Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02389702 2002-05-O1
WO 01/34589 PCT/CA00/01293
SEMI-SYNTHESIS OF BACCATIN III FROM
9-DIHYDRO-13-ACETYLBACCATIN III
FIELD OF THE INVENTION
The present invention relates to a semi-synthetic process to convert a
naturally occurring
taxane into a suitable starting material for the synthesis of paclitaxel and
related compounds.
Specifically, the present invention relates to a process for the conversion of
9-dihydro-13-
acetylbaccatin III into baccatin III which can then be used as starting
material for the
synthesis of taxane derivatives such as paclitaxel, docetaxel, cephalomannine
and other
taxanes structurally related to baccatin III.
BACKGROUND OF THE INVENTION
The taxane family of terpenes is considered to be an exceptionally promising
group of cancer
chemotherapeutic agents. Many taxane derivatives, including paclitaxel,
docetaxel,
taxcultine canadensol are highly cytotoxic and possess strong in vivo
activities in a number
of leukemic and other tumor systems. Paclitaxel, and a number of its
derivatives, have been
shown to be effective against advanced breast and ovarian cancers in clinical
trials (W.P.
MacGuire et al., Annals of Internal Medicine, vol 111, pg. 273, 1989). They
have also
exhibited promising activity against a number of other tumor types in
preliminary
investigations. Paclitaxel has recently been approved in the U. S. and Canada
for the
treatment of ovarian cancers (Rose et al., in "The Alkaloids", A. Brossi, Ed.,
Academic Press,
New York, Paclitaxel: A Review of its preclinical in vivo Antitumor Activity.
Anti-Cancer
Drugs 3, 311-321 1992; and Suffness, M., Paclitaxel: from discovery to
therapeutic use.
Ann. Rep. In Med. Chem., 28, 305-314, 1993). Taxanes are believed to exert
their
antiproliferative effect by inducing tubulin polymerization, which forms
extremely stable and
nonfunctional microtubules (Schiff, et al., Promotion of Microtubule Assembly
in vitro by
Paclitaxel. Nature, 277, 665-667, 1979). However, a major problem with the
clinical studies
is the limited availability of paclitaxel and its derivatives.
Taxanes are natural products which can be isolated from yew trees. The first
taxane to be
characterized was paclitaxel (also known as taxolTM) which was isolated and
purified from
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CA 02389702 2002-05-O1
WO 01/34589 PCT/CA00/01293
the bark of the Pacific yew in 1971. The only available natural source of
paclitaxel to date
are several species of a slow growing yew (genus Taxus), wherein paclitaxel is
found in very
low concentrations (less than 400 parts per million) in these trees.
Furthermore the extraction
is difficult, the process is expensive and the yield of paclitaxel is low
(Huang et al, J. Nat.
Prod. 49 665, 1986, reported a yield of 0.00025% of a crude paclitaxel
fraction from Taxus
brevifolia bark).
0
0
Ph"NH O
Ph~O
OH OH
Ph
\\O O
Paclitaxel
Paclitaxel can be isolated from the bark of Taxus brevifolia, the pacific yew
tree, or from
Taxus baccata, its European relative. Since removal of the bark destroys the
trees and
endangers the species, isolation of taxanes from the stems and needles of
various Taxus
species offers hope that the supply of taxanes, in particular paclitaxel,
would become more
abundant.
The preparation of paclitaxel derivatives, some of which have been reported to
demonstrate
enhanced chemotherapeutic activity, ultimately depends upon the supply of the
parent
compound - baccatin III. The structure of baccatin III has the basic
diterpenoid structure of
paclitaxel without the side chain at the C-13 position.
2
2~i=11-2(701 ~ ~ CA000129.
CA 02389702 2002-05-O1
HO'
B aCCBtin III ' . . . . . , .
Haccatin III is an important starting material in paclitaxel- semi-
synthesis:., Therefore -the
significance of baccatin III will l~cely iuacrease as more clinical.studie..s
are performedW sing
paclitaxel and its derivatives. It appears that water soluble~paclitaxel-hive
compounds; writhe .: : . w , .
slightly modified C-13 side chains, may be more desirable; as cancer-
chemo_therapeutic agents - . . ..:,
than the naturally occulting paclitaxel, which has vlo~a: water solubility: -:
.1'his~ further _. .
increases the urgent need for a source of baccatin TJT as~ a: starting
material to synthesize both . .
pa.clitaxel and second yr third generation paclitaxel-li>'e: compounds..: In-
particular; there is a. . .:" ~ ~ . ..
need for an improved method of isolating and/or synthesizing Bac~atiawlln.~ :
. . ~ '.- . . _.. : . : -° .
The majority of research to date has been concerned, with-
;the~.develvpme~t.vf, ~techniqucs':to~>.~.
increase the availability of either paclitaxe! or baccatin:III:.:Tfiese.-
techrtiques>have .included_ : ; .. ..
improvements to the isvlatavn and purification pmcesses~.(U.S:: Patent~.5,4.
07;6.74 :and:.U.S. :. ' w . - _
Patent 5,380,916), to the total synthesis (U.5. Patent No: :5;.405.972 and
JAGS, 1995; ~7. 553- ' . . ' :.'
659) aad partial synthesis (from more abundant paclitax'el precursozs) ~ and
balsa isolation from ~ -
a variety of cell culture systems (U.S. Patent No.5,019,504). vIn additio~o~,
an endophytic~ fungi :' :~. ..
isolated fmm bald cypress (Taxodium distichum) was~reported~to produce.ve-
ry_~small achounts - ~ . .
of paelitaxel (Strobel, R. et al., Microbiology, 142, ZZZ3-ZZZ6;.199~_~ . ._ _
. . ~ . ' . ..~ . ' . '.. . . .
2O
Due to tic structural complexity of paclitaxel, partial 'synthesis is a
far:rriore.viable~apprvach' .
to providing adequate supplies of pacl itaxel and paclitaxel .precursors and
derivatives ~ than
total synthesis. The first successful semi-synthesis of paclitaxel was
developed by Deni9 et
al, (U.S. Patent No. 4,924,011 re-issued as 34,277), using the starting
material 10-
deacetylbaccatin III which tea be extracted in relatively high yield from the
needles of
3
Em~f .zei t :26/11/~(~11 2:14 Errnf .nr .: l~ P.~
AMENDED SHEET
CA 02389702 2002-05-O1
WO 01/34589 PCT/CA00/01293
specific species.
OH
HO''~, H~O
HO O
Ph I/
O O
10-Deacetylbaccatin III
Most of the research to date, regarding the semi-synthesis of paclitaxel has
involved 10-
deacetylbaccatin III. The conversion of 10-deacetylbaccatin III into
paclitaxel is typically
achieved by protecting the hydroxy at C-7, attachment of an acetyl group at
the C-10
position, attachment of a C-13 (3-amido ester side chain at the C-13 position
through
esterification of the C-13 alcohol with the ~3-lactam moiety, and deprotecting
C-7. Since the
supply of 10-deacetylbaccatin III is limited, alternative semi-synthetic
starting materials
should be considered.
Research has recently centred on semi-synthesis of paclitaxel from 10-
deacetylbaccatin III
because it is the major metabolite obtained from specific species of the
European Yew (Taxus
baccata). However to date, the yields of 10-deacetylbaccatin III have been
unsatisfactory,
ranging from 50-165 mg taxane per kilogram of starting material (i.e.
providing yields of
between 0.005 to 0.017%). Hence there is an urgent need for novel semi-
synthetic
techniques to produce higher yields of paclitaxel precursors, such as baccatin
III, for
subsequent use in the production of paclitaxel and paclitaxel derivatives.
Another taxane, 9-
dihydro-13-acetylbaccatin, is produced as a major metabolite in a certain
Taxus species and
has been used as a paclitaxel precursor (Canadian Patent Application Nos.
2,188,190 and
2,204,197). Relatively large amounts of a 7-protected baccatin III or baccatin
III itself can
be produced using the reported processes. The significance of this recent work
is that the
yield of 9-dihydro-13-acetylbaccatin III, which varies from 0.5 to 2.5 g per
kilogram of dry
4
2~-11-2001 CA 02389702 2002-05-O1 CA0001293
plant depending on the collection site, is much higher -than the yield, of !0-
dea.catylbac~atia
III that can be isolated from natural sources. Using the 9-dihydrol3-
acetylbaccatin III is the
synthetic process of the Canadian Patent Application No. 2.188,190 it was
possible to obtain
7 protected baccatin III in 209b yield. The synthetic. steps required: for
this conversion
included pmteeting the hpdrvxy group al the C-7 position or C-9 , yr both C-7
and C-9
sequentially; oxidizing the resulting gmLip at the C-9 position; and
deacylatiag the ester at ..
pvaitions . .C-13. Baccatin III could then be obtaS.ned_ by, deacylating_
the.:ester~-at the -G7
position. Another process far the prceparatlvn of taxol and its. analogs._fmm -
dihydm-13- . .
acetylbaccatin III has been described by Liu, J. (WO 98/50378).., - . - : . ._
., . ._ - . _ w. .
'The present invention provides a new method of converting.9-dihyc~v-13-
acetplbaccatin-III. - - .
into baccatia III which can then be used as a precursor.: to ~paclitaucc! ;
and: paditaxel . : . , . .
inta~mediates and derivatives. The method allows the production,of.relatively
large~amounts : :. .
of baccatin III without the use of protecting groups at any step itt the
process. . . . . . . . . ~ - . .
i5
SZtMMA.RY OF 'THE INVENTION . . . . : . _ . . .' .: : . . ' _ : . . ..
The present invention is directed towards a method..,of ~FOducing.-.baccatin
III. .-_i~rom :9.- ;, ,. . ~.. , ; .
d~ydro-13-acetylbaccafin III, which is a naturally occutrittg taxane.produced
in:high yields: . _ . .. .
in Taxus carcadensis. The baccatin III generated by this..method,. can then be
usedyas:a
starting material for the synthesis of paclitaxel and paclitaxel intermediates
and deiivatiyes : . ... ., . .
Accordingly, it is an object of this invention to provide -a. reproducable -
method- for the. semi- . ,- -_ , . .
synthesis of baccatin III from the naturally occurring-compound, 9-dihydm-13-
acetylbaccatin, . , -. ,
I>:1, isolated from plant matter derived from the Taxus.genus;of plants: ..
..~ . .. ~ _ _ . ' _ . : - ~ .
In accordance with an aspect of the present invention there is-provided a
method for the serni-
synthesis of baccatin III, and other intermediates, that proceeds with, higher
yields -than ~ -
currently lrnowa methods.
5
EmPf.zeit:L6/11/2001 2:14 EmPf.nr.:109 P.006
AMENDED SHEET
CA 02389702 2002-05-O1
WO 01/34589 PCT/CA00/01293
In accordance with another aspect of the present invention there is provided a
process for the
preparation of Baccatin III from a compound of formula (X)
0
v
~OH
AcO~
Ph
O O
S
X
which comprises the steps of
(i) oxidizing the hydroxy groups, on a compound of Formula X, at the C-7 and C-
9 positions;
(ii) reducing the resulting ketone at the C-7 position;
(iii) epimerizing the resulting 7a-hydroxy; and
(iv) deacylating the ester at the C-13 position.
In accordance with another aspect of the present invention there is provided
compounds of
the general Formula I:
6
CA 02389702 2002-05-O1
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0
O ~O R
Ac0°~',~~~~ a _W ,O
OH O
Ph
O
O
Formula I
wherein R is an alpha-hydroxy or ketone group.
In accordance with another aspect of the present invention there is provided
compounds of
the general Formula II:
0
O R1 p
Ac0 R2
OH O HO 20
Ph
O
Formula II
wherein R1 is a hydroxy or ketone group and R2 is a hydroxy or O-acetyl group.
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CA 02389702 2002-05-O1
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DETAILED DESCRIPTION OF INVENTION
The present invention relates to a high yield process for converting 9-dihydro-
13-
acetylbaccatin III (an abundant taxane found in T. canadensis needles), into
baccatin III,
which can subsequently be used as starting material for the synthesis of
paclitaxel and related
compounds.
The starting material for use in this invention is vegetal material, selected
from a group of
plants commonly referred to as taxads. The most suitable plants of this group
are the species
Taxus. Amongst the Taxus species, Taxus canadensis is a preferred source for
use in the
semi-synthetic method claimed in the present invention and differs from other
yews both in
its physical appearance (it is a small ramping evergreen bush), and in the
composition of
some of its taxanes. Paclitaxel, cephalomannine and 10-deacetylbaccatin III
can be isolated
from Taxus canadensis and are also found in most, if not all, other yews.
Taxus canadensis
is, however, the only yew presently known to accumulate a significant quantity
of 9-dihydro-
13-acetylbaccatin III in its needles, wherein it is found in concentrations 3 -
7 times greater
than paclitaxel (Zamir L. O
et al. Tetrahedron Letters 33, 5173, 1992). The only other yew where 9-dihydro-
13-
acetylbaccatin III is found albeit in traces is the Chinese yew (Taxus
chinenesis) (Zhang, S.;
Chen, W. M.; Chen, Y. H. Yaoxue Xuebao 1992, 27, 268).
0
O OH OH
13
O °~,... ~ H O
O - O
OH O
Ph
O
O
9-dihydro-13-acetylbaccatin III
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CA 02389702 2002-05-O1
WO 01/34589 PCT/CA00/01293
The methods disclosed herein are equally effective when using the roots or
bark of the Taxus
bushes but the preferred source is the needles, which are in abundant supply
and one of the
most renewable parts of the plant.
S A number of different methods have described the isolation and purification
of 9-dihydro-13-
acetylbaccatin III ( Zamire et al. Tetrahedron Lett. 1992, 33, 5173,
Gunawardana G. P. et al.,
J. Nat. Prod. 1992, 55, 1686 and Zamir et al. Can. J. Chem. 1995, 73, 655,). 9-
Dihydro-13-
acetylbaccatin III is particularly useful as a synthetic starting material
since it can be isolated
from plant material using simple recrystallizations (Canadian Patent No.
2,213,952) instead
of the numerous silica gel column and HPLC techniques commonly used for
isolation of
other starting materials, eg. 10-deacetylbaccatin III. In this way, 9-dihydro-
13-acetylbaccatin
III can be obtained in relatively high yield with relative ease, thereby
rendering it an ideal
starting material for many semi-synthetic pathways.
9-Dihydro-13-acetylbaccatin III, l, is the major component isolated from the
needles of the
Canadian yew taxus canadensis, as referenced in J. Nat. Prod., 53: 1249, 1990;
Tetrahedron
Lett., 33: 5235, 1992; Tetrahedrozz Lett., 33: 5173, 1992; Carz. J. Chem., 73:
655, 1995;
Phytochemistzy, 41: 803, 1996; .I. Nat. Prod., 55: 1686, 1992. Typically 1.0
kg of dry
needles will afford from 0.5 to 1.0 g of pure, isolated 9-dihydro-13-
acetylbaccatin III but can
possibly vary up to ranges of 2.0 to 2.5 g per kg of dry plant material. Its
efficient
transformation to baccatin III, 2, would be beneficial since this would be
another large source
of starting material for the semi-synthesis of paclitaxel.
Ac ' OH OH Ac ;~ O pH
:;J ~ ; ~J
%; ~~ i-
Ac0'. ~s.-~ H ~,,.0 HOs' ~~~~ H _ O
HO HO
OBz OAc OBz OAc
1 2
13-Acetyl-9-dihydrobaccatin III Baccatin III
The major concern in creating this transformation was the selective oxidation
of the 9a-OH.
A mild oxidizing agent, such as pyridinium dichromate (PDC), was used to
oxidize 13
acetyl-9-dihydrobaccatin III. Using these reaction conditions, the 7(3-OH was
preferentially
9
CA 02389702 2002-05-O1
WO 01/34589 PCT/CA00/01293
oxidized over the 9a-OH and the oxetane ring was opened affording 13-acetyl-9-
dihydro-D-
seco-5, 6-dehydrobaccatin III, 3, (Scheme 1). Jones' oxidation of the 9-
dihydro-13-
acetylbaccatin III, 1, afforded 13-acetyl-7-oxobaccatin III, 4, wherein both
the 9a-OH and the
7~3-OH are oxidized simultaneously. Similar to the investigation, of the
oxidation products of
paclitaxel, realized by Kingston (J. Org. Chem., 1998, SI, 797), 13-acetyl-7-
oxobaccatin III
is unstable and converts readily to the D-seco derivative, 13-acetyl-D-seco-
5,6-
dehydrobaccatin III, 5. When the reaction mixture, after oxidation, was
quenched under non-
aqueous conditions with KHC03, dried over MgS04, and filtered through silica
gel with
ether, very little conversion to the D-seco derivative resulted and the
desired 13-acetyl-7-
oxobaccatin III was obtained in 69% yield. Alternatively, if
Ac0 OH O
/
Ac0''', = H ~
HO ='OAc
Ac ; OH OH pgz OH
3
Ac0'~~ = H~O
HO
OBz OAc
Ac0 O O Ac0 O O
/ ~ /
Ac0'~~ : H _ O Ac0'~~ HO = H~R
HO OAc OBz OH
OBz
5 R = OAc
Sa R=OH
Scheme 1
the reaction mixture, after oxidation, was diluted with EtOAc, washed with
aqueous
NaHC03, then brine, dried over MgSOa , filtered, evaporated and the product
purified on
silica gel with EtOAc, then a 100% conversion to the D-seco derivative, 5, was
observed.
However, 13-acetyl-7-oxobaccatin III was stable when the purification step on
silica gel was
CA 02389702 2002-05-O1
WO 01/34589 PCT/CA00/01293
omitted, despite the aqueous work-up.
The reduction of 13-acetyl-7-oxobaccatin III, 4, (Scheme 2) was then
investigated. By taking
advantage of the greater reactivity of the 7-oxo group, 4 was reduced using
NaBH4 in
methanol at 4 °C. As a result, 13-acetyl-7-epibaccatin III, 13, was
produced exclusively in
82% yield. The NOESY data show a correlation between the C-7 proton and the
methyl
group at C-19, and a correlation between the 7-OH and the proton at C-3,
thereby confirming
a C-7a-OH. This result was undesired since the original 7~3-OH confirmation
was expected.
Kingston et al. (J. Org. Chem., 58: 3798, 1993) were able to successfully
convert 7-epi-taxol
to taxolTM via its 7-(S-methylthiocarboxy)taxol derivative. A more direct
route was used in
the present invention which employed the equilibrium reaction between the two
epimers at C-
7 under basic conditions, which takes advantage of the equilibrium reaction
previously
studied by Fang et al. (Synthetic Comm., 27: 2305, 1997). A similar technique
had been
found to be useful in the preparation of 6a-hydroxypaclitaxel, as referenced
in Tetrahedron
Lett., 1998, 39, 4967. Treatment of compound 13 in toluene with the bulky base
1,8-
diazabicyclo[5,4,0]undec-7-ene at 80°C, yielded a single product, the
7(3-OH, 14, in 29%
yield along with recovered starting material in 57% yield. Although the
reaction is not
quantitative, the yield based on recovered starting material was 67% which is
quite
acceptable. According to Fang et al. (Synthetic Comm., 1997, 27: 2305), 7(3-
epimerization is
enhanced when the 13-OAc has been cleaved. In an attempt to improve the yield
of the 7a to
the 7(3-epimerization, the hydrolysis of the 13-OAc of 13 was performed prior
to
epimerization step. When 13 was treated successively with NaBH4 in the
THF/O.OSM
phosphate buffer, pH 7.0, 2/1 at 23 °C, two compounds were obtained,
neither of which were
identified as the expected 7-epibaccatin III. The major compound was
identified as the
hydrolysis of the 4-OAc from 13, which was then hydrolyzed at the C-13
position to afford
the minor compound. Therefore, the final step in the synthesis was the
hydrolysis of the C-13
acetyl. Compound 14 was reductively cleaved with NaBH4 in THF/0.05 M potassium
phosphate buffer; pH 7,2/1 at 23 °C to afford 2 in 48%yield. The NMR
spectroscopic
properties of 2 were identical to those for baccatin III known in the
literature (In Progress in
the Chemistry of Organic Natural Products, Springer-Verlag, New York, 61: 1,
1993).
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CA 02389702 2002-05-O1
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Ac0 OH OH Ac0 O p Ac ; O OH
/ a / b /
v'~ ' . O 69% v = O 82% ~~''
Ac0 HO OB OAc yield Ac0 HO = H OAc Yield Ac0 HO = H OAc O
OBz OBz
1 4 13
c
67% yield
Ac0 O OH Ac0 O OH
/y ~ /
HO~~~ -~p 49% ~~'~ __
HO . H v yield Ac0 HO = H
OBz OAc ~Bz OAc
2 14
Reagents: (a) Jones' reagent, acetone, 23 °C, 30 min; (b) NaBH4, 4
°C, 1 h; (c) DBU, toluene,
80 °C, 1.5 h; (d) NaBH4, TI~'/0.0~ M KP04 buffer, pH 7, 2:1, 23
°C.
Scheme 2
Paclitaxel derivatives are useful for their antitumor activity, particularly
for the treatment of
the same cancers for which paclitaxel has been shown active, including human
lung tumors,
melanoma, leukemia, mammary tumors, and colon cancer. The taxane intermediates
of the
present invention can be used in the treatment of the same cancers for which
paclitaxel and
other taxanes have been used.
Paclitaxel has been shown to exhibit a very unique mechanism of action, in
that it promotes
the assembly of microtubules, but inhibits their disassembly, thereby
interfering with the G2
and M phases of cell cycles and division. In vitro studies have shown that
microtubules, once
polymerized, in the presence of paclitaxel resist depolymerization by other
agents such as
CaCl2 or cold temperature, which normally depolymerize microtubules.
A microtubule assembly study can be conducted, using compounds of the general
Formula I
and Formula II, according to the in vitro procedures disclosed, for example in
Parness et al.,
12
CA 02389702 2002-05-O1
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J. ('ell Biol. 91:479, 1981. In these studies, conditions can be established
whereby a dynamic
steady state exists between tubulin assembling into microtubules and the
disassembly at the
other end. This dynamic steady state can be measured spectrophotometrically,
observing the
absorbance of the solution at 350 nm. It has been shown that paclitaxel binds
specifically and
reversibly to the protein, stabilizing the microtubules in the polymerized
form. This effect
can be visualized by following the increase in absorbance of the solution at
350 nm. Cells
treated with taxolTM, or derivatives thereof, which exhibit chemotherapeutic
activity, usually
die as they are effectively blocked in mitosis.
The present invention also provides pharmaceutical compositions comprising one
or more
compounds of general Formula I and Formula II, as disclosed in the claims, in
combination
with one or more pharmaceutically acceptable, inert or physiologically active,
diluents or
adjuvants.
The present invention also provides pharmaceutical compositions comprising one
or more
compounds that can be prepared by further chemical manipulations of the
compounds of
general Formula I and Formula II (for example: deacetylation at C-13 and
attaching an
appropriate side chain at C-13), in combination with one or more
pharmaceutically
acceptable, inert or physiologically active, diluents or adjuvants.
The compounds of the instant invention can be freeze dried and, if desired,
combined with
other pharmaceutically acceptable excipients to prepare formulations for
administration.
These compositions may be presented in any form appropriate for the
administration route
envisaged. The parenteral and the intravenous route are the preferential
routes for
administration.
Compounds of general Formula I and Formula II, as disclosed in the claims, may
be
administered orally, topically, parenterally, by inhalation or spray or
rectally in dosage unit
formulations containing conventional non-toxic pharmaceutically acceptable
carriers,
adjuvants and vehicles. The term parenteral as used herein includes
subcutaneous injections,
intravenous, intramuscular, intrasternal injection or infusion techniques. In
addition, there is
provided a pharmaceutical formulation comprising a compound of general Formula
I or
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CA 02389702 2002-05-O1
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Formula II and a pharmaceutically acceptable carrier. One or more compounds of
general
Formula I and Formula II may be present in association with one or more non-
toxic
pharmaceutically acceptable carriers and/or diluents and/or adjuvants and if
desired other
active ingredients. The pharmaceutical compositions containing compounds of
general
Formula I and Formula II may be in a form suitable for oral use, for example,
as tablets,
troches, lozenges, aqueous or oily suspensions, dispersible powders or
granules, emulsion
hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use may be prepared according to any known to
the art for the
manufacture of pharmaceutical compositions and such compositions may contain
one or
more agents selected from the group consisting of sweetening agents,
flavouring agents,
colouring agents and preserving agents in order to provide pharmaceutically
elegant and
palatable preparations. Tablets contain the active ingredient in admixture
with non-toxic
pharmaceutically acceptable excipients which are suitable for the manufacture
of tablets.
These excipients may be for example, inert diluents, such as calcium
carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate: granulating and
disintegrating
agents for example, corn starch, or alginic acid: binding agents, for example
starch, gelatin or
acacia, and lubricating agents, for example magnesium stearate, stearic acid
or talc. The
tablets may be uncoated or they may be coated by known techniques to delay
disintegration
and absorption in the gastrointestinal tract and thereby provide a sustained
action over a
longer period. For example, a time delay material such as glyceryl monosterate
or glyceryl
distearate may be employed.
Formulations for oral use may also be presented as hard gelatin capsules
wherein the active
ingredient is mixed with an inert solid diluent, for example, calcium
carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient
is mixed with
water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
Aqueous suspensions contain active materials in admixture with excipients
suitable for the
manufacture of aqueous suspensions. Such excipients are suspending agents, for
example
sodium carboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose,
sodium
alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia: dispersing or
wetting agents
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may be a naturally-occurring phosphatide, for example, lecithin, or
condensation products of
an alkylene oxide with fatty acids, for example polyoxyethyene stearate, or
condensation
products of ethylene oxide with long chain aliphatic alcohols, for example
hepta-
decaethyleneoxycetanol, or condensation products of ethylene oxide with
partial esters
derived from fatty acids and a hexitol such as polyoxyethylene sorbitol
monooleate, or
condensation products of ethylene oxide with partial esters derived from fatty
acids and
hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous
suspensions
may also contain one or more preservatives, for example ethyl, or n-propyl p-
hydroxy
benzoate, one or more colouring agents, one or more flavouring agents or one
or more
sweetening agents, such as sucrose or saccharin.
Oily suspensions may be formulated by suspending the active ingredients in a
vegetable oil,
for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral
oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for example
beeswax, hard
paraffin or cetyl alcohol. Sweetening agents such as those set forth above,
and flavouring
agents may be added to provide palatable oral preparations. These compositions
may be
preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous
suspension by the
addition of water provide the active ingredient in admixture with a dispersing
or wetting
agent, suspending agent and one or more preservatives. Suitable dispersing or
wetting agents
and suspending agents are exemplified by those already mentioned above.
Additional
excipients, for example sweetening, flavouring and colouring agents, may also
be present.
Pharmaceutical compositions of the invention may also be in the form of oil-in-
water
emulsions. The oils phase may be a vegetable oil, for example olive oil or
arachis oil, or a
mineral oil, for example liquid paraffin or mixtures of these. Suitable
emulsifying agents
may be naturally-occurring gums, for example gum acacia or gum tragacanth,
naturally-
occurring phosphatides, for example soy bean, lecithin, and esters or partial
esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and
condensation
products of the said partial esters with ethylene oxide, for example
polyoxyethylene sorbitan
monoleate. The emulsions may also contain sweetening and flavouring agents.
CA 02389702 2002-05-O1
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Syrups and elixirs may be formulated with sweetening agents, for example
glycerol,
propylene glycol, sorbitol or sucrose. Such formulations may also contain a
demulcent, a
preservative and flavouring and colouring agents. The pharmaceutical
compositions may be
in the form of a sterile injectable aqueous or oleaginous suspension. This
suspension may be
formulation according to known art using those suitable dispersing or wetting
agents and
suspending agents which have been mentioned above. The sterile injectable
preparation may
also be sterile injectable solution or suspension in a non-toxic parentally
acceptable diluent or
solvent, for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and
solvents that may be employed are water, Ringer's solution and isotonic sodium
chloride
solution. In addition, sterile, fixed oils are conventionally employed as a
solvent or
suspending medium. For this purpose any bland fixed oil may be employed
including
synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid
find use in the
preparation of injectables.
The compounds) of general Formula I and Formula II may be administered,
together or
separately, in the form of suppositories for rectal administration of the
drug. These
compositions can be prepared by mixing the drug with a suitable non-irritating
excipient
which is solid at ordinary temperatures but liquid at the rectal temperature
and will therefore
melt in the rectum to release the drug. Such materials are cocoa butter and
polyethylene
glycols.
Compounds) of general Formula I and Formula II may be administered, together
or
separately, parenterally in sterile medium. The drug, depending on the vehicle
and
concentration used, can either be suspended or dissolved in the vehicle.
Advantageously,
adjuvants such as local anaesthetics, preservatives and buffering agents can
be dissolved in
.the vehicle.
The mode, dosage and schedule of administration of paclitaxel in human cancer
patients has
been studied extensively (see Ann. Int. Med. 111:273 1989). For the compounds
of this
invention, the dose to be administered, whether a single dose, multiple dose,
or a daily dose,
will vary with the particular compound being used. Factors to consider when
deciding upon a
dose regimen include potency of the compound, route of administration, size of
the recipient
16
CA 02389702 2002-05-O1
WO 01/34589 PCT/CA00/01293
and the nature of the patient's condition.
The dosage to be administered is not subject to defined limits, but it will
usually be an
effective amount. It will usually be the equivalent, on a molar basis of the
pharmacologically
active free form produced from a dosage formulation upon the metabolic release
of the active
free drug to achieve its desired pharmacological and physiological effects.
An oncologist skilled in the art of cancer treatment will be able to
ascertain, without undue
experimentations, appropriate protocols for effective administration of the
compounds of this
present invention by referring to the earlier studies of taxolTM and its
derivatives.
To assist in understanding the current invention, the following non-limiting
examples are
provided. The examples describe the chemical transformation of this baccatin
III precursor,
9-dihyro-13-acetylbaccatin III, into baccatin III and baccatin III derivatives
which in turn can
be transformed into paclitaxel and other biologically active precursors and
derivatives. The
following examples should not be construed as specifically limiting the
present invention,
variations presently known or later developed, which would be in the
understanding of one
skilled in the art and considered to fall within the scope of the present
invention as described
herein.
17
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EXAMPLES
General Experimental Procedures.
All the Nuclear Magnetic Resonance (NMR) spectra were obtained at room
temperature with
a UNITY-50 spectrometer operating at 499.83 MHz for 1H, 125.697 MHz for 1 3 C
and
76.75 MHz for 2H. The NMR spectra were obtained on 5-10 mg dissolved in 0.3-
0.4 mL
of CDC13. The solvent was used as an external reference (77.0 ppm for 1 3 C
and 7.25 ppm
for 1H and 2H). Chemical shifts are expressed in part per million (ppm).
Coupling
constants (J) are given in Hertz, where s, bs, t, d, dd, q and m indicate
singlet, broad singlet,
triplet, doublet, doublet of doublet, quadruplet and multiplet, respectively.
Positive ion Fast
Atom Bombardment Mass Spectra (FAB-MS) were obtained with a Vacuum Generators
ZAB-HS double-focussing instrument using a xenon beam having 8 kV energy at 1
mA
equivalent neutral current. Flash chromatography was performed on Silica gel
60, 230-400
mesh (EM Science). Thin layer chromatography was conducted on Silica Gel 60
FZS4 pre-
coated TLC plates, 0.25 mm (EM Science). Analytical high performance liquid
chromatography (HPLC) was performed on a Waters 600E delivery system coupled
to a PDA
996 detector. Preparative and semi-preparative HPLC were carried out on a
Waters Delta
Prep 3000 instrument coupled to UV 486 Tunable Absorbance detector set at 227
nm
(Waters, Montreal, Quebec, Canada). Analytical HPLC was performed with two
Whatman
partisil 10 ODS-2 analytical columns in series (4.6 x 250 mm). Semi-
preparative HPLC was
performed with two Whatman partisil 10 ODS-2 Mag 9 semi-preparative columns in
series
(9.4 x 250 mm). Preparative HPLC was performed with one partisil 10 ODS-2 MAG-
20
preparative column (22 x 500 mm).
Example 1: Preparation of 13-acetyl-9-dihydro-D-seco-5, 6-dehydrobaccatin III,
3, from 13-
acetyl-9-dihydrobaccatin III, 1
13-Acetyl-9-dihydrobaccatin III (15 mg; 0.024 mmol) in CHZC12 (3 mL) was
treated with
pyridinium dichromate (29 mg; 0.077 mmol) at 23 °C for 4.5 h. The
mixture was diluted in
CHZC12 and washed with brine, dried over MgS04, filtered and evaporated in
vacuo. The
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WO 01/34589 PCT/CA00/01293
residue was purified by semi-preparative HPLC (25-100% CH3CN in H20, 50 min
gradient at
a flow rate of 3 mL/min) affording 13-acetyl-9-dihydro-D-seco-5,6-
dehydrobaccatin III (2
mg, 13%, R, = 32.97 min). HRFABMS m/z 651.2420 (M + Na+), calculated for
C33Hao0izNa, 651.2418; 1H NMR (CDC13, 500 MHz) 8 8.10 (d, J = 7.6 Hz, 2H, H-
2,6 of
OBz), 7.61 (t, J = 7.3 Hz, 1H, H-4 of OBz), 7.50 (t, J = 7.5 Hz, 2H, H-3,5 of
OBz), 6.69 (d, J
= 10.3 Hz, 1H, H-5), 6.20 (d, J = 10.3 Hz, 1H, H-10), 5.88 (d, J = 10.8 Hz,
1H, H-6), 5.75 (br
m, 1H, H-13), 5.73 (br d, 1H, H-2), 4.38 (d, J = 12.2 Hz, 1H, H-20a), 4.37 (d,
J = 12.7 Hz,
1H, H-20b), 4.32 (d, J = 11.7 Hz, 1H, OH-9), 4.19 (t, J = 11.2 Hz, 1H, H-9),
3.42 (d, J = 5.8
Hz, 1H, H-3), 3.24 (s, 1H, OH-4), 2.73 (dd, J = 15.1, 3.4 Hz, 1H, H-14a), 2.46
(dd, J = 15.6,
10.3 Hz, 1H, H-14b), 2.16 (s, 3H, OAc), 2.10 (s, 3H, OAc), 1.84 (s, 3H, OAc),
1.72 (s, 3H,
H-18), 1.63 (s, 3H, H-19), 1.58 (s, 3H, H-17), 1.09 (s, 3H, H-16); '3C NMR
(CDCI3, HMQC
data) b 206.1 (C-7), 170.9 (CO-OAc), 170.5 (CO-OAc), 169.9 (CO-OAc), 166.8 (CO-
OBz),
152.8 (C-S), 138.1 (C-12), 136.7 (C-11), 133.8 (C-4 of OBz), 130.1 (C-2,6 of
OBz), 129.3
(C-1 of OBz), 128.9 (C-3,5 of OBz), 124.6 (C-6), 77.6 (C-1), 75.2 (C-9), 74.8
(C-4), 73.3 (C-
10), 72.3 (C-2), 71.2 (C-13), 69.0 (C-20), 53.9 (C-3), 51.3 (C-8), 41.8 (C-
15), 36.5 (C-14),
29.3 (C-16), 21.3 (OAc), 21.1 (OAc), 20.3 (OAc), 20.2 (C-17), 19.5 (C-19),
15.7 (C-18).
Example 2: Preparation of 13-acetyl-7-epibaccatin III, 13, from 13-acetyl-7-
oxobaccatin
III, 4
13-Acetyl-7-oxobaccatin III (46 mg; 0.073 mmol) was dissolved in methanol (10
mL) and
treated with NaBH4 (6 mg; 0.16 mmol) at 4 ° C, for 2 h. The reaction
was quenched by
dilution with brine. The solution was extracted with dichloromethane, and the
combined
organic layers were dried over MgS04, filtered and evaporated in vacuo. The
residue was
purified by chromatography on silica gel (EtOAc/hexane, 50/50) affording 13-
acetyl-7-
epibaccatin III (38 mg, 82%). HRFABMS m/z 651.2420 (M + Na+), calculated for
CssHaoOizNa, 651.2418; 1H NMR (CDCI3, 500 MHz) b 8.08 (dd, J = 7.8, 0.7 Hz,
2H, H-2,6
of OBz), 7.63 (t, J = 7.3 Hz, 1H, H-4 of OBz), 7.50 (t, J = 7.5 Hz, 2H, H-3,5
of OBz), 6.83 (s,
1H, H-10, 6.13 (br t, J = 8.0 Hz, 1H, H-13), 5.75 (d, J = 7.3 Hz, 1H, H-2),
4.95 (dd, J = 9.3,
3.2 Hz, 1H, H-5), 4.66 (d, J = 11.7 Hz, 1H, OH-7), 4.40 (d, J = 8.5 Hz, 1H, H-
20a), 4.35 (d, J
= 8.5 Hz, 1H, H-20b), 3.96 (d, J = 7.3 Hz, 1H, H-3), 3.70 (ddd, J = 11.7, 4.6,
1.7 Hz, 1H, H-
7), 2.41 (s, 3H, OAc), 2.36 (ddd, J = 15.9, 9.5, 2.0 Hz, 1H, H-6a), 2.27 (om,
1H, H-6b), 2.27
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WO 01/34589 PCT/CA00/01293
(om, 2H, H-14a/b), 2.21 (s, 3H, OAc), 2.20 (s, 3H, OAc), 1.87 (d, J = 1.2 Hz,
3H, H-18), 1.65
(s, 3H, H-19), 1.19 (s, 3H, H-16), 1.15 (s, 3H, H-17); 13C NMR (CDCl3, HMQC
data) 8
207.3 (C-9), 171.6 (CO-OAc), 170.2 (CO-OAc), 169.4 (CO-OAc), 167.0 (CO-OBz),
140.6
(C-12), 133.9 (C-4 of OBz), 133.0 (C-I 1), 130.0 (C-2,6 of OBz), 129.3 (C-1 of
OBz), 128.6
(C-3,5 of OBz), 82.5 (C-5), 82.1 (C-4), 79.0 (C-1), 78.1 (C-10), 77.5 (C-20),
75.4 (C-7), 75.2
(C-2), 69.6 (C-13), 57.5 (C-8), 42.3 (C-15), 40.2 (C-3), 35.8 (C-14), 35.2 (C-
6), 25.8 (C-16),
22.3 (OAc), 20.8 (OAc), 20.8 (OAc), 20.6 (C-17), 15.9 (C-19), 15.0 (C-18).
Example 3: Preparation of 13-acetylbaccatin 111, 14, from 13-acetyl-7-
epibaccatin III, 13
To a solution of 13-acetyl-7-epi-baccatin III (7 mg; 0.011 mmol) in toulene
(0.5 mL) was
added 1,8-diazabicyclo[5,4,0]undec-7-ene (55 ~,L; 0.37 mmol) and the mixture
was stirred at
80°C for 1.5 h. The reaction mixture was then diluted in EtOAc and
washed with dilute HCI,
a saturated solution of NaHC03 and brine. The organic layer was then dried
over MgS04,
filtered and evaporated in vacuo. The residue was purified by semi-preparative
HPLC (25-
100%) CH 3 CN in H 2 O, 50 min. gradient at a flow rate of 3 mL/min) affording
13-
acetylbaccatin III (2 mg, 29%, Rt = 33.58 min) and recovered 13-acetyl-7-epi-
baccatin III (4
mg, 57%, Rt = 37.98 min). HRFABMS m/z 651.2420 (M + Na+), calculated for
C 3 3 H 4 o O 12 Na, 651.2418; 1H NMR (CDCI 3 , 500 MHz) 8 8.07 (d, J = 7.6
Hz, 2H, H-2,6
of OBz), 7.61 (t, J = 7.5 Hz, 1H, H-4 of OBz), 7.48 (t, J = 7.4 Hz, 2H, H-3,5
of OBz), 6.30 (s,
1H, H-10), 6.18 (dd, J = 9.5, 8.2 Hz, 1H, H-13), 5.66 (d, J = 7.2 Hz, 1H, H-
2), 4.97 (dd, J =
9.8, 1.6 Hz, 1H, H-S), 4.43 (br m, 1H, H-7), 4.30 (d, J = 8.1 Hz, 1H, H-20a),
4.16 (d, J = 8.5
Hz, 1H, H-20b), 3.82 (d, J = 6.9 Hz, 1H, H-3), 2.55 (ddd, J = 14.7, 9.4, 6.2
Hz, 1H, H-6a),
2.46 (br d, J = 3.0 Hz, 1H, OH-7), 2.32 (s, 3H, OAc), 2.27 (om., 1H, H-6b),
2.24 (om, 2H, H-
14a/b), 2.24 (s, H, OAc), 2.20 (s, 3H, OAc), 1.90 (d, J = I.1 Hz, 3H, H-18),
1.67 (s, 3H, H-
19), 1.23 (s 3H, H-16), 1.13 (s, H, H-17); 13C NMR (CDCl3, HMQC data) 8 203.4
(C-9),
170.9 (CO-OAc), 169.6 (CO-OAc), 169.2 (CO-OAc), 166.6 (CO-OBz), 142.5 (C-12),
134.2
(C-4 of OBz), 132.2 (C-11), 130.3 (C-2,6 of OBz), 129.3 (C-I of OBz), 128.8 (C-
3,5 of
OBz), 84.6 (C-5), 80.5 (C-4), 78.7 (C-1), 76.3 (C-20), 75.8 (C-10), 75.1 (C-
2), 72.4 (C-7),
69.7 (C-13), 58.1 (C-8), 45.6 (C-3), 42.6 (C-15), 35.6 (C-14), 35.6 (C-6),
26.7 (C-16), 22.4
(OAc), 21.4 (C-17), 21.1 (OAc) 20.9 (OAc) 15.0 (C-18), 9.4 (C-19).
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Example 4: Preparation of Baccatin Ill, 2, from 13-acetylbaccatin III, 19
To a solution of 13-acetylbaccatin III (10 mg; 0.016 mmol) in THF (0.6 mL) was
added 0.05
M potassium phosphate buffer, pH 7.0, (0.3 mL). The opaque solution was
treated with
NaBH4 (2 x 5 mg; 0.13 mmol) at 23 C over a period of 10 h. The reaction was
quenched by
dilution with brine. The solution was extracted with dichloromethane, and the
combined
organic layers were dried over MgS04, filtered and evaporated in vacuo. The
residue was
purified by semi-preparative HPLC (25-100% CH3CN in H20, 50 min gradient at a
flow rate
of 3 mL/min) affording Baccatin III (4.5 mg, 48%, Rt = 28.45 min). The NMR
spectroscopic properties of 2 were identical for those known for baccatin III.
These and other objects, as well as the nature, scope and utilization of this
invention, will
become readily apparent to those skilled in the art from the following
description and the
appended claims.
21
