ([3H]-107), followed by purification on a reversed phase semipreparative HPLC using a C-18 column (Scheme 21).66 Photoaffinity label [3H]-109 was assessed to possess >99.9% radiochemical purity and a high specific radioactivity (34 Ci/mmol). 

Initially, researchers focused on the separation of paclitaxel from cephalomannine, a paclitaxel analogue that shares the C-13 ester side-chain and, therefore, exhibits cytotoxic activity. Cephalomannine was found to elute close to paclitaxel and caused interferences for determination and purification purposes. Lately, the main taxane targets are baccatin III and 10-deacetyl-baccatin III (10-DAB III). Both compounds lack the C-13 side-chain (Fig. 1) thus, they do not show antitumour activity. Both baccatins, especially 10-DAB III, serve as synthons for the synthesis of paclitaxel or analogues. 7-Epi-paclitaxel differs from paclitaxel only in the stereochemistry of the hydroxy group in the C-7 position. 7-Epi-paclitaxel, a product of paclitaxel epimerization, shows cytotoxic activity and difficulty in separation from paclitaxel. The deacetyl derivatives of paclitaxel, 7-epi-paclitaxel, baccatin III, and cephalomannine, are also quite often the... 

The isolation of paclitaxel exemplifies that most preparative separations must be downsized to a level where a limited number of individual compounds are present to ease the final purification steps. This downsizing of the separation problem can be done by crude separations or by a cascade of consecutive chromatographic separation steps. One finally ends up at a point where a multicomponent mixture with a broad concentration range of the different substances has to be fractionated to a series of mixtures. This approach is described in Fig. 4.4. In general, a mixture can be split into three types of fractions, which each represent a specific separation problem. These three fractions exemplify possible separation scenarios that differ with regard to the ratio of target products and impurities. 

Paclitaxel and related compounds have also been found in various Taxus species in addition to the Pacific yew, occurring in roots, stems, wood, and needles as well as bark. Yew extracts contain a complex mixture of taxanes, with paclitaxel usually constituting less than 20% of the total taxanes. Isolation of paclitaxel from these mixtures is a difficult purification problem and contributed to the slow development of this compound as a drug. The most valuable material in this mixture for semisynthesis is 10-deacetylbaccatin-III. Microbial strains were isolated from soil samples containing C-13 deacylase and C-lO-deacetylase enzyme activities that are able to convert mixtures of taxanes to 10-deacetylbaccatin-III, thereby increasing the amount and ease of isolation of this precursor for semisynthesis (Scheme 17.14). Treatment of ethanol extracts, prepared either from whole plants of a variety of renewable yew cultivars or from material derived from the bark of... 

Sparreboom A, Huizing MT, Boesen JJB, Nooijen WJ, van Tellingen O, Beijnen JH (1995) Isolation, Purification, and Biological Activity of Mono- and Dihydroxylated Paclitaxel Metabolites from Human Feces. Cancer Chemother Pharmacol 36 299... 

Another approach in taxoid preparative separation included solid-phase extraction (alumina, silica, or RP-8 cartridges) followed by preparative TLC on silica gel plates with quaternary mobile phase consisting of dichloromethane-dioxane-acetone-methanol (83 5 10 2, v/v). In this way, 10-DAB III, paclitaxel, and cephalomannine as well as two further taxoids could be easily isolated with relatively high efficiencies from yew materials (Fig. 2). Multiple development technique or fiuther separation of the isolated taxoid fractions (especially less polar ones) on RP-2 silica bond stationary phase with methanol-water mixtures as mobile phases was applied for purification of the compounds isolated. 10-DAB III isolated in this way was relatively pure, as was shown in reversed-phase (RP)-HPLC analysis (Fig. 3). 

The natural substance Paclitaxel (Taxol ) can be extracted from the bark of Pacific yew and exhibits a positive effect when used in treating various types of cancer such as ovary, breast, lung and prostate carcinoma. As the above-mentioned natural source is not sufficient to cover global demand, presently a combination of extraction (Baccatin III from the needles of English yew) and synthesis labor- and cost-extensive chromatographic purification processes °° is used. 

Paditaxel, a complex terpene molecule with antimitotic activity (commercialized as Taxol ), is used for various cancer treatments, including ovarian and breast cancer treatments. The isolation of this naturally occurring compound requires the harvesting of a large amount of trees and involves difficult purification. A semisynthetic route has been developed based on the coupling of baccatin III (a diterpenoid that can be isolated from yew leaves) with the chiral side-chain (2R,3S)-N-benzoyl-3-phenylisoserine ethyl ester. The latter could be obtained from the diastereoselective microbial reduction of racemic 2-keto-3-(N-benzoylamino)-3-phenylpropionic acid ethyl ester using whole cells of Hansemda sp. (Figure 13.12). Despite concomitant production of the undesired anti diastereomers (2R,3R)-N-benzoyl-3-phenylisoser-ine ethyl ester and (2S,3S)-N-benzoyl-3-phenylisoserine ethyl ester (up to 20%) due to nonperfect selectivity, the C-13 side-chain synthon required for paclitaxel... 

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