Title of Invention

A PROCESS FOR PREPARING 10-DEACETYLBACCATIN III

Abstract A process is provided for the semi-synthesis and isolation of taxane intermediates useful in the preparation of paclitaxel and docetaxel, in particular, the semi-synthesis and isolation of 10-deacetylbaccatin III, the semi-synthesis of a mixture of 10- deacetylbaccatin III and baccatin III, and protected derivatives thereof, from a mixture of taxanes.
Full Text BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the semi-synthesis of taxane
intermediates useful in the preparation of paclitaxel and docetaxel, in particular, the
semi-synthesis and isolation of 10-deacetylbaccatin III, the semi-synthesis of a mixture
of 10-deacetylbaccatin III and baccatin III, and derivatives thereof, from a mixture of
taxanes.
Description of the Related Art
The taxane family of terpenes has received much attention in the
scientific and medical community because members of this family have demonstrated
broad spectrum anti-leukemic and tumor-inhibitory activity. A well-known member of
this family is paclitaxel (1, Taxol).

Paclitaxel was first isolated from the bark of the pacific yew tree {Taxus
brevifolia) in 1971, and has proved to be a potent natural anticancer agent. For
example, paclitaxel has been found to have activity against different forms of leukemia
and against solid tumors in the breast, ovary, brain, and lung in humans.
This activity has stimulated an intense research effort over recent years,
including the search for other taxanes having similar or improved properties, and the
development of synthetic pathways for making taxanes such as paclitaxel. One result
from this research effort was the discovery of a synthetic analog of paclitaxel, docetaxel
(2, more commonly known as taxotere). As disclosed in U.S. Patent No. 4,814,470,
taxotere has been found to have very good anti-tumor activity and better bio-availability

than paclitaxel. Taxotere is similar in structure to paclitaxel, having t-butoxycarbonyl
instead of benzoyl on the amino group at the 3' position, and a hydroxyl group instead
of the acetoxy group at the C-10 position.

Taxanes are structurally complicated molecules, and the development of
commercially viable synthetic methods to make taxanes has been a challenge. A
number of semi-synthetic pathways have been developed, which typically begin with
the isolation and purification of a naturally occurring material and then its conversion to
the taxane of interest. For example, paclitaxel and taxotere may be prepared semi-
synthetically from 10-deacetylbaccatin III or baccatin III as set forth in U.S. Patent No.
4,924,011 to Denis et al. and U.S. Patent No. 4,924,012 to Colin et al. or by the reaction
of a p-lactam and a suitably protected 10-deacetylbaccatin III or baccatin III derivative
as set forth in U.S. Patent No. 5,175,315 to Holton et al. or U.S. Patent Application No.
10/683,865, which application is assigned to the assignee of the present invention. 10-
deacetylbaccatin III (10-DAB, 3) and baccatin III (BACC III, 4) can be separated from
mixtures extracted from natural sources such as the needles, stems, bark or heartwood
of numerous Taxus species and have the following structures.

Although, much of the research towards the semi-synthesis of paclitaxel
and taxotere has involved 10-deacetylbaccatin III as the starting material, other taxanes
from the Taxus species, such as 9-dihydro-13-acetylbaccatin III (9-DHB, 5), present in
the Canadian yew (Taxus Canadensis), cephalomannine (6), 10-deacetyl taxol (10-

DAT, 7), 7-xylosyl taxol (8), 10-deacetyl-7-xylosyl taxol (9), and a number of 7-epi-
taxanes have been collected and identified.


As disclosed in U.S. Patent Application No. 10/695,416, which
application is assigned to the assignee of the present invention, U.S. Patent Nos.
6,576,777 and 6,222,053 to Zamir et al. and U.S. Patent Nos. 6,175,023 and 6,179,981
to Liu et al., docetaxel and paclitaxel (and suitable starting materials for the synthesis
thereof) may also be prepared semi-synthetically from 9-dihydro-13-acetylbaccatin III.
In addition, U.S. Patent Nos. 5,202,448 and 5,256,801 to Carver et al.,
U.S. Patent No. 5,449,790 to Zheng et al. and U.S. Patent No. 6,281,368 to McChesney
et al. disclose processes for converting certain taxanes (namely, paclitaxel,
cephalomannine, 10-deacetyl taxol and certain 10-deacetyl taxol derivatives) present in
partially purified taxane mixtures into 10-deacetylbaccatin III and baccatin III, which
may subsequently be utilized in the foregoing semi-synthetic pathways.
Although there have been many advances in the field, there remains a
need for new and improved processes for the preparation of taxane intermediates and
their conversion to paclitaxel and docetaxel, in particular, for the preparation of such
taxane intermediates from crude and partially purified mixtures comprising a plurality
of taxanes. The present invention addresses these needs and provides further related
advantages.
BRIEF SUMMARY OF THE INVENTION
According to one aspect of the present invention, the present invention
relates to the semi-synthesis and isolation of taxane intermediates useful in the
preparation of paclitaxel and docetaxel, in particular, the semi-synthesis and isolation of
10-deacetylbaccatin III, and protected derivatives thereof, from a mixture of taxanes.
These processes may thus be utilized to convert a plurality of taxanes present in a crude
taxane extract or in a waste taxane solution into taxanes, and taxane derivatives, that
can be used to further synthesize paclitaxel and docetaxel. Representative waste taxane
solutions may comprise (1) pooled waste stream fractions collected following the
chromatographic separation and collection of paclitaxel enriched fractions from a crude
or partially purified taxane extract, and/or (2) pooled waste mother liquors collected
following the recrystallization of a crude or partially purified taxane extract.
Generally stated, each processes according to this aspect of the present
invention comprise an initial step of cleaving the ester linkages at the C-10 and C-13
positions of each taxane in the initial mixture having an ester linkage at one or both of
the C-10 and C-13 positions. Following such initial step, a series of further protection,
chromatographic separation, oxidation and deprotection steps are utilized to prepare 10-

deacetylbaccatin III, and protected derivatives thereof. 10- deacetylbaccatin III can
then be converted to paclitaxel or docetaxel. —
More specifically, in a first embodiment, the present invention provides
a process for preparing 10-deacetylbaccatin III from an initial mixture of taxanes,
wherein the initial mixture comprises 9-dihydro-13-acetylbaccatin III, and at least one
additional taxane selected from paclitaxel, 10-deacetylbaccatin III, baccatin III,
cephalomannine, 10-deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, the
process comprising the steps of:
(1) cleaving the ester linkages at the C-10 and C-13 positions of
each taxane in the initial mixture having an ester linkage at one or both of the C-10 and
C-13 positions to yield a first intermediate mixture of C-10 and C-13 deprotected
taxanes;
(2) separating the taxanes in the first intermediate mixture
having a keto substituent at the C-9 position from the taxanes in the first intermediate
mixture having a hydroxy group at the C-9 position to yield 10-deacetylbaccatin III and
a second intermediate mixture of C-9 hydroxy taxanes;
(3) protecting the hydroxy groups at the C-7 and C-10 positions
of each taxane in the second intermediate mixture to yield a third intermediate mixture
of C-7 and C-10 protected taxanes;
(4) oxidizing the hydroxy group at the C-9 position of each
taxane in the third intermediate mixture to yield a fourth intermediate mixture of C-9
oxidized taxanes; and
(5) deprotecting the hydroxy groups at the C-7 and C-10
positions of each taxane in the fourth intermediate mixture to yield 10-
deacetylbaccatin III.
In a second embodiment, the order of steps (2) and (3) above are
reversed and the present invention provides a process for preparing 10-deacetylbaccatin
III from an initial mixture of taxanes, wherein the initial mixture comprises 9-dihydro-
13-acetylbaccatin III, and at least one additional taxane selected from paclitaxel, 10-
deacetylbaccatin III, baccatin III, cephalomannine, 10-deacetyl taxol, 7-xylosyl taxol
and 10-deacetyl-7-xylosyl taxol, the process comprising the steps of:
(1) cleaving the ester linkages at the C-10 and C-13 positions of
each taxane in the initial mixture having an ester linkage at one or both of the C-10 and
C-13 positions to yield a first intermediate mixture of C-10 and C-13 deprotected
taxanes;

(2) protecting the hydroxy groups at the C-7 and C-10 positions
of each taxane in the first intermediate mixture having a hydroxy group at one or both
of the C-7 and C-10 positions to yield a second intermediate mixture of C-7 and C-10
protected taxanes;
(3) separating the taxanes in the second intermediate mixture
having a keto substituent at the C-9 position from the taxanes in the second intermediate
mixture having a hydroxy group at the C-9 position to yield C-7 and C-10 protected 10-
deacetylbaccatin III and a third intermediate mixture of C-9 hydroxy taxanes;
(4) oxidizing the hydroxy group at the C-9 position of each
taxane in the third intermediate mixture to yield a fourth intermediate mixture of C-9
oxidized taxanes; and
(5) deprotecting the hydroxy groups at the C-7 and C-10
positions of each taxane in the fourth intermediate mixture to "yield 10-
deacetylbaccatin III.
In a third embodiment, the present invention provides a process for
preparing paclitaxel or docetaxel from an initial mixture of taxanes, wherein the initial
mixture comprises 9-dihydro-13-acetylbaccatin III, and at least one additional taxane
selected from paclitaxel, 10-deacetylbaccatin III, baccatin III, cephalomannine, 10-
deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, the process comprising
the steps of:
(1) cleaving the ester linkages at the C-10 and C-13 positions of
each taxane in the initial mixture having an ester linkage at one or both of the C-10 and
C-13 positions to yield a first intermediate mixture of C-10 and C-13 deprotected
taxanes;
(2) separating the taxanes in the first intermediate mixture
having a keto substituent at the C-9 position from the taxanes in the first intermediate
mixture having a hydroxy group at the C-9 position to yield 10-deacetylbaccatin III and
a second intermediate mixture of C-9 hydroxy taxanes;
(3) protecting the hydroxy groups at the C-7 and C-10 positions
of each taxane in the second intermediate mixture to yield a third intermediate mixture
of C-7 and C-10 protected taxanes;
(4) oxidizing the hydroxy group at the C-9 position of each
taxane in the third intermediate mixture to yield a fourth intermediate mixture of C-9
oxidized taxanes;

(5) deprotecting the hydroxy groups at the C-7 and C-10
positions of each taxane in the fourth intermediate mixture to yield 10-deacetylbaccatin
III; and
(6) converting the 10-deacetylbaccatin III obtained from steps
(2) and (5) to paclitaxel or docetaxel.
In a fourth embodiment, the present invention provides a process for
preparing paclitaxel or docetaxel from an initial mixture of taxanes, wherein the initial
mixture comprises 9-dihydro-13-acetylbaccatin III, and at least one additional taxane
selected from paclitaxel, 10-deacetylbaccatin III, baccatin III, cephalomannine, 10-
deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, the process comprising
the steps of:
(1) cleaving the ester linkages at the C-10 and C-13 positions of
each taxane in the initial mixture having an ester linkage at one or both of the C-10 and
C-13 positions to yield a first intermediate mixture of C-10 and C-13 deprotected
taxanes;
(2) protecting the hydroxy groups at the C-7 and C-10 positions
of each taxane in the first intermediate mixture having a hydroxy group at one or both
of the C-7 and C-10 positions to yield a second intermediate mixture of C-7 and C-10
protected taxanes;
(3) separating the taxanes in the second intermediate mixture
having a keto substituent at the C-9 position from the taxanes in the second intermediate
mixture having a hydroxy group at the C-9 position to yield C-7 and C-10 protected 10-
deacetylbaccatin III and a third intermediate mixture of C-9 hydroxy taxanes;
(4) oxidizing the hydroxy group at the C-9 position of each
taxane in the third intermediate mixture to yield a fourth intermediate mixture of C-9
oxidized taxanes; ._
(5) deprotecting the hydroxy groups at the C-7 and C-10
positions of each taxane in the fourth intermediate mixture to yield 10-deacetylbaccatin
III; and
(6) converting the C-7 and C-10 protected 10-deacetylbaccatin
III obtained from step (3) and the 10-deacetylbaccatin III obtained from step (5) to
paclitaxel or docetaxel.
According to another aspect of the present invention, the present
invention relates to the semi-synthesis of taxane intermediates useful in the preparation
of paclitaxel and docetaxel, in particular, the semi-synthesis yields a mixture of 10-
deacetylbaccatin IH and baccatin III, and derivatives thereof, from a mixture of taxanes.

The mixture of 10-deacetylbaccatin III and baccatin III can then be converted to
paclitaxel or docetaxel.
According to this aspect of the invention, the processes comprise an
initial combined step of protecting the hydroxy group at the C-7 position of each taxane
in the initial mixture having a hydroxy group at the C-7 position and cleaving the ester
linkage at the C-13 and/or C-10 positions of each taxane in the initial mixture having an
ester linkage at the C-13 and/or C-10 positions.
More specifically, in a fifth embodiment, the present invention provides
a process for preparing 10-deacetylbaccatin III and baccatin III from an initial mixture
of taxanes, wherein the initial mixture comprises 9-dihydro-13-acetylbaccatin III or
cephalomannine, and at least one additional taxane selected from paclitaxel, 10-
deacetylbaccatin III, baccatin III, 9-dihydro-13-acetylbaccatin III, cephalomannine, 10-
deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, the process comprising
the steps of:
(1) protecting the hydroxy group at the C-7 position of each
taxane in the initial mixture having a hydroxy group at the C-7 position and cleaving
the ester linkage at the C-13 and C-10 positions of each taxane in the initial mixture
having an ester linkage at the C-13 and C-10 positions to yield a first intermediate
mixture of C-7 protected taxanes;
(2) oxidizing the hydroxy group at the C-9 position of each
taxane in the first intermediate mixture having a hydroxy group at the C-9 position to
yield a second intermediate mixture of C-7 protected taxanes; and
(3) deprotecting the hydroxy group at the C-7 position of each
taxane in the second intermediate mixture to yield 10-deacetylbaccatin III and
baccatin III.
In a six embodiment, the present invention provides a process for
preparing paclitaxel or docetaxel from an initial mixture of taxanes, wherein the initial
mixture comprises 9-dihydro-13-acetylbaccatin III or cephalomannine, and at least one
additional taxane selected from paclitaxel, 10-deacetylbaccatin III, baccatin III, 9-
dihydro-13-acetylbaccatin III, cephalomannine, 10-deacetyl taxol, 7-xylosyl taxol and
10-deacetyl-7-xylosyl taxol, the process comprising the steps of:
(1) protecting the hydroxy group at the C-7 position of each
taxane in the initial mixture having a hydroxy group at the C-7 position and cleaving
the ester linkage at the C-13 and C-10 positions of each taxane in the initial mixture
having an ester linkage at the C-13 and C-10 positions to yield a first intermediate
mixture of C-7 protected taxanes;

(2) oxidizing the hydroxy group at the C-9 position of each
taxane in the first intermediate mixture having a hydroxy group at the C-9 position to
yield a second intermediate mixture of C-7 protected taxanes;
(3) deprotecting the hydroxy group at the C-7 position of each
taxane in the second intermediate mixture to yield 10-deacetylbaccatin III and baccatin
III; and
(4) converting the 10-deacetylbaccatin III and baccatin III to
paclitaxel or docetaxel,
wherein the step of converting the 10-deacetylbaccatin III and baccatin III to paclitaxel
or docetaxel further comprises protecting the hydroxy group at the C-7 position of each
of the 10-deacetylbaccatin III and baccatin III.
In further embodiments of the foregoing processes, the initial mixture
comprises: (1) 9-dihydro-13-acetylbaccatin III, and at least two additional taxanes
selected from paclitaxel, 10-deacetylbaccatin III, baccatin III, cephalomannine, 10-
deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol; (2) 9-dihydro-13-
acetylbaccatin III, and at least three additional taxanes selected from paclitaxel, 10-
deacetylbaccatin III, baccatin III, cephalomannine, 10-deacetyl taxol, 7-xylosyl taxol
and 10-deacetyl-7-xylosyl taxol; or (3) 9-dihydro-13-acetylbaccatin III, paclitaxel, 10-
deacetylbaccatin III, baccatin III, cephalomannine, 10-deacetyl taxol, 7-xylosyl taxol
and 10-deacetyl-7-xylosyl taxol.
In other further embodiments of the foregoing processes, the initial
mixture of taxanes is a waste taxane solution comprising one or more of the following:
(1) pooled waste stream fractions collected during a chromatographic separation of a
crude or partially purified taxane extract; and (2) pooled waste mother liquors collected
during a recrystallization of a crude or partially purified taxane extract. In more
specific embodiments: (1) the waste taxane solution comprises pooled waste stream
fractions collected during a chromatographic separation of a crude taxane extract; (2)
the waste taxane solution comprises pooled waste stream fractions collected during
chromatographic separations of both crude and partially purified taxane extracts and
pooled waste mother liquors collected during recrystallizations of both crude and
partially purified taxane extracts; or (3) the crude and partially purified taxane extracts
are obtained from taxane-containing materials from the genus Taxus.
These and other aspects of the invention will be apparent upon reference
to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
As used herein, the following terms have the following meanings.
"Silica matrix" is a solid media containing a silicate which is used as an
adsorbent or column material in chromatographic separations, including (but not limited
to) ordinary silica, Florisil, porous silica gels or any physical formulation of a silicate
for use in chromatographic procedures.
"Taxane-containing material" refers to selected parts of a plant, plant
tissues, cell cultures, microorganisms or extracts with extractable taxanes, including
paclitaxel, 10-deacetylbaccatin III (10-DAB), baccatin III (BACC III), 9-dihydro-13-
acetylbaccatin III (9-DHB), cephalomannine, 10-deacetyl taxol (10-DAT), 7-xylosyl
taxol and 10-deacetyl-7-xylosyl taxol.
"Crude taxane extract" refers to a composition obtained from a taxane-
containing material by treating the taxane-containing material with at least one solvent.
"Partially purified taxane extract" refers to a paclitaxel enriched
composition obtained from the chromatographic separation and/or recrystallization of a
crude or partially purified taxane extract.
"Waste stream fractions" refers to fractions collected following the
chromatographic separation and collection of paclitaxel enriched fractions from a crude
or partially purified taxane extract by, for example, the process of U.S. Patent No.
6,136,989.
"Waste mother liquors" refers to mother liquors collected following the
recrystallization of a crude or partially purified taxane extract by, for example, the
process of U.S. Patent No. 6,136,989.
"Hydroxy-protecting group" refers to a readily cleavable group bonded
to the oxygen of a hydroxy (-OH) group. Examples of hydroxy-protecting groups
include, without limitation, formyl, acetyl (Ac), benzyl (PhCH2), 1-ethoxyethyl (EE),
methoxymethyl (MOM), (methoxyethoxy)methyl (MEM), (p-
methoxyphenyl)methoxymethyl (MPM), tert-butyldimethylsilyl (TBS), tert-
butyldiphenylsilyl (TBPS), tert-butoxycarbonyl (tBoc, t-Boc, tBOC, t-BOC),
tetrahydropyranyl (THP), triphenylmethyl (Trityl, Tr), 2-methoxy-2-methylpropyl,
benzyloxycarbonyl (Cbz), dichloroacetyl, trichloroacetyl (OCCCI3), 2,2,2-
trichloroethoxycarbonyl (Troc), benzyloxymethyl (BOM), tert-butyl (t-Bu), triethylsilyl
(TES), trimethylsilyl (TMS), triisopropylsilyl (TIPS), propionyl, isopropionyl, pivalyl,
dimethylisopropylsilyl, diethylisopropylsilyl, methyldiphenylsilyl, dimethylphenylsilyl,

tert-butyldiphenylsilyl, tribenzylsilyl, triphenylsilyl, trichloroethoxycarbonyl, benzyl,
para-nitrobenzyl, para-methoxybenzyl, benzoyl, methoxyethyl, para-methoxyphenyl,
tetrahydrofuranyl, alkylsulfonyl and arylsulfonyl. The related term "protected hydroxy
group" refers to a hydroxy group that is bonded to a hydroxy-protecting group. General
examples of protected hydroxy groups include, without limitation, -O-alkyl, -O-acyl,
acetal, and -O-ethoxyethyl, where some specific protected hydroxy groups include,
formyloxy, acetoxy, propionyloxy, chloroacetoxy, bromoacetoxy, dichloroacetoxy,
trichloroacetoxy, trifluoroacetoxy, methoxyacetoxy, phenoxyacetoxy, benzoyloxy,
benzoylformoxy, p-nitro benzoyloxy, ethoxycarbonyloxy, methoxycarbonyloxy,
propoxycarbonyloxy, 2,2,2-trichloro ethoxycarbonyloxy, benzyloxycarbonyloxy, tert-
butoxycarbonyloxy, 1-cyclopropyl ethoxycarbonyloxy, phthaloyloxy, butyryloxy,
isobutyryloxy, valeryloxy, isovaleryloxy, oxalyoxy, succinyloxy and pivaloyloxy,
phenylacetoxy, phenylpropionyloxy, mesyloxy, chlorobenzoyloxy, para-
nitrobenzoyloxy, para-tert-butyl benzoyloxy, capryloyloxy, acryloyloxy,
methylcarbamoyloxy, phenylcarbamoyloxy, naphthylcarbamoyloxy, and the like.
Hydroxy-protecting groups and protected hydroxy groups are described in, e.g., C. B.
Reese and E. Haslam, "Protective Groups in Organic Chemistry," J. G. W. McOmie,
Ed., Plenum Press, New York, N.Y., 1973, Chapters 3 and 4, respectively, and T. W.
Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis," Second Edition,
John Wiley and Sons, New York, N.Y., 1991, Chapters 2 and 3.
The following Table shows the chemical structure of some hydroxy-
protecting groups, as well as nomenclature used to identify those chemical structures.


"Alkyl" refers to a hydrocarbon structure wherein the carbons are
arranged in a linear, branched, or cyclic manner, including combinations thereof.
Lower alkyl refers to alkyl groups of from 1 to 5 carbon atoms. Examples of lower
alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl and the like.
"Cycloalkyl" is a subset of alkyl and includes cyclic hydrocarbon groups of from 3 to
13 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl,
cyclopentyl, norbomyl, adamantyl and the like. When an alkyl residue having a
specific number of carbons is named, all geometric isomers having that number of
carbons are intended to be encompassed; thus, for example, "butyl" is meant to include
n-butyl, sec-butyl, isobutyl and t-butyl; propyl includes n-propyl and isopropyl.
"Alkenyl" refers to an alkyl group having at least one site of
unsaturation, i.e., at least one double bond.

"Alkynyl" refers to an alkyl group having at least one triple bond
between adjacent carbon atoms.
"Alkoxy" and "alkoxyl" both refer to moieties of the formula -O-alkyl.
Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy,
cyclohexyloxy and the like. Lower-alkoxy refers to groups containing one to four
carbons. The analogous term "aryloxy" refers to moieties of the formula -O-aryl.
"Acyl" refers to moieties of the formula -C(=0)-alkyl. One or more
carbons in the acyl residue may be replaced by nitrogen, oxygen or sulfur as long as the
point of attachment to the parent remains at the carbonyl. Examples include acetyl,
benzoyl, propionyl, isobutyryl, t-butoxycarbonyl, benzyloxycarbonyl and the like.
Lower-acyl refers to groups containing one to four carbons.
"Aryl" refers to phenyl or naphthyl. Substituted aryl refers to mono- and
poly- substituted phenyl or naphthyl. Exemplary substituents for aryl include one or
more of halogen, hydroxyl, alkoxy, aryloxy, heteroaryloxy, amino, alkylamino,
dialkylamino, mercapto, alkylthio, arylthio, heteroarylthio, cyano, carboxyl,
alkoxycarbonyl where the alkoxy portion contains 1 to 15 carbons, aryloxycarbonyl
where the aryloxy portion contains 6 to 20 carbon, or heteroarylcarbonyl where the
heteroaryl portion contains 3 to 15 carbon atoms.
"Heteroaryl" refers to a 5- or 6-membered heteroaromatic ring
containing 1-3 heteroatoms selected from O, N, or S; a bicyclic 9- or 10-membered
heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, or S; or a
tricyclic 13- or 14-membered heteroaromatic ring system containing 0-3 heteroatoms
selected from O, N, or S. Exemplary aromatic heterocyclic rings include, e.g.,
imidazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole,
quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole and pyrazole.
"Halogen" refers to fluoro, chloro, bromo or iodo.
"Keto" refers to =0.
II. Process for Preparing and isolating 10-DAB from a Mixture of Taxanes
As noted above, one aspect of the present invention relates to the semi-
synthesis of taxane intermediates useful in the preparation of paclitaxel and docetaxel,
in particular, the semi-synthesis of 10-deacetylbaccatin III, and protected derivatives
thereof, from an initial mixture of taxanes.
The taxanes present in the initial mixture, namely, 9-dihydro-13-
acetylbaccatin III, paclitaxel, 10-deacetylbaccatin III, baccatin III, cephalomannine, 10-

deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, can be represented by
the following baccatin molecular framework:

wherein, R1 is -OH, -OAc, N-(2-methyl-2-butenoyl)-(2R, 3S)-3-phenylisoserine or N-
benzoyl-(2R, 3S)-3-phenylisoserine, R2 is -OH or -OAc, R3 is -OH or =O, and R4 is
- OH or xylosyl. For example, when R1 is -OAc, R2 is -OAc, R3 is -OH and R4 is -
OH, the foregoing structure represents 9-dihydro-13-acetylbaccatin III, and when R1 is
-OH, R2 is -OAc, R3 is =O and R4 is -OH, the foregoing structure represents baccatin
III.
As described in more detail below, 10-deacetylbaccatin III may be
prepared from such an initial mixture of taxanes through steps of cleavage, protection,
oxidation and deprotection of certain R1, R2, R3 and R4 substituents of the taxanes
present in the initial mixture and the chromatographic separation of various taxanes
present in the mixture following certain of such steps. For example, in a first
embodiment, the present invention provides a process for preparing 10-deacetylbaccatin
III from an initial mixture of taxanes, wherein the initial mixture comprises 9-dihydro-
13-acetylbaccatin III, and at least one additional taxane selected from paclitaxel, 10-
deacetylbaccatin III, baccatin III, cephalomannine, 10-deacetyl taxol, 7-xylosyl taxol
and 10-deacetyl-7-xylosyl taxol, the process comprising the steps of:
(1) cleaving the ester linkages at the C-10 and C-13 positions
of each taxane in the initial mixture having an ester linkage at one or both of the C-10
and C-13 positions to yield a first intermediate mixture of C-10 and C-13 deprotected
taxanes;
(2) separating the taxanes in the first intermediate mixture
having a keto substituent at the C-9 position from the taxanes in the first intermediate
mixture having a hydroxy group at the C-9 position to yield 10-deacetylbaccatin III and
a second intermediate mixture of C-9 hydroxy taxanes;
(3) protecting the hydroxy groups at the C-7 and C-10 positions
of each taxane in the second intermediate mixture to yield a third intermediate mixture
of C-7 and C-10 protected taxanes;

(4) oxidizing the hydroxy group at the C-9 position of each
taxane in the third intermediate mixture to yield a fourth intermediate mixture of C-9
oxidized taxanes; and
(5) deprotecting the hydroxy groups at the C-7 and C-10
positions of each taxane in the fourth intermediate mixture to yield 10-
deacetylbaccatin III.
In a second embodiment, the order of steps (2) and (3) are reversed, and
the present invention provides a process for preparing 10-deacetylbaccatin III from an
initial mixture of taxanes, wherein the initial mixture comprises 9-dihydro-13-
acetylbaccatin III, and at least one additional taxane selected from paclitaxel, 10-
deacetylbaccatin III, baccatin III, cephalomannine, 10-deacetyl taxol, 7-xylosyl taxol
and 10-deacetyl-7-xylosyl taxol, the process comprising the steps of:
(1) cleaving the ester linkages at the C-10 and C-13 positions of
each taxane in the initial mixture having an ester linkage at one or both of the C-10 and
C-13 positions to yield a first intermediate mixture of C-10 and C-13 deprotected
taxanes;
(2) protecting the hydroxy groups at the C-7 and C-10 positions
of each taxane in the first intermediate mixture having a hydroxy group at one or both
of the C-7 and C-10 positions to yield a second intermediate mixture of C-7 and C-10
protected taxanes;
(3) separating the taxanes in the second intermediate mixture
having a keto substituent at the C-9 position from the taxanes in the second intermediate
mixture having a hydroxy group at the C-9 position to yield C-7 and C-10 protected 10-
deacetylbaccatin III and a third intermediate mixture of C-9 hydroxy taxanes;
(4) oxidizing the hydroxy group at the C-9 position of each
taxane in the third intermediate mixture to yield a fourth intermediate mixture of C-9
oxidized taxanes; and
(5) deprotecting the hydroxy groups at the C-7 and C-10
positions of each taxane in the fourth intermediate mixture to yield 10-
deacetylbaccatin in.
In general, such cleavage, protection, separation, oxidation and
deprotection steps comprise the following methods.

General Method of Cleavage
In one embodiment, the ester linkages at the C-10 and C-13 positions of
a taxane in the initial mixture can be cleaved using a base. Suitable bases include, but
are not limited to, sodium carbonate, sodium bicarbonate, potassium tert-butoxide,
lithium tert-butoxide, LiHMDS, n-butyl lithium, lithium hydroxide, methyl lithium, or a
mixture of n-BuLi/K-t-OBu. Typically, K-t-OBu, Li-t-OBu, LiHMDS, n-BuLi, LiOH
or CH3Li can be used. More typically, the step of cleaving the ester linkages at the C-
10 and C-13 positions of each taxane in the initial mixture comprises contacting the
initial mixture with K-t-OBu.
In another embodiment, the ester linkages at the C-10 and C-13 positions
of a taxane in the initial mixture can be cleaved using a reducing salt. The term
"reducing salt" refers to a reducing agent in the presence of a Lewis acid. Suitable
reducing agents include, but are not limited to, tetrabutylammonium borohydride,
lithium borohydride, sodium triacetoxy borohydride, sodium hydride and sodium
borohydride. Suitable Lewis acids include, but are not limited to, SbCl5, ZnCl2, CuCl2,
PbCl2, GeCl2, SnBr2, Snl2 and CoBr2.
For example, in one embodiment, a mixture of taxanes having ester
linkages at one or both of the C-10 and C-13 positions are dissolved in an organic
solvent, such as DCM (dichloromethane), THF (tetrahydrofuran), DMF (dimethyl
formamide) or DMSO (dimethyl sulfoxide), and cooled to a low temperature under
argon atmosphere. To this solution is added a suitable base, and the reaction is stirred
until complete consumption of the starting material as evidenced by TLC. The reaction
is then worked up as usual and, after purification of the crude mixture by column
chromatography using mixtures of DCM/ethyl acetate, the pure C-10 and C-13
deprotected product was obtained.
In another embodiment, the mixture of taxanes having ester linkages at
one or both of the C-10 and C-13 positions are dissolved in an organic solvent and a
minimum volume of water is added. To this mixture, a suitable reducing agent is added
in small portions with vigorous stirring and a catalytic amount of a Lewis acid is added.
After completion of the addition, the reaction mixture is stirred for an additional 15
min, then NH4Cl is added as a concentrated aqueous solution, the layers are then
separated and the aqueous phase is extracted with DCM. The combined organic extract
is dried and evaporated to give the crude C-10 and C-13 deprotected product.
Purification by dry-flash chromatography using DCM/MeOH affords the purified C-10
and C-13 deprotected product.

General Method of Protection
Generally stated, the hydroxy groups at the C-7 and C-10 positions of a
taxane can be selectively protected by contacting the intermediate mixture with a base
and a hydroxy-protecting group in an organic solvent. The intermediate mixture can be
the immediate reaction mixture resulted from an initial cleavage of the ester groups at
the C-10 and C-13 position and comprises taxanes having C-9 keto and C-9 hydroxy
groups. Alternatively, the intermediate mixture comprises only those taxanes having C-
9 hydroxy groups after a separation step.
The hydroxy groups at the C-7 and C-10 positions of a taxane can be
selectively protected using any of a variety of hydroxy protecting groups, such as
alkylating agents, acylating agents and silylating agent. For example, the C-7 and C-10
hydroxy groups may be silylated using any of a variety of common silylating agents
including, but not limited to, tri(hydrocarbonyl)silyl halides and tri(hydrocarbonyl)silyl
trifiates. The hydrocarbonyl moieties of these compounds may be substituted or
unsubstituted and preferably are substituted or unsubstituted alkyl or acyl. More
specifically, the C-7 and C-10 hydroxy groups can be selectively silylated, for example,
using silylating agents such as tribenzylsilyl chloride, trimethylsilyl chloride,
triethylsilyl chloride, dimethylisopropylsilyl chloride, dimethylphenylsilyl chloride and
the like. Alternatively, selective acylation of the C-7 and C-10 hydroxy groups can be
achieved using any of a variety of common acylating agents, including, but not limited
to substituted and unsubstituted carboxylic acid derivatives, e.g., carboxylic acid
halides, anhydrides, dicarbonates, isocyanates and haloformates. Typically, the C-7
and C-10 hydroxy groups can be selectively acylated, for example, with di-tert-butyl
dicarbonate, dibenzyl dicarbonate, diallyl dicarbonate, 2,2,2-trichloroethyl
chloroformate, benzyl chloroformate or dichloroacetyl chloride or another common
acylating agent. More typically, tert-butoxycarbonyl, benzyloxycarbonyl, 2,2,2-
trichloroethoxycarbonyl, dichloroacetyl and acetyl are used.
In the present invention, these protecting reactions are carried out in the
presence of a base, such as, for example, Li-t-OBu, K-t-OBu, n-BuLi, a mixture of n-
BuLi/K-t-OBu, LiOH, pyridine, DMAP or TEA. In a specific embodiment, the base is
DMAP and the hydroxy-protecting group is tert-butoxycarbonyl.
Exemplary reaction conditions are as follows: a mixture of taxanes is
dissolved in an organic solvent, such as anhydrous DCM (dichloromethane) or THF
(tetrahydrofuran) or DMF (dimethyl formamide) or DMSO (dimethyl sulfoxide) under
an argon atmosphere at low temperature. To this solution is added DMAP
(dimethylaminopyridine) or any other base, such as Li-t-OBu or K-t-OBu, followed by

an acylating agent, such as di-tert-butyl dicarbonate, or an alkylating agent, such as
triethyl silyl chloride or any other chloride containing a hydroxy-protecting group. The
mixture is left at low to room temperature until complete consumption of the starting
material, as visualized by TI.C. The mixture is then quenched with cold water and
extracted thrice with DCM. The organic layer is washed with water and then with brine
to remove unwanted salts. The organic layer may then be dried and evaporated under
vacuum, and the residue recrystallized or column chromatographed with DCM/EtOAc
mixtures to afford C-7 and C-10 protected taxanes.
General Method of Separation
Normal phase silica chromatography may be utilized to separate a
mixture of taxanes into the following two groups: taxanes having a keto substituent at
the C-9 position (i.e., protected 10-DAB derivatives) and taxanes having a hydroxy
group at the C-9 position. As used herein, silica chromatography generally refers to the
process of contacting a sample dissolved in a feed solvent with a silica matrix then
eluting the silica matrix with an eluting solvent to obtain a fraction enriched with a
desired component.
The dimensions of the silica column are selected according to the
quantity and purity of the solids to be separated. In one embodiment of a pilot scale
process, about 250 grams of solids are dissolved in about 0.75 liters of feed solvent
which is then chromatographed over a Silica column of about 1.5-inches x 10-feet. In
another embodiment, about 40-50 kg of solids are dissolved in about 100-200 liters of
feed solvent, and chromatographed over a Silica column of about 18-inches x 10-feet.
It has also been shown that a layer of about 1-15 cm of Celite, preferably
about 2-8 cm, on top of the silica column is recommended as a column prefilter which
substantially decreases the loading time of the sample. It has further been shown that
the optimal eluting solvent for the Silica column can be a hexane/acetone or DCM/ethyl
acetate or DCM/methanol mixtures.
General Method of Oxidation
Generally stated, the hydroxy group at the C-9 position of each taxane in
an intermediate mixture can be selectively oxidized by contacting the intermediate
mixture with an oxidizing agent selected from the group consisting of 4-
(dimethylamino)pyridinium chlorochromate, pyridinium chlorochromate, chromium
(IV) oxide-silica gel, chromium (IV) oxide-acetic acid, bromine, dimethyl sulfoxide-

dicyclohexylcarbodiimide, and manganese dioxide with dichloro(p-cymene)-ruthenium
(II). In specific embodiments, the oxidizing agent is chromium (IV) oxide-silica gel.
For example, C-7 protected taxanes are dissolved in an organic solvent
and treated with an oxidant at low to room temperature conditions. The reaction is
stirred until all the starting material is consumed, as evidenced by TLC. The reaction is
then worked up as usual to yield a mixture of C-9 oxidized taxanes. Such mixture can
be further purified by column chromatography or crystallized from a suitable solvent.
General Method of Deprotection
Generally stated, the hydroxy groups at the C-7 and C-10 positions of
each taxane in an intermediate mixture are separately deprotected.
C-10 protected taxanes may be deprotected using a base. Suitable bases
include sodium carbonate, sodium bicarbonate, potassium tert-butoxide, lithium tert-
butoxide, LiHMDS, n-butyl lithium, lithium hydroxide, methyl lithium or a mixture of
n-BuLi/K-t-OBu. For example, C-10 protected taxanes are dissolved in an organic
solvent and cooled to a low temperature under argon atmosphere. To this solution is
added a suitable base, and the reaction is stirred until complete consumption of the
starting material as evidenced by TLC. The reaction is then worked up as usual and,
after purification of the crude mixture by column chromatography using mixtures of
DCM/ethyl acetate, the pure C-10 deprotected product was obtained.
C-7 protected taxanes may be deprotected using an acid. Suitable acids
include HF, HCl, TFA and acetic acid. For example, C-7 protected taxanes are
dissolved in pyridine or an organic solvent at room temperature and treated with an
acid, such as HF, HCl, TFA or acetic acid. The reaction is stirred at this temperature
until complete consumption of the starting materials, as evidenced by TLC. The
reaction is worked up as usual to give the C-7 deprotected product, which could be
further purified by column chromatography or crystallized from a suitable solvent.
In a specific embodiment, the step of deprotecting the hydroxy groups at
the C-7 and C-10 positions of each taxane in an intermediate mixture comprises: (1)
deprotecting the hydroxy groups at the C-10 positions of each taxane in an intermediate
mixture comprises contacting the intermediate mixture with a base selected from the
group consisting of LiOH, n-BuLi, Li-t-OBu, CH3Li, K-t-OBu and LiHMDS, and (2)
deprotecting the hydroxy groups at the C-7 position of each taxane in the fourth
intermediate mixture comprises contacting the fourth intermediate mixture with an acid
selected from the group consisting of HF, TFA, HC1 and acetic acid.

III. Process for Preparing 10-DAB and BACC HI from a Mixture of Taxanes
As noted above, another aspect of the present invention relates to the
semi-synthesis of taxane intermediates useful in the preparation of paclitaxel and
docetaxel, in particular, the semi-synthesis of 10-deacetylbaccatin III and baccatin III,
and derivatives thereof, from an initial mixture of taxanes.
The taxanes present in the initial mixture, namely, 9-dihydro-13-
acetylbaccatin III, paclitaxel, 10-deacetylbaccatin III, baccatin III, cephalomannine, 10-
deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, contain the following
baccatin molecular framework:

wherein R1 is -OH, -OAc, N-(2-methyl-2-butenoyl)-(2R, 3S)-3-phenylisoserine or N-
benzoyl-(2R, 3S)-3-phenylisoserine, R2 is -OH or -OAc, R3 is -OH or =O, and R4 is -
OH or xylosyl. For example, when R1 is -OAc, R2 is -OAc, R3 is -OH and R4 is -OH,
the foregoing structure represents 9-dihydro-13-acetylbaccatin III, and when R1 is -OH,
R2 is -OAc, R3 is =O and R4 is -OH, the foregoing structure represents baccatin III.
As described in more detail below, 10-deacetylbaccatin III and baccatin
III may be prepared from such an initial mixture of taxanes through the protection,
cleavage, oxidation and deprotection of certain R1, R2, R3 and R4 substituents of the
taxanes present in the initial mixture. For example, the present invention provides a
process for preparing 10-deacetylbaccatin III and baccatin III from an initial mixture of
taxanes, wherein the initial mixture comprises 9-dihydro-13-acetylbaccatin III or
cephalomannine, and at least one additional taxane selected from paclitaxel, 10-
deacetylbaccatin III, baccatin III, 9-dihydro-13-acetylbaccatin III, cephalomannine, 10-
deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, the process comprising
the steps of:
(1) protecting the hydroxy group at the C-7 position of each
taxane in the initial mixture having a hydroxy group at the C-7 position and cleaving
the ester linkage at the C-13 position of each taxane in the initial mixture having an
ester linkage at the C-13 position to yield a first intermediate mixture of C-7 protected
taxanes;

(2) oxidizing the hydroxy group at the C-9 position of each
taxane in the first intermediate mixture having a hydroxy group at the C-9 position to
yield a second intermediate mixture of C-7 protected taxanes; and
(3) deprotecting the hydroxy group at the C-7 position of each
taxane in the second intermediate mixture to yield 10-deacetylbaccatin III and baccatin
The combined step of protection and cleavage can be carried out
according to the following method:
Combined Step of Protection and Cleavage
The hydroxy group at the C-7 position of a taxane can be selectively
protected using any of a variety of hydroxy protecting groups, such as acetal, ketal,
silyl, and removable acyl protecting groups. For example, the C-7 hydroxy group may
be silylated using any of a variety of common silylating agents including, but not
limited to, tri(hydrocarbonyl)silyl halides and tri(hydrocarbonyl)silyl triflates. The
hydrocarbonyl moieties of these compounds may be substituted or unsubstituted and
preferably are substituted or unsubstituted alkyl or acyl. More specifically, the C-7
hydroxy group can be selectively silylated, for example, using silylating agents such as
tribenzylsilyl chloride, trimethylsilyl chloride, triethylsilyl chloride,
dimethylisopropylsilyl chloride, dimethylphenylsilyl chloride and the like.
Alternatively, selective acylation of the C-7 hydroxy group can be achieved using any
of a variety of common acylating agents, but not limited to substituted and
unsubstituted carboxylic acid derivatives, e.g., carboxylic acid halides, anhydrides,
dicarbonates, isocyanates and haloformates. More specifically, the C-7 hydroxy group
can be selectively acylated, for example, with di-tert-butyl dicarbonate, dibenzyl
dicarbonate, diallyl dicarbonate, 2,2,2-trichloroethyl chloroformate, benzyl
chloroformate or dichloroacetyl chloride or another common acylating agent.
In the present invention, these protecting reactions are carried out in the
presence of a base, such as, for example, Li-t-OBu, K-t-OBu, n-BuLi, a mixture of n-
BuLi/K-t-OBu, LiOH, pyridine, DMAP or TEA. In addition to aiding in the protection
of the C-7 hydroxy group, depending upon the amount of base utilized, the base also
cleaves any ester linkage at the C-13 position and, may also cleave any ester linkage at
the C-10 position.
Exemplary reaction conditions are as follows: a mixture of taxanes is
dissolved in anhydrous DCM (dichloromethane) or THF (tetrahydrofuran) or DMF
(dimethyl formamide) or DMSO (dimethyl sulfoxide) under an argon atmosphere at
low temperature. To this solution is added DMAP (dimethylaminopyridine) or any

other lithium or potassium base, such as Li-t-OBu, K-t-OBu, n-BuLi, a mixture of n-
BuLi/K-t-OBu, or LiOH, followed by an acylating agent, such as di-tert-butyl
dicarbonate, or an alkylating agent, such as triethyl silyl chloride or any other chloride
containing a hydroxy-protecting group. The mixture is left at low to room temperature
until complete consumption of the starting material, as visualized by TLC. In addition,
excess amounts of the base may be added to this mixture, in the same pot, to ensure
cleavage of any ester linkages at the C-13 and/or C-10 positions. The mixture is then
quenched with cold water and extracted thrice with DCM. The organic layer is washed
with water and then with brine to remove unwanted salts. The organic layer may then
be dried and evaporated under vacuum, and the residue recrystallized or column
chromatographed with DCM/EtOAc mixtures to afford a mixture of C-7 protected
taxanes.
The step of oxidation can be carried out in a similar manner as described
above.
The step of deprotection can be carried out in the following method:
General Method of Deprotection
C-7 protected taxanes may be deprotected using an acid. Suitable acids
include HF, HC1, TFA and acetic acid.
For example, C-7 protected taxanes are dissolved in pyridine or an
organic solvent at room temperature and treated with an acid, such as HF, HC1, TFA or
acetic acid. The reaction is stirred at this temperature until complete consumption of
the starting materials, as evidenced by TLC. The reaction is worked up as usual to give
the deprotected product, which could be further purified by column chromatography or
crystallized from a suitable solvent.
IV. Initial Mixture of Taxanes
As noted above, the processes of the present invention may be utilized
for high yield and large scale conversion of taxanes present in a waste taxane solution
into taxanes intermediates, namely, 10-deacetylbaccatin III and baccatin III protected
derivatives thereof, that can be used to further synthesize paclitaxel and docetaxel.
Such a waste taxane solution may comprise (1) pooled waste stream fractions collected
following the chromatographic separation and collection of paclitaxel enriched fractions
from a crude or partially purified taxane extract, and/or (2) pooled waste mother liquors
collected following the recrystallization of a crude or partially purified taxane extract.

Such waste taxane solutions may be obtained by a number of different
methods, such as, for example, the methods disclosed in U.S. Patent No. 6,136,989 to
Foo et al., and other references cited therein, which patent is incorporated herein by
reference in its entirety. A representative method of obtaining a waste taxane solution,
which comprises pooled waste stream fractions, comprises the following extraction and
column chromatography steps.
Starting Taxane-Containing Material
A suitable taxane-containing material is any tissue that contains a high
taxane content. Examples of suitable taxane-containing material include tissues from
various species of Yew plants comprising the genus Taxus, most preferably the roots
and needles of ornamental Yew plants such as T. canadensis, T. x media spp Hicksii, T.
x dark green spreader and Hill., T. chinensis, T. wallichiana, T. cuspidata, T. globosa,
T. sumatrana, T. marei and T. floridana, and the bark of T. brevifolia or T. yunnanensis.
Other suitable material include cultures of plant tissues obtained from a Taxus species.
In a typical practice, such as set forth in U.S. Patent No. 6,139,989, the
taxane-containing material is either pulverized, chipped or otherwise ground into small
pieces so as to increase efficiency of a solvent extraction. The taxane-containing
material may also optionally be dried. Taxane-containing cell culture, cells,
microorganisms and fermentation broths will typically be concentrated prior to solvent
extraction. Cells and microorganisms can be processed as whole cells or cell paste or
pulver.
Extraction
The taxane-containing material may be initially extracted by contacting
the material with an organic solvent, usually for a prolonged period of at least 8 hours
and typically for about 3 days with or without physical agitation to promote formation
of a crude organic extract containing a plurality of taxanes. The extraction may employ
any of the solvent systems that are known to be used for the extraction of paclitaxel,
including but not limited to, acetone, methanol, ethanol, ethyl acetate, methylene
chloride, chloroform, mixtures thereof, and mixtures containing an aqueous component
of up to 60%. These solvents are typically added in an amount of about 4-20 liter per
kg of the taxane-containing material to prepare the crude organic extract. Reference is
made for example, to U.S. Patent No. 6,136,989 and the publications cited therein
which provide a non-exclusive description of several solvent systems that may be used
to prepare an organic extract containing a plurality of taxanes.

In one embodiment, the organic solvent is a polar organic solvent,
typically an alcohol. For some embodiments, methanol is preferred because of its low
cost, ease of removal and efficiency of taxane extraction. In one embodiment, about 6-
15 liters of methanol is added for every kg of taxane-containing material to be
extracted. The extraction is accelerated by agitating the taxane-containing material, for
example, by stirring or percolating the methanol with the taxane-containing material for
about 1-5 days at a temperature between room temperature and about 60°C, most
typically at about 40°C. When the taxane-containing material contains a paclitaxel
content of at least 0.005%, methanol extraction for three days as described above
recovers at least 90% of the available paclitaxel from the taxane-containing material, in
addition to a plurality of other taxanes, to form a crude methanol extract containing
about 0.1-0.5% paclitaxel and having an overall solid content of about 0.5-5% (w/v).
The large volume of methanol extract thus obtained is optionally
concentrated, typically about 10-30 fold by evaporation to obtain a methanol extract
concentrate having a solid content of about 100-400 g/L.
Liquid-Liquid Extraction
The crude organic extract may be subsequently enriched for taxanes by
performing 1-3 liquid-liquid extractions by mixing the organic extract with a non-
miscible, organic solvent to form a two phase system wherein one phase contains the
plurality of taxanes. Generally, the two phase system includes a polar phase.
Optionally, the taxane-containing phase is selected and concentrated by evaporation to
form a concentrated extract having a solid content of about 100-400 g/L and a paclitaxel
purity of about 1-4%. In some embodiments, water is included to help remove
preferentially water soluble materials and the less polar solvent is selected to remove
undesirable compounds such as waxes, lipids, pigments, and sterols that are found in
different amounts depending on the taxane-containing material used. Typical solvents
for liquid-liquid partitioning include hexane, hexanes, and methylene chloride.
Methylene chloride has generally been found to be suitable for liquid-liquid extraction
of taxane-containing material especially when the solvent used for the crude organic
extract is an alcohol.
The concentrated extract obtained is optionally evaporated and the
residue is re-dissolved in a solvent for loading onto a silica chromatography matrix.
Other example methods of performing a liquid-liquid extraction are
illustrated in U.S. Patent Nos. 5,475,120, 5,380,916, and 5,670,673 to Rao and
references cited therein, and also in U.S. Patent Nos. 5,618,538 and 5,480,639 to

ElSohly et al. and references cited therein. These methods or variants thereof may
alternatively be used in lieu of the embodiments described. Furthermore, liquid-liquid
extraction may be omitted altogether when a plant extract containing high taxane levels
is obtained by other methods such as for example, by intervening precipitation,
crystallization or chromatography steps. One example of such a method is found in
PCT Publication Nos. WO 98/07712 by Zamir et al, which uses a precipitation step
immediately after obtaining an initial organic extract to obtain a paclitaxel fraction that
may be about 1% or higher.
Silica Gel Column Chromatography
As further set forth in U.S. Patent No. 6,136,989, the concentrated
extract may be further purified by normal phase silica chromatography. As used herein,
silica chromatography generally refers to the process of contacting a sample dissolved
in a feed solvent with a silica matrix then eluting the silica matrix with an eluting
solvent to obtain a fraction enriched with a desired component.
The dimensions of the first silica column are selected according to the
quantity and purity of the solids to be separated. In one embodiment of a pilot scale
process, about 250 grams of solids are dissolved in about 0.75 liters of feed solvent
which is then chromatographed over a Silica column of about 1.5-inches x 10-feet. In
another embodiment, about 40-50 kg of solids are dissolved in about 100-200 liters of
feed solvent, and chromatographed over a Silica column of about 18-inches x 10-feet.
It has also been shown that a layer of about 1-15 cm of Celite, preferably
about 2-8 cm, on top of the silica column is recommended as a column prefilter which
substantially decreases the loading time of the sample. It has further been shown that
the optimal eluting solvent for the Silica column should be a hexane/acetone mixture at
a ratio of about 3:1 or a DCM/ethyl acetate mixture at a ratio of about 7:3. The "heart
cut" fractions containing at least 2% paclitaxel are pooled and further purified, for
example, according to the process set forth in U.S. Patent No. 6,136,989. The
remaining waste stream fractions, which contain a plurality of taxanes, including,
paclitaxel, 10-deacetylbaccatin III (10-DAB), baccatin III (BACC III), 9-dihydro-13-
acetylbaccatin III (9-DHB), cephalomannine, 10-deacetyl taxol (10-DAT), 7-xylosyl
taxol and 10-deacetyl-7-xylosyl taxol are pooled into a waste taxane solution for further
processing according to the present invention.

Further Purification Steps
As set forth in more detail in U.S. Patent No. 6,139,989, the paclitaxel
enriched "heart cut" fractions obtained from the foregoing chromatography step may be
further purified through one or more additional chromatographic or recrystallization
steps. Any waste stream fractions or waste mother liquors collected during such
additional purification steps may also be pooled and added to the waste taxane solution
for further processing according to the present invention.
V. Process for Preparing Paclitaxel or Docetaxel
As noted above, the 10-deacetylbaccatin III, and protected derivatives
thereof, prepared from an initial mixture of taxanes according to the foregoing
processes may be utilized to further synthesize paclitaxel and docetaxel. In this way, in
a third embodiment, the present invention provides an overall process for preparing
paclitaxel or docetaxel from an initial mixture of taxanes, wherein the initial mixture
comprises 9-dihydro-13-acetylbaccatin III, and at least one additional taxane selected
from paclitaxel, 10-deacetylbaccatin III, baccatin III, cephalomannine, 10-deacetyl
taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, the process comprising the steps
of:
(1) cleaving the ester linkages at the C-10 and C-13 positions of
each taxane in the initial mixture having an ester linkage at one or both of the C-10 and
C-13 positions to yield a first intermediate mixture of C-10 and C-13 deprotected
taxanes;
(2) separating the taxanes in the first intermediate mixture
having a keto substituent at the C-9 position from the taxanes in the first intermediate
mixture having a hydroxy group at the C-9 position to yield 10-deacetylbaccatin III and
a second intermediate mixture of C-9 hydroxy taxanes;
(3) protecting the hydroxy groups at the C-7 and C-10 positions
of each taxane in the second intermediate mixture to yield a third intermediate mixture
of C-7 and C-10 protected taxanes;
(4) oxidizing the hydroxy group at the C-9 position of each
taxane in the third intermediate mixture to yield a fourth intermediate mixture of C-9
oxidized taxanes;
(5) deprotecting the hydroxy groups at the C-7 and C-10
positions of each taxane in the fourth intermediate mixture to yield 10-deacetylbaccatin
III; and

(6) converting the 10-deacetylbaccatin III obtained from steps
(2) and (5) to paclitaxel or docetaxel.
In addition, in a fourth embodiment, the present invention provides a
process for preparing paclitaxel or docetaxel from an initial mixture of taxanes, wherein
the initial mixture comprises 9-dihydro-13-acetylbaccatin III, and at least one additional
taxane selected from paclitaxel, 10-deacetylbaccatin III, baccatin III, cephalomannine,
10-deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, the process
comprising the steps of:
(1) cleaving the ester linkages at the C-10 and C-13 positions of
each taxane in the initial mixture having an ester linkage at one or both of the C-10 and
C-13 positions to yield a first intermediate mixture of C-10 and C-13 deprotected
taxanes;
(2) protecting the hydroxy groups at the C-7 and C-10 positions
of each taxane in the first intermediate mixture having a hydroxy group at one or both
of the C-7 and C-10 positions to yield a second intermediate mixture of C-7 and C-10
protected taxanes;
(3) separating the taxanes in the second intermediate mixture
having a keto substituent at the C-9 position from the taxanes in the second intermediate
mixture having a hydroxy group at the C-9 position to yield C-7 and C-10 protected 10-
deacetylbaccatin III and a third intermediate mixture of C-9 hydroxy taxanes;
(4) oxidizing the hydroxy group at the C-9 position of each
taxane in the third intermediate mixture to yield a fourth intermediate mixture of C-9
oxidized taxanes;
(5) deprotecting the hydroxy groups at the C-7 and C-10
positions of each taxane in the fourth intermediate mixture to yield 10-deacetylbaccatin
III; and
(6) converting the C-7 and C-10 protected 10-deacetylbaccatin
III obtained from step (3) and the 10-deacetylbaccatin III obtained from step (5) to
paclitaxel or docetaxel.
10-deacetylbaccatin III, and protected 10-deacetylbaccatin III, may be
converted to paclitaxel and docetaxel by a number of different methods, such as, for
example, the methods disclosed in U.S. Patent Nos. 4,924,011, 4,924,012, 5,175,315
and 5,466,834, which patents are incorporated herein by reference in their entirety, and
U.S. Patent Application No. 10/683,865, which application is assigned to the assignee
of the present invention and is incorporated herein by reference in its entirety.

In a further embodiment, a mixture of 10-deacetylbaccatin III and
baccatin III prepared from an initial mixture of taxanes according to the foregoing
process can be converted to paclitaxel and docetaxel. To that end, the present invention
provides an overall process for preparing paclitaxel or docetaxel from an initial mixture
of taxanes, wherein the initial mixture comprises 9-dihydro-13-acetylbaccatin III or
cephalomannine, and at least one additional taxane selected from paclitaxel, 10-
deacetylbaccatin III, baccatin III, 9-dihydro-13-acetylbaccatin III, cephalomannine, 10-
deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, the process comprising
the steps of:
(1) protecting the hydroxy group at the C-7 position of each
taxane in the initial mixture having a hydroxy group at the C-7 position and cleaving
the ester linkage at the C-13 position of each taxane in the initial mixture having an
ester linkage at the C-13 position to yield a first intermediate mixture of C-7 protected
taxanes;
(2) oxidizing the hydroxy group at the C-9 position of each
taxane in the first intermediate mixture having a hydroxy group at the C-9 position to
yield a second intermediate mixture of C-7 protected taxanes;
(3) deprotecting the hydroxy group at the C-7 position of each
taxane in the second intermediate mixture to yield 10-deacetylbaccatin III and baccatin
III; and
(4) converting the 10-deacetylbaccatin IE and baccatin III to
paclitaxel or docetaxel,
wherein the step of converting the 10-deacetylbaccatin III and baccatin III to paclitaxel
or docetaxel further comprises protecting the hydroxy group at the C-7 position of each
of the 10-deacetylbaccatin III and baccatin III.
10-deacetylbaccatin III and baccatin III may be converted to paclitaxel
and docetaxel by a number of different methods, such as, for example, the methods
disclosed in U.S. Patent Nos. 4,924,011, 4,924,012, 5,175,315 and 5,466,834, which
patents are incorporated herein by. reference in their entirety, and U.S. Patent
Application No. 10/683,865, which application is assigned to the assignee of the present
invention and is incorporated herein by reference in its entirety.
EXAMPLES
The following Examples disclose specific processes for synthesizing and
isolating 10-deacetylbaccatin III, or a mixture of 10-deacetylbaccatin III and baccatin
III, and protected derivatives thereof, from a solution containing a plurality of taxanes,

and their subsequent conversion to paclitaxel and docetaxel. Unless otherwise noted,
all scientific and technical terms have the meanings as understood by one of ordinary
skill in the art.
EXAMPLE 1
Extraction of Taxanes from Taxus Canadensis
Needles from the Canadian yew (Taxus Canadensis) were collected in
Quebec. The dried needles (3 kg) were extracted by percolation with methanol at room
temperature three times using 10L, 6L and 6L volumes of methanol in a glass container
equipped with a filter at the bottom with a tap. The extraction with each subsequent
volume of methanol was left for 24 hours and the mixture was filtered into an
erlenmeyer flask by opening the tap at the bottom to give a crude extract. The crude
methanolic extracts were combined and concentrated to give about 1.1L of a crude
methanol extract concentrate.
EXAMPLE 2A
Filtration of the Crude Extract
A silica/charcoal filter was prepared as follows. Norit SA3 charcoal (0.5
kg: 100 mesh - Aldrich) was mixed with celite (0.5 kg: AC 2098T - Anachemia) and
placed into a coarse scintered glass funnel. The charcoal-celite mixture was soaked
with dichloromethane:methanol (9:1) and washed with an additional 1.0 L of the same
solvent. The crude methanol extract concentrate was filtered on this bed of charcoal
and then washed with 1.5 L of dichloromethane:methanol (9:1). The collected mixture
was evaporated under vacuum using a rotovap and the residue was left under high
vacuum for one hour using a vacuum pump to remove all traces of methanol.
EXAMPLE 2B
Liquid-Liquid Extraction
The crude methanolic extract concentrate was partitioned with a mixture
containing methanol (400 ml), water (800 ml) and hexane (1100 ml) in a 5L separatory
funnel. After allowing for the solvents to partition, the top layer with dark green color
was tested and discarded, the lower aqueous phase was extracted with methylene
chloride two times. The methylene chloride extracts from two partitions were

combined and then concentrated to generate 270 ml of DCM extract concentrate
containing the plurality of taxanes.
EXAMPLE 3
Silica Gel Column Chromatography
318 g of silica gel (40-63 µm) was used to pack a lab 2-feet long column
and 70 ml of the DCM extract concentrate (~21g solid) was loaded onto the column
followed by DCM/EtOAc elution: 7L of DCM/EtOAc 7:3 and 3L of DCM/EtOAc 1:1.
100 fractions were collected, each having a 100 ml volume. According to HPLC and
TLC analyses, fractions were combined into five groups: paclitaxel containing
fractions, cephalomannine and paclitaxel containing fractions, 9-DHB containing
fractions, baccatin III containing fractions and 10-DAB containing fractions. The last
four fractions (i.e., the waste stream fractions) can be combined into a pooled waste
stream solution containing a plurality of taxanes, or can be used individually in further
synthetic conversions. In the present case, after the paclitaxel containing fractions were
eluted from the column, the remaining fractions were collected and pooled to form a
waste taxane solution, which was further utilized in the following steps.
EXAMPLE 4
SYNTHESIS AND ISOLATION OF 10-DEACETYLBACCATIN III
FROM A WASTE TAXANE SOLUTION
Cleavage of C-10 and C-13 Ester Linkages
As noted above, any ester linkages at C-10 and C-13 positions of the
taxanes in the waste taxane solution obtained from the above process can be cleaved by
using reagents such as K-t-OBu, Li-t-OBu, LiHMDS, n-BuLi, LiOH and CH3Li, or
NaBH4 and NaH in the presence of a Lewis acid, such as ZnCl2. For example, the
waste taxane solution was dissolved in THF and cooled to -40 °C under argon
atmosphere. To the stirred solution at this temperature was added any one of the above
reagents, such as, for example, K-t-OBu and the reaction was monitored by TLC. The
reaction was further stirred at this temperature for a period between 30 minutes to 6 hrs
until complete consumption of the starting material as evidenced by TLC. Additional
amounts of the base (e.g., K-t-OBu) were added at this temperature, and the reaction
was stirred for an additional hour, as necessary for complete consumption of the starting
material. The reaction mixture was then worked up as usual. Evaporation of the

solvent afforded a crude first intermediate mixture of C-10 and C-13 deprotected
taxanes that could be used directly in the next step of the synthesis or could be further
purified by column chromatography using mixtures of DCM/ethyl acetate and/or
crystallized from a suitable solvent.
Protection of C-7 and C-10 Hydroxy Groups
The first intermediate mixture of C-10 and C-13 deprotected taxanes was
dissolved in THF and stirred at -40 °C under argon atmosphere. To this stirred solution
at -40 °C was added a base (such as DMAP, pyridine, TEA or any other base, such as
LiOH, Li-t-OBu, n-BuLi, K-t-OBu, or a mixture of n-BuLi/K-t-OBu) followed by
addition of a hydroxy-protecting group agent (such as triethyl silyl chloride or any other
alkylating agent, or acetic anhydride, acetyl chloride, di-tert-butyl dicarbonate or any
other acylating agent). The reaction was stirred at this temperature for a period between
30 minutes to 6 hrs until complete consumption of the initial starting material as
evidenced by TLC and HPLC analysis. Additional amounts of the base and alkylating
or acylating agent were added at this temperature, as necessary for complete
consumption of the starting materials. The reaction mixture was then worked up as
usual and the solvent removed to give a second intermediate mixture of C-7 and C-10
protected taxanes that could be used directly in the next step of the synthesis or purified
by either column chromatography using mixtures of DCM/EtOAc or crystallized from a
suitable solvent.
Chromatographic Separation
318 g of silica gel (40-63 µm) was used to pack a lab 2-feet long column
and the second intermediate mixture was loaded onto the column followed by
DCM/EtOAc elution: 7L of DCM/EtOAc 7:3 and 3L of DCM/EtOAc 1:1. 100
fractions were collected, each having a 100 ml volume. According to HPLC and TLC
analyses, fractions were combined into two groups: fractions containing taxanes having
a keto substituent at the C-9 position (i.e., protected 10-DAB derivatives) and taxanes
having a hydroxy group at the C-9 position. The fractions containing taxanes having a
hydroxy group at the C-9 position were pooled to yield a third intermediate mixture of
C-9 hydroxy taxanes, which was further utilized in the following synthetic steps.
Oxidation of C-9 Hydroxy Groups
The third intermediate mixture of C-9 hydroxy taxanes was oxidized
using an oxidizing agent to obtain a ketone at the C-9 position. This oxidation can be

achieved using a variety of oxidants under mild conditions, including 4-
(dimethylamino)pyridinium chlorochromate, pyridinium chlorochromate, chromium
(IV) oxide-silica gel, chromium (IV) oxide-acetic acid (Fieser reagent) or acidic media,
bromine, dimethyl sulfoxide-dicyclohexylcarbodiimide, and manganese dioxide with
dichloro(p-cymene)-ruthenium (II).
The third intermediate mixture of C-9 hydroxy taxanes was dissolved in
an organic solvent (such as acetone) and cooled to near 0 °C with continuous stirring.
The oxidant was added to this solution at low temperature and the reaction was stirred
for a period between 30 minutes to 6 hrs until complete consumption of the starting
materials as evidenced by TLC. After completion of the reaction, the reaction was
worked up as usual to afford a crude fourth intermediate mixture of C-9 oxidized
taxanes that could be further purified (using silica gel column chromatography with
mixtures of DCM/EtOAc for elution or crystallization from a suitable solvent) or used
directly for the next step of the synthesis.
Deprotection of C-7 and C-10 Hydroxy Groups
As noted above, deprotection of the C-7 and C-10 hydroxy groups in the
fourth intermediate mixture of C-9 oxidized taxanes can be accomplished by using
reagents such as LiOH, n-BuLi, Li-t-OBu, CH3Li, K-t-OBu, and LiHMDS for the
deprotection of the C-10 hydroxy groups, and HF, HC1, TFA and acetic acid for the
deprotection of the C-7 hydroxy groups.
The fourth intermediate mixture of C-9 oxidized taxanes was dissolved
in THF and cooled to -40 °C under argon atmosphere. To the stirred solution at this
temperature was added any one of the above bases, such as, for example, K-t-OBu and
the reaction was monitored by TLC. The reaction was further stirred at this temperature
for a period between 30 minutes to 6 hrs until complete consumption of the starting
material as evidenced by TLC. The reaction mixture was then worked up as usual.
Evaporation of the solvent afforded a crude mixture of C-10 deprotected taxanes that
could be further deprotected at the C-7 position as described below, or could be further
purified by column chromatography using mixtures of DCM/ethyl acetate and/or
crystallized from a suitable solvent prior to deprotection at the C-7 position.
The crude mixture of C-10 deprotected taxanes was dissolved in
pyridine, DCM or an organic solvent at room temperature. To this solution was added
HF or TFA. After complete consumption of the starting material as evidenced by TLC,
the reaction was worked up and purified by flash chromatography using mixtures of

DCM/EtOAc to afford 10-deacetylbaccatin III, which could further be crystallized from
a suitable solvent to give a product of >99% purity.
EXAMPLE 5
SYNTHESIS OF PACLITAXEL AND DOCETAXEL FROM 10-DEACETYLBACCATIN III
The 10-deacetylbaccatin III obtained from the prior steps is then
converted to paclitaxel and/or docetaxel according to, for example, the process set forth
in U.S. Patent No. 5,466,834, which application is incorporated herein by reference in
its entirety.
EXAMPLE 6
SYNTHESIS OF 10-DEACETYLBACCATIN III AND BACCATIN III
FROM WASTE TAXANE SOLUTION
Protection of C-7 Hydroxy Groups and Cleavage of C-13 Ester Linkages
The waste taxane solution obtained from the above process was
dissolved in THF and stirred at -40 °C under argon atmosphere. To this stirred solution
at -40 °C was added a base (such as DMAP, pyridine, TEA or any other lithium or
potassium base, such as LiOH, Li-t-OBu, n-BuLi, K-t-OBu or a mixture of n-BuLi/K-t-
OBu) followed by addition of a hydroxy-protecting group agent (such as triethylsilyl
chloride or any other alkylating agent, or acetic anhydride, acetyl chloride, di-tert-butyl
dicarbonate or any other acylating agent). The reaction was stirred at this temperature
for a period between 30 minutes to 6 hrs until complete consumption of the initial
starting material as evidenced by TLC and HPLC analysis. Additional amounts of the
base and alkylating or acylating agent were added at this temperature, as necessary for
complete consumption of the starting materials and to ensure cleavage of the C-13 ester
linkages. Further amounts of the base may be added to ensure cleavage of the C-10
ester linkages. The reaction mixture was then worked up as usual and the solvent
removed to give a crude first intermediate mixture of C-7 protected taxanes that could
be used directly in the next step of the synthesis or purified by either column
chromatography using mixtures of DCM/EtOAc or crystallized from a suitable solvent.
Oxidation of C-9 Hydroxy Groups
The first intermediate mixture of C-7 protected taxanes was oxidized
using an oxidizing agent to obtain a ketone at the C-9 position. This oxidation for the

first intermediate mixture of C-7 protected taxanes can be achieved using a variety of
oxidants under mild conditions, including 4-(dimethylamino)pyridinium
chlorochromate, pyridinium chlorochromate, chromium (IV) oxide-silica gel, chromium
(IV) oxide-acetic acid (Fieser reagent) or acidic media, bromine, dimethyl sulfoxide-
dicyclohexylcarbodiimide, and manganese dioxide with dichloro(p-cymene)-ruthenium
(II).
The first intermediate mixture of C-7 protected taxanes was dissolved in
an organic solvent (such as acetone) and cooled to near 0°C with continuous stirring.
The oxidant was added to this solution at low temperature and the reaction was stirred
for a period between 30 minutes to 6 hrs until complete consumption of the starting
materials as evidenced by TLC. After completion of the reaction, the reaction was
worked up as usual to afford a crude second intermediate mixture of C-7 protected
taxanes that could be further purified (using silica gel column chromatography with
mixtures of DCM/EtOAc for elution or crystallization from a suitable solvent) or used
directly for the next step of the synthesis.
Deprotection of C-7 Hydroxy Groups
As noted above, deprotection of the C-7 hydroxy groups in the second
intermediate mixture of C-7 protected taxanes can be accomplished by using an acid,
such as, HF, HC1, TFA or acetic acid.
The second intermediate mixture of C-7 protected taxanes was dissolved
in pyridine or an organic solvent at room temperature. To this solution was added HF,
or any other acid such as HC1, TFA or acetic acid. After complete consumption of the
starting material, as evidenced by TLC, the reaction was worked up as usual and
purified by flash chromatography using mixtures of DCM/EtOAc to afford a mixture of
10-deacetylbaccatin III and baccatin III, that could further be crystallized from a
suitable solvent to give a product of >99% purity.
EXAMPLE 7
SYNTHESIS OF PACLITAXEL AND DOCETAXEL FROM 10-DEACETYLBACCATIN III
AND BACCATIN III
The mixture of 10-deacetylbaccatin III and baccatin III is then converted
to paclitaxel and/or docetaxel according to, for example, the process set forth in U.S.
Patent No. 5,466,834, which application is incorporated herein by reference in its
entirety.

All of the above U.S. patents, U.S. patent application publications, U.S.
patent applications, foreign patents, foreign patent applications and non-patent
publications referred to in this specification and/or listed in the Application Data Sheet,
are incorporated herein by reference, in their entirety.

WE CLAIM:
1. A process comprising : preparing 10-deacetylbaccatin III by
(1) providing an initial mixture comprising 9-dihydro-13-acetylbaccatin III, and at least one additional
taxane selected from paclitaxel, 10-deacetylbaccatin III, baccatin III, cephalomannine, 10-deacetyl
taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol;
(2) prior to steps 3, 4 and 5, cleaving the ester linkages at the C-10 and C-13 positions of each taxane
in the initial mixture having an ester linkage at one or both of the C-10 and C-13 positions to yield a
first intermediate mixture of C-10 and C-13 deprotected taxanes;
(3) separating the taxanes in the first intermediate mixture having a keto substituent at the C-9
position from the taxanes in the first intermediate mixture having a hydroxy group at the C-9 position
to yield 10-deacetylbaccatin III and a second intermediate mixture of C-9 hydroxy taxanes;
(4) protecting the hydroxy groups at the C-7 and C-10 positions of each taxane in the second
intermediate mixture to yield a third intermediate mixture of C-7 and C-10 protected taxanes;
(5) oxidizing the hydroxy group at the C-9 position of each taxane in the third intermediate mixture to
yield a fourth intermediate mixture of C-9 oxidized taxanes; and
(6) deprotecting the hydroxy groups at the C-7 and C-10 positions of each taxane in the fourth
intermediate mixture to yield 10-deacetylbaccatin III.
2 The process as claimed in claim 1 wherein the step of cleaving the ester linkages at the C-10
and C-13 positions of each taxane in the initial mixture comprises contacting the initial mixture with:
a base selected from the group consisting of K-t-OBu, Li-t-OBu, LiHMDS, n-BuLi, LiOH and CH3Li;
or
a reducing salt comprising a reducing agent selected from NaBH4 and NaH and a Lewis acid.
3. The process as claimed in claim 2 wherein the step of cleaving the ester linkages at the C-10
and C-13 positions of each taxane in the initial mixture comprises contacting the initial mixture with
K-t-OBu.
4. The process as claimed in claim 1 wherein the step of protecting the hydroxy groups at the C-
7 and C-10 positions of each taxane in the second intermediate mixture comprises contacting the

second intermediate mixture with a base and a hydroxy-protecting group in an organic solvent, and
wherein:
the base is selected from the group consisting of DMAP, pyridine, TEA, LiOH, Li-t-OBu, n-BuLi, K-
t-OBu and a mixture of n-BuLi/K-t-OBu; and
the hydroxy-protecting group is selected from the group consisting of alkylating agents and acylating
agents.
5. The process as claimed in claim 4 wherein the hydroxy-protecting group is selected from the
group consisting of tert-butoxycarbonyl, benzyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl,
dichioroacetyl and acetyl.
6. The process as claimed in claim 5 wherein the base of DMAP and the hydroxy-protecting
group is tert-butoxycarbonyl.
7. The process as claimed in claim 1 wherein the step of oxidizing the hydroxy group at the C-9
position of each taxane in the third intermediate mixture comprises contacting the third intermediate
mixture with an oxidizing agent selected from the group consisting of 4-(dimethylamino)pyridinium
chlorochromate, pyridinium chlorochromate, chromium (IV) oxide-silica gel, chromium (IV) oxide-
acetic acid, bromine, dimethyl sulfoxide-dicyclohexylcarbodiimide, and manganese dioxide with
dichloro(p-cymene)-ruthenium (II).
8. The process as claimed in claim 7 wherein the oxidizing agent is chromium (IV) oxide-silica
gel.
9. The process as claimed in claim 1 wherein the step of deprotecting the hydroxy groups at the
C-7 and C-10 positions of each taxane in the fourth intermediate mixture comprises the steps of:
deprotecting the hydroxy groups at the C-10 positions of each taxane in the fourth intermediate
mixture; and
deprotecting the hydroxy groups at the C-7 positions of each taxane in the fourth intermediate
mixture.

10. The process as claimed in claim 9 wherein the step of deprotecting the hydroxy groups at the
C-10 positions of each taxane in the fourth intermediate mixture comprises contacting the fourth
intermediate mixture with a base selected from the group consisting of LiOH, n-BuLi, Li-t-OBu,
CH3Li, K-t-OBu and LiHMDS.
11. The process as claimed in claim 9 wherein the step of deprotecting the hydroxy groups at the
C-7 position of each taxane in the fourth intermediate mixture comprises contacting the fourth
intermediate mixture with an acid selected from the group consisting of HF, TFA, HCI and acetic
acid.
12. The process as claimed in claim 1 comprising converting the 10-deacetylbaccatin III obtained
from steps (2) and (5) to paclitaxel and docetaxel.
13. A process comprising:
preparing 10-deacetylbaccatin III from an initial mixture comprising 9-dihydro-13-acetylbaccatin III,
and at least one additional taxane selected from paclitaxel, 10-deacetylbaccatin III, baccatin III,
cephalomannine, 10-deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, the process
including
(1) prior to steps 3, 4 and 5, cleaving the ester linkages at the C-10 and C-13 positions of each taxane
in the initial mixture having an ester linkage at one or both of the C-10 and C-13 positions to yield a
first intermediate mixture of C-10 and C-13 deprotected taxanes;
(2) protecting the hydroxy groups at the C-7 and C-10 positions of each taxane in the first
intermediate mixture having a hydroxy group at one or both of the C-7 and C-10 positions to yield a
second intermediate mixture of C-7 and C-10 protected taxanes;
(3) separating the taxanes in the second intermediate mixture having a keto substituent at the C-9
position from the taxanes in the second intermediate mixture having a hydroxy group at the C-9
position to yield C-7 and C-10 protected 10-deacetylbaccatin III and a third intermediate mixture of
C-9 hydroxy taxanes;
(4) oxidizing the hydroxy group at the C-9 position of each taxane in the third intermediate mixture to
yield a fourth intermediate mixture of C-9 oxidized taxanes; and
(5) deprotecting the hydroxy groups at the C-7 and C-10 positions of each taxane in the fourth
intermediate mixture to yield 10-deacetylbaccatin III.

14. The process as claimed in claim 13 wherein the step of cleaving the ester linkages at the C-10
and C-13 positions of each taxane in the initial mixture comprises contacting the initial mixture with:
a base selected from the group consisting of K-t-OBu, Li-t-OBu, LiHMDS, n-BuLi, LiOH and CH3Li;
or
a reducing salt comprising a reducing agent selected from NaBH4 and NaH and a Lewis acid.
15. The process as claimed in claim 14 wherein the step of cleaving the ester linkages at the C-10
and C-13 positions of each taxane in the initial mixture comprises contacting the initial mixture with
K-t-OBu.
16. The process as claimed in claim 13 wherein the step of protecting the hydroxy groups at the
C-7 and C-10 positions of each taxane in the first intermediate mixture comprises contacting the first
intermediate mixture with a base and a hydroxy-protecting group in an organic solvent, and wherein:
the base is selected from the group consisting of DMAP, pyridine, TEA, LiOH, Li-t-OBu, n-BuLi, K-
t-OBu and a mixture of n-BuLi/K-t-OBu; and
the hydroxy-protecting group is selected from the group consisting of alkylating agents and acylating
agents.
17. The process as claimed in claim 16 wherein the hydroxy-protecting group is selected from the
group consisting of tert-butoxycarbonyl, benzyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl,
dichloroacetyl and acetyl.
18. The process as claimed in claim 17 wherein the base is DMAP and the hydroxy-protecting
group is tert-butoxycarbonyl.
19. The process as claimed in claim 13 wherein the step of oxidizing the hydroxy group at the C-9
position of each taxane in the third intermediate mixture comprises contacting the third intermediate
mixture with an oxidizing agent selected from the group consisting of 4-(dimethylamino)pyridinium
chlorochromate, pyridinium chlorochromate, chromium (IV) oxide silica gel, chromium (IV) oxide
acetic acid, bromine, dimethyl sulfoxide-dicyclohexylcarbodiimide, and manganese dioxide with
dichloro(p-cymene)-ruthenium (II).

20. The process as claimed in claim 19 wherein the oxidizing agent is chromium (IV) oxide-silica
gel.
21. The process as claimed in claim 13 wherein the step of deprotecting the hydroxy groups at the
C-7 and C-10 positions of each taxane in the fourth intermediate mixture comprises the steps of:
deprotecting the hydroxy groups at the C-10 positions of each taxane in the fourth intermediate
mixture; and
deprotecting the hydroxy groups at the C-7 position of each taxane in the fourth intermediate mixture.
22. The process as claimed in claim 21 wherein the step of deprotecting the hydroxy groups at the
C-10 positions of each taxane in the fourth intermediate mixture comprises contacting the fourth
intermediate mixture with a base selected from the group consisting of LiOH, n-BuLi, Li-t-OBu,
CH3Li, K-t-OBu and LiHMDS.
23. The process as claimed in claim 21 wherein the step of deprotecting the hydroxy groups at the
C-7 position of each taxane in the fourth intermediate mixture comprises contacting the fourth
intermediate mixture with an acid selected from the group consisting of HF, TFA, HCI and acetic
acid.
24. The process as claimed in claim 13 comprising converting the 10-deacetylbaccatin III
obtained from steps (2) and (5) to paclitaxel or docetaxel.
25. The process as claimed in claim 1 wherein the initial mixture comprises 9-dihydro-13-
acetylbaccatin III, and at least two additional taxanes selected from paclitaxel, 10-deacetylbaccatin III,
baccatin III, cephalomannine, 10-deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol.
26. The process as claimed in claim 1 wherein the initial mixture comprises 9-dihydro-13-
acetylbaccatin III, and at least three additional taxanes selected from paclitaxel, 10-deacetylbaccatin
III, baccatin III, cephalomannine, 10-deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol.

27. The process as claimed in claim 1 wherein the initial mixture comprises 9-dihydro-13-
acetylbaccatin III, paclitaxel, 10-deacetylbaccatin III, baccatin III, cephalomannine, 10-deacetyl taxol,
7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol.
28. The process as claimed in claim 1 wherein the initial mixture of taxanes is a waste taxane
solution comprising one or more of the following:
pooled waste stream fractions collected during a chromatographic separation of a crude or partially
purified taxane extract; and
pooled waste mother liquors collected during a recrystallization of a crude or partially purified taxane
extract.
29. The process as claimed in claim 28 wherein the waste taxane solution comprises pooled waste
stream fractions collected during a chromatographic separation of a crude taxane extract.
30. The process as claimed in claim 28 wherein the waste taxane solution comprises pooled waste
stream fractions collected during chromatographic separations of both crude and partially purified
taxane extracts and pooled waste mother liquors collected during recrystallizations of both crude and
partially purified taxane extracts.
31. The process as claimed in claim 28 wherein the crude or partially purified taxane extracts are
obtained from taxane-containing materials from the genus Taxus.
32. A process comprising:
preparing 10-deacetylbaccatin III and baccatin III from an initial mixture comprising 9-dihydro-13-
acetylbaccatin III or cephalomannine, and at least one additional taxane selected from paclitaxel, 10-
deacetylbaccatin III, baccatin III, 9-dihydro-13-acetylbaccatin III, cephalomannine, 10-deacetyl taxol,
7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol, including the steps of:
protecting the hydroxy group at the C-7 position of each taxane in the initial mixture having a
hydroxy group at the C-7 position and cleaving the ester linkage at the C-13 position of each taxane
in the initial mixture having an ester linkage at the C-13 position to yield a first intermediate mixture
of C-7 protected taxanes;

oxidizing the hydroxy group at the C-9 position of each taxane in the first intermediate mixture
having a hydroxy group at the C-9 position to yield a second intermediate mixture of C-7 protected
taxanes; and
deprotecting the hydroxy group at the C-7 position of each taxane in the second intermediate mixture
to yield 10-deacetylbaccatin III and baccatin III.
33. The process as claimed in claim 32 wherein the step of protecting the hydroxy group at the C-
7 position of each taxane in the initial mixture and cleaving the ester linkage at the C-13 position of
each taxane in the initial mixture comprises cleaving the ester linkage at the C-10 position of at least
one taxane in the initial mixture having an ester linkage at the C-10 position.
34. The process as claimed in claim 32 wherein:
the step of protecting the hydroxy group at the C-7 position of each taxane in the initial mixture and
cleaving the ester linkage at the C-13 position of each taxane in the initial mixture comprising
contacting the initial mixture with a base and a hydroxy-protecting group in an organic solvent;
the base is selected from the group consisting of DMAP, pyridine, TEA, LiOH, Li-t-OBu n-BuLi, K-
t-OBu and a mixture of n-BuLi/K-t-OBu; and
the hydroxy-protecting group is selected from the group consisting of alkylating agents and acylating
agents.
35. The process as claimed in claim 34 wherein the hydroxy-protecting group is selected from the
group consisting of tert-butoxycarbonyl, benzyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl,
dichloroacetyl and acetyl.
36. The process as claimed in claim 35 wherein the base is DMAP and the hydroxy-protecting
group is tert-butoxycarbonyl.
37. The process as claimed in claim 32 wherein cleaving the ester linkage at the C-13 position of
each taxane in the initial mixture comprises contacting the initial mixture with a base.
38. The process as claimed in claim 32 wherein the step of oxidizing the hydroxy group at the C-9
position of each taxane in the first intermediate mixture comprises contacting the first intermediate

mixture with an oxidizing agent selected from the group consisting of 4-(dimethylamino)pyridinium
chlorochromate, pyridinium chlorochromate, chromium (IV) oxide-silica gel, chromium (IV) oxide-
acetic acid, bromine, dimethyl sulfoxide-dicyclohexylcarbodiimide, and manganese dioxide with
dichloro(p-cymene)-ruthenium (II).
39. The process as claimed in claim 38 wherein the oxidizing agent is chromium (IV) oxide-silica
gel.
40. The process as claimed in claim 32 wherein the step of deprotecting the hydroxy group at the
C-7 position of each taxane in the second intermediate mixture comprises contacting the second
intermediate mixture with an acid.
41. The process as claimed in claim 40 wherein the acid is selected from the group consisting of
HF, TFA, HCI and acetic acid.
42. The process as claimed in claim 32 wherein the initial mixture comprises 9-dihydro-13-
acetylbaccatin III or cephalomannine, and at least two additional taxanes selected from paclitaxel, 10-
deacetylbaccatin III, baccatin III, 9-dihydro-13-acetylbaccatin III, cephalomannine, 10-deacetyl taxol,
7-xyloxyl taxol and 10-deacetyl-7-xyloxyl taxol.
43. The process as claimed in claim 32 wherein the initial mixture comprises 9-dihydro-13-
acetylbaccatin III or cephalomannine and at least three additional taxanes selected from paclitaxel,
10-deacetylbaccatin III, baccatin III, 9-dihydro-13-acetylbaccatin III, cephalomannine, 10-deacetyl
taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol.
44. The process as claimed in claim 32 wherein the initial mixture comprises 9-dihydro-13-
acetylbaccatin III, paclitaxel, 10-deacetylbaccatin III, baccatin III, cephalomannine, 10-deacetyl taxol,
7-xylosyl taxol and 10-deacetyl-7-xylosyl taxol.
45. The process as claimed in claim 32 wherein the initial mixture of taxanes is a waste taxane
solution comprising one or more of the following:

pooled waste stream fractions collected during a chromatographic separation of a crude or partially
purified taxane extract; and
pooled waste mother liquors collected during a recrystallization of a crude or partially purified taxane
extract.
46. The process as claimed in claim 45 wherein the waste taxane solution comprises pooled waste
stream fractions collected during a chromatographic separation of a crude taxane extract.
47. The process as claimed in claim 45 wherein the waste taxane solution comprises pooled waste
stream fractions collected during chromatographic separations of both crude and partially purified
taxane extracts and pooled waste mother liquors collected during recrystallizations of both crude and
partially purified taxane extracts.
48. The process as claimed in claim 45 wherein the crude and partially purified taxane extracts are
obtained from taxane-containing materials from the genus Taxus.
49. The process as claimed in claim 32 comprising protecting the hydroxy group at the C-7
position of each of the 10-deacetylbaccatin III and baccatin III and converting the protected 10-
deacetylbaccatin III and baccatin III to paclitaxel or docetaxel.


A process is provided for the semi-synthesis and isolation of taxane intermediates
useful in the preparation of paclitaxel and docetaxel, in particular, the semi-synthesis and
isolation of 10-deacetylbaccatin III, the semi-synthesis of a mixture of 10-
deacetylbaccatin III and baccatin III, and protected derivatives thereof, from a mixture of
taxanes.

Documents:

03483-kolnp-2006 abstract.pdf

03483-kolnp-2006 claims.pdf

03483-kolnp-2006 correspondence others.pdf

03483-kolnp-2006 description (complete).pdf

03483-kolnp-2006 form-1.pdf

03483-kolnp-2006 form-3.pdf

03483-kolnp-2006 form-5.pdf

03483-kolnp-2006 international publication.pdf

03483-kolnp-2006 international search authority report.pdf

3483-KOLNP-2006-ABSTRACT 1.1.pdf

3483-KOLNP-2006-AMANDED CLAIMS.pdf

3483-KOLNP-2006-CORRESPONDENCE-1.1.pdf

3483-KOLNP-2006-CORRESPONDENCE.pdf

3483-KOLNP-2006-DESCRIPTION (COMPLETE) 1.1.pdf

3483-KOLNP-2006-EXAMINATION REPORT.pdf

3483-KOLNP-2006-FORM 1-1.1.pdf

3483-KOLNP-2006-FORM 13-1.1.pdf

3483-KOLNP-2006-FORM 13.pdf

3483-KOLNP-2006-FORM 18-1.1.pdf

3483-kolnp-2006-form 18.pdf

3483-KOLNP-2006-FORM 2.pdf

3483-KOLNP-2006-FORM 3-1.1.pdf

3483-KOLNP-2006-FORM 3-1.2.pdf

3483-KOLNP-2006-FORM 5.pdf

3483-KOLNP-2006-GPA.pdf

3483-KOLNP-2006-GRANTED-ABSTRACT.pdf

3483-KOLNP-2006-GRANTED-CLAIMS.pdf

3483-KOLNP-2006-GRANTED-DESCRIPTION (COMPLETE).pdf

3483-KOLNP-2006-GRANTED-FORM 1.pdf

3483-KOLNP-2006-GRANTED-FORM 2.pdf

3483-KOLNP-2006-GRANTED-SPECIFICATION.pdf

3483-KOLNP-2006-OTHERS-1.1.pdf

3483-KOLNP-2006-OTHERS.pdf

3483-KOLNP-2006-PA.pdf

3483-KOLNP-2006-PETITION UNDER RULE 137.pdf

3483-KOLNP-2006-REPLY TO EXAMINATION REPORT-1.1.pdf

3483-KOLNP-2006-REPLY TO EXAMINATION REPORT.pdf


Patent Number 251855
Indian Patent Application Number 3483/KOLNP/2006
PG Journal Number 15/2012
Publication Date 13-Apr-2012
Grant Date 12-Apr-2012
Date of Filing 22-Nov-2006
Name of Patentee PHYTOGEN LIFE SCIENCES INC.
Applicant Address 1527 CLIVEDEN AVENUE, DELTA BRITISH COLUMBIA V3M 6P7
Inventors:
# Inventor's Name Inventor's Address
1 NAIDU RAGINA 3768 BRANDON STREET, BURNABY BRITISH COLUMBIA V5G 2P2
2 FOO SAMUEL SIANG KIANG 6587 CARTIER STREET,VANCOUVER BRITISH COLUMBIA V 6P 4 S1
PCT International Classification Number C07D305/14; C07D305/
PCT International Application Number PCT/US2005/014080
PCT International Filing date 2005-04-22
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10/838,653 2004-05-04 U.S.A.
2 10/831,648 2004-04-23 U.S.A.