Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-11-28
2001-12-25
Trinh, Ba K. (Department: 1625)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C549S511000
Reexamination Certificate
active
06333419
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed to the separation of paclitaxel from its analogue cephalomannin starting from extracts of plants of the genus Taxus or their cell cultures. In particular, paclitaxel is separated from cephalomannin through chromatography on direct-phase silica gel columns.
BACKGROUND ART
Paclitaxel, formerly called “Taxol” is an exceptionally promising anticancer agent. It was isolated from the bark of
Taxus brevifolia
by Wani et al. in 1971 (J. Am. Chem. Soc. 93, 2325, 1971) and its structure was defined using chemical methods and X-ray crystallographic analysis.
Paclitaxel has been approved by the Food and Drug Adminstration for the treatment of breast and ovarian cancer and is currently in clinical trials for the treatment of lung and colon cancers (for example, see W. P. McGuire and E. K. Rowinsky, Paclitaxel in Cancer Treatment, M. Dekker, New York 1995, pages 1 to 337).
A primary natural source for paclitaxel is the bark of the Pacific Yew tree,
Taxus brevifolia
. It has also been found that paclitaxel is present in the epigeal parts and the roots of other yew species, including the European yew (
Taxus baccata
), Asian yews (
Taxus wallichiana
and
Taxus chinensis
), and yew trees cultivated for ornamental purposes (for example,
Taxus media
).
The method of isolation of paclitaxel from any natural resource is complex and expensive, partly because of the relatively low concentrations in vegetable materials but also because of the presence of one of its congeners, cephalomannin. The contents of and ratios between paclitaxel and cephalomannin vary in vegetable materials depending on the species and the part of the plant in question. In general, it has been found that the content of paclitaxel and cephalomannin ranges from 0.001% to 0.08% and 0.001% to 0.22% respectively (K. M. Witherup et al., J. Nat. Prod., 53,1249, 1990; R. G. Kelsey et., J. Nat. Prod., 55, 912, 1992; N. C. Wheeler et al., J. Nat. Prod., 55,432, 1992). In particular, the
Taxus media
species which, being renewable vegetable material, is the most commonly used raw material for the preparation of paclitaxel, contains on the average the highest concentration of cephalomannin in comparison with the other species.
Even the paclitaxel production techniques based on yew cell cultures, which have recently been given a substantial boost to obviate the conventional extraction of expensive vegetable material, yield a relevant quantity of cephalomannin in addition to paclitaxel.
The only structural difference between paclitaxel and cephalomannin involves the side chain portion of the compound, thus giving rise to similar chemical properties. The two compounds, therefore, possess very similar chromatographic properties and clean separation of these related compounds is difficult. A number of chromatographic methods, mainly based on the use of inverted-phase chromatography or expensive bonded-phase columns, have been proposed (J. H. Cardellina, J. Liq. Chromatogr., 12, 2117, 1989), but these cannot be easily adapted to a large commercial scale operation. For this reason, the availability of methods allowing the separation of paclitaxel and cephalomannin remains a topic of great practical importance.
In the past, paclitaxel and cephalomannin separation methods, based on the different reactivity of the two compounds to oxidants, were proposed. It was found that the double olefin bond existing in the tiglic residue of cephalomannin could be oxidized by reaction with osmium tetroxide (D. G. I. Kingston et al. , J. Nat. Prod., 55, 259, 1992) or ozone (J. T. Beckvermit et al., J. Org. Chem., 61, 9038, 1996), while paclitaxel did not undergo any chemical transformation during oxidation reactions. Another approach considered the treatment of mixtures of paclitaxel and cephalomannin with bromine (J. M. Rimoldi et al., J. Nat. Prod., 59, 167, 1996). Treatment with bromine, performed under controlled temperature and time reaction conditions, causes the formation of dibromocephalomannin, while paclitaxel is not affected by this chemical reagent. These methods, however, have a drawback in their use of such toxic reagents as osmium tetroxide and, in any case, result in the destruction or transformation of cephalomannin into its derivatives, from which cephalomannin can be regenerated only through difficult synthetic processes. There is still a need, therefore, for in inexpensive, simple, safe and effective separation of cephalomannin from paclitaxel. Accordingly, the primary objective of this invention is to provide a simple method to separate paclitaxel and cephalomannin from their mixtures or yew extracts.
SUMMARY OF THE INVENTION
The present invention provides a method for separating paclitaxel from cephalomannin and other related compounds. In particular, this method comprises obtaining a starting material that contains paclitaxel and cephalomannin; dissolving the starting material in any one of a number of particularly defined solvents to form a mixture; subjecting the mixture to column chromatography to obtain an eluted fraction of paclitaxel, an eluted fraction of cephalomannin and a residue; and separately drying the paclitaxel and cephalomannin fractions to obtain separate crystalline forms of paclitaxel and cephalomannin, respectively. The solvent is preferably butyl formate or butyl or benzyl acetate.
DETAILED DESCRIPTION OF THE INVENTION
Surprisingly, it was found that highly pure palitaxel and cephalomannin could be obtained with great yields by chromatographic separation on one direct phase silica gel column using one solvent as the elutant. Preferably, this solvent has the following general formula:
where R
1
is hydrogen or methyl and R
2
preferably contains four carbon atoms, that is, it may be n-butyl, isobutyl, sec-butyl or t-butyl. These solvents are not frequently used in routine chromatographic purification on column, but provide surprisingly good practical results in the specific case of the paclitaxel-cephalomannin separation.
The starting material of this invention may be a mixture of cephalomannin and paclitaxel alone in any ratio or extract of fresh or dry Taxus roots, leaves, branches, seeds or their mixtures. The method of this invention may also involve an extract obtained from a cell culture. These starting materials are generally known to one of ordinary skill in the art so that they do not need to be further mentioned herein.
The extract in question may be a raw or a purified extract—the latter having been treated with conventional solvents and subjected to preliminary chromatographic purification. Again, these techniques are well know to the skilled artisan so that no further mention need be made herein. The starting material may be in the form of a solid, a syrup or a semisolid gummy material, depending on the experimental conditions used for its preparation. The material may simply be subjected to column chromatography following its dissolution in one of the solvents described herein.
The chromatographic purification of the invention utilizes a simple, direct-phase silica gel in a quantity of about 50 to 100 parts in weight of the starting material, depending on its composition.
Column chromatography with the solvents of the invention is fast, does not require high pressures and is performed in normal gravity conditions.
Table 1 shows the behavior of cephalomannin and paclitaxel in the thin-layer chromatographic analysis using silica gel plates and a series of the solvents of the invention which allow a satisfactory separation of the two compounds.
TABLE 1
Rf values of paclitaxel and cephalomannin on silica gel plates
Solvent
R
1
R
2
Paclitaxel
Cephalomannin
n-Butyl formate
H
n-C
4
H
9
0.17
0.12
i-Butyl formate
H
i-C
4
H
9
0.20
0.15
t-Butyl formate
H
t-C
4
H
9
0.20
0.13
n-Butyl formate
CH
3
n-C
4
H
9
0.36
0.28
s-Butyl formate
CH
3
s-C
4
H
9
0.35
0.24
i-Butyl acetate
CH
3
i-C
4
H
9
0.31
0.19
t-Butyl acetate
CH
3
t-C
4
H
9
0.19
0.09
Benzyl acetate
CH
3
PhCH
2
0.28
0.16
The use of the solvents described in this invention to isolate paclitaxel and cephaloman
Gabetta Bruno
Zini Gianfranco
Indena SpA
Pennie & Edmonds LLP
Trinh Ba K.
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