Note: Descriptions are shown in the official language in which they were submitted.
CA 02307548 2001-O1-25
PROCESS FOR CONVERTING 9-DIHYDRO-13-ACETYLBACCATIN III INTO
TAXOL AND DERIVATIVES THEREOF
FIELD OF THE INVENTION
The present invention relates to a process for converting 9-dihydro-13-
acetylbaccatin
III into taxane, and is particularly concerned with a process for converting 9-
dihydro-13-
acetylbaccatin III into taxol, baccatin III, 10-deacetyIbaccatin III and their
intermediates.
As used herein, taxol is a registered trademark.
BACKGROUND OF THE INVENTION
to Paclitaxel (taxol), represented by the following structural formula:
O
H3C
O O OH
\ /
,o s
O NH O
. ~s
\ '~:
OH O HO
/ \ O ~ CHI
O O
is a potent antitumor compound. Paclitaxel exhibits a unique mechanism for
preventing the
growth of cancer cells by affecting the microtubules, which play an important
role in cell
division and other cell functions. A.t the beginning of cell division. a large
number of
microtubules are produced, and as the division reaches an end, the
microtubules are normally
CA 02307548 2001-O1-25
broken down. Taxol prevents microtubules from breaking down, which has the
effect of
clogging up cancer cells to an extent that the cells cease to grow and divide.
Taxol is clinically effective :for the treatment of refractory human ovarian
and breast
cancer, and has exhibit promising activity against a number of other types of
cancers such as
liver, peritoneal, cervical, prostate, colon, and esophageal cancers.
Taxol was primarily extracted from the bark of the Pacific yew Taxes brevi_
folia.
Unfortunately, the yew grows very slow, approximately eight inches per year,
and therefore
the tree is a limited source of taxol. This has lead researchers to seek
alternative means for
to producing taxol and analogs thereof which may display superior antitumor
activity.
SUMMARY OF THE INVENTION
In one aspect of the present invention, there is provided a process for
preparing a
I5 taxane comprising the step of oxidizing the C-9 position of 9-dihydro-13-
acetylbaccatin III
with a suitable oxidizing reagent such as tetra-n-propylammonium perruthenate,
Collin's
reagent or activated dimethylsulfoxide (DMSO).
In another aspect of the present invention, there is provided a process for
preparing
2o taxol and a derivative thereof comprising the steps of
(a) protecting the C-7 hydroxy group of 9-dihydro-I3-acetylbaccatin III with a
suitable protecting group to obtain a protected product; and
(b) oxidizing the C-9 hydroxy group of the protected product.
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Preferably, the protecting group is selected from the group consisting of
benzyl, substituted
benzyl, benzylformate, substituted benzylformate, tosyl, substituted tosyl,
dihydropyran,
methoxymethyl, benzoyl and substituted benzoyl.
The process may fi.trther include the steps of deacetylating the C-13
position, and
deprotection of the C-7 position to obtain baccatin III. Alternatively,
following the C-13
deacetylation, the C-10 position can be deacetylated and the C-7 position can
be deprotected
to obtain 10-deacetylbaccatin III. Or alternatively, following the C-13
deacetylation, a
suitable side chain can be added to the C-13 position and the resulting
intermediate selectively
deprotected to obtain a desired product such as taxol. To obtain taxol, the
deprotection is
1u done at the C-7 and 2' positions. Suitable side chains are (2R, 2S)-N-
benzoyl-O-(1-
ethoxyethyl)-3-phenylisose;rine and (3R, 4S)-3-(1-ethoxyethoxy)-4-(phenyl)-N-
benzoyl-2-
azetidinone.
In accordance with another aspect of the present invention, there is provided
a
compound having the following chemical structure:
Ac:O OH
OR
10 9 /
0\ -
o"
W m.. ~3
H3C H~iO
HC~ _
/ \ ~O ~-CHa
(I)
wherein R is selected from the grotup consisting of benzyl, substituted
benzyl, benzylformate,
substituted benzylformate, tosyl, substituted tosyl, dihydropyran,
methoxymethyl, benzoyl,
2o substituted benzoyl, benzoylmethyl and substituted benzoylmethyl.
3
SUSST1TUTE SHEET (RULE 26)
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WO 98/50378 PCT/CA98100401
In accordance with another aspect of the present invention, there is provided
a
compound of the formula:
Ac0
~o s
O
O
/ \ O ~CHa
O O (II)
wherein R is selected from the group consisting of benzyl, substituted benzyl,
benzylformate,
substituted benzylformate, tosyl, substituted tosyl, dihydropyran,
methoxymethyl, benzoyl,
substituted benzoyl, benzoylmethyl and substituted benzoylmethyl.
1o DETAILED DESCRIPTION OF THE INVENTION
Table 1 lists definitions and chemical structures of compounds as used herein,
and
those definitions and chemical structures are to be applied throughout the
present
specification.
4
SUBSTITUTE SHEET (RULE 26)
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Table 1: Definitions ,And Chemical Structures
Name Definition/chemical structure
Functional group an atom or group of atoms having a characteristic
chemical
reactivit
Benzyl
CHz
Substituted benzyi a benzyl group as defined above substituted with
one or more
functional rou s
Dihydropyran
o
Benzylformate o
CHz C \
O-
Substituted benzylformatea benzyiformate as defined above substituted
with one or m
r
o
e
functional ~rou (s
Methoxymethyl -cH2-o - cH3
Benzoylmethyl p
-
C -CH
z
/
Substituted benzoylmethyla benzoylmethyl as defined above substituted
with one or more
functional rou s
Tosyl
-02S ~ / CHI
Substituted tosyl a tosyl as defined above substituted with one
or more fianctional
~roup(s)
SUBSTITUTE SHEET (RULE 26)
CA 02307548 2001-O1-25
The starting material, 9-dihydro-13-acetylbaccatin III, can be obtained by
various
means including by extraction of TczxuS species. Briefly, the isolation
process entails
collecting plant material such as stems and needles, and grounding and
extracting the
material with methanol. The extraction is carried through for about 24 hours,
and the
resulting mixture is filtered and the extract collected. The extract is
concentrated to
about 10°l0 of its original volume by evaporation, and further diluted
with water. The
aqueous solution is extracted several times with hexane to give an aqueous
layer and a
non-aqueous layer. The aqueous layer is extracted several times with
chloroform or
dichloromethane. The chloroform or dichloromethane extract is concentrated to
dryness, and the residue is dissolved in a mixture of chloroform, methanol and
acetone
(10:1:0.5), and fractionated by dry column chromatography to obtain fractions
of taxol
and 9-dihydro-13-acetylbaccatin ID. The fractions are combined, extracted and
the 9-
dihydro-13-acetylbaccatin is crystallized out.
t>
Taxol and derivatives thereof may be synthesized from 9-dihydro-13-
acetylbaccatin III
by a number of protection, oxidation, reaction and deprotection steps. For
example, referring
to the processes illustrated below in Schemes 1-3, 9-dihydro-13-acetylbaccatin
III is first
protected at the C-7 position by adding a protecting group such as a
methoxybenzyl or tosyi
20~ group to form a protected intermediate such as compound 2 7 or 16. Other
suitable
protecting groups are substituted benzyl, dihydropyran, benzylformate (CBZ),
substituted
benzylformate, methoxymethyl (MOM), benzoylmethyl (BOM) and substituted
benzoylmethyl.
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The protected intermediate is oxidized at the C-9 position with a suitable
oxidizing
agent such as tetra-n-prop5~lammonium perruthenate (TPAP), Collin's reagent
(chromium
trioxide and pyridine in dichloromethane) or activated methyl sulfoxide (DMSO)
to give an
oxidized intermediate such as compound 3, 8 or 17. The oxidized intermediate
may be
deacetylated at the C-13 position by reaction with, for example butyliithium
in hexane or
methyllithium, to give a bac:catin LII analog such as compound 4 or 9 .
Depending of the
desired product, the baccatin III analog can either be deprotected with, for
example 2,3-
dichloro-5,6-dicyanobenzoyuinone (DDQ) to yield baccatin III (15) or further
reacted to
eventually obtain 10-deacetylbaccatin III ( 13 ) or taxol ( 18) or other
desirable products.
To obtain 10-deace~:ylbaccatin III , the baccatin III analog (4) is next
deacetylated at
the C-10 position by reaction with, for example triethylamine in sodium
hydride, to
compound 12 which is further deprotected. To obtain taxol, the C-13 taxol side
chain is
added to the baccatin III analog by reaction with, for example (2R, 2S)-N-
benzoyl-O-( 1-
t5 ethoxyethyl)-3-phenylisoserine and lithium hexamethyldisilazide, to give a
taxol analog such
as compound 5 or 10. The taxol analog is next deprotected at the 2' by
reaction with, for
example 1% hydrochloric a~~ed, and C-7 position by reaction with, for example
triethylamine
and UV light, to give taxol. Other suitable C-13 side chains such as (3R, 4S)-
3-(1-
ethoxyethoxy)-4-(phenyl)-Td-benzoyl-2-azetidinone may also be used to obtain
taxol analogs.
It will be appreciate~~ by one skilled in the art that other reagents and
reactants may be
used to obtain the same results, and that protection, oxidation and
deprotection steps may be
carried out in varying order; or number of steps, as necessary, and that
schemes 1-3 are
intended to include such variations.
SUBSTITUTE SHEET (RULE 26)
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WO 98150378 PCT/CA98/00401
Ac0 OH SCHEME 1 AcO OH / \
OH OCHz~OMe
s
s
_ CH~O / \ CH:CI
Ac01°' ~3
ACO nn", ~3 ,i,
HO H = / O NaH, n-Bu4Nl HO H ~/ O
OAc \ ~ OAc 2
/ \ O
TPAP
Ac0 O NMO
OCHz / \ OMe
RT
s
HO m, is ", CH3Li Ac0 O
OCH2 / ~ OMe
H - / O - 44 degree s
HO -
4 OAc
/ \ O AcOm, ~3 "' _
H ~ WO
O HO
DDQ ~~ OAc
/ \
CHZC12-H20 3
(18:1 ) ESN / \ NH 4 O
NaH
/ \ _
Ac0 O ~H LiHMDS
OH OEE
s
Of E~O~, ,Ph
HOin'. i3 ",
N 5
\'Ph
HO = ~ 0 0O
OAc
/ \ O
O
HO O OCHz / \ OMe HO O OH
s s
HO ii" ~3 ",
HO u"
DDQ =
HO ~ O CH2Ch-H20 HO H , O
OAc (20:1 ) OAc
/ \ O / \ O
O O
12 13
SUBSTITUTE SHEET (RULE 26)
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WO 98/50378 PCT/CA98/00401
SCHEME 1 (cont'd)
0 0
Ac0 OCH2 ~ ~ OMe
NH O s
/ \ ~ ",
O n~" ~3
OEE a -
HO ~/
OAc
/ ~ O
1% HCI
O O
/ \ Ac0 OCHz ~ ~ OMe
NH O
s
/ \
_ p m , ~3 ",
OH H~ 'O
HO
OAc
/ ~ O
DDQ
CHzCh-H20 (20:1 )
O
Ac0 O
/ \ OH
NH O s
/ \
_ O m" ~ 3 "'
OH
HO -
OAc
18
9
SUBSTITUTE SHEET (RULE 26)
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WO 98/50378 PCT/CA98/00401
SCHEME 2
Ac0 OH
OH
Ac0 OH OSO2 ~ ~ CH3
CH3 ~ ~ S02C1
,,, _
Ac0 "". ~s
O NaH, n-Bu4Nl Ac0 i"" is , 7
HO
OAc H ~ ~p
O HO
OAc
O ~ ~ O
O
1
TPAP
NMO
RT
Ac0 O OS02 ~ ~ CH3
i
Ac0 m. ~ 3 ,,,
H~ '
i
HO
OAc
~ O
O
n-BuLi
-40 degree
9
SUBSTITUTE SHEET (RULE 26)
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WO 98/50378 PCT/CA98/00401
Ac0 O OSO~~~CH3
o SCHEME 2 (cont'd)
HO ~~~~ is ~,, \ / NH O
H = ~O
HO = ' \ / off
OAc -
/ \ p OEE
LiHMDS
O
9
DDS
CH2Clz Hz0 - Ac0 O OS02 ~ ~ CH3
(18:1) \ / NH o s
\ / ~O m, ~3 ~.,
Ac0 O OH OEE ~.~ _ ~O ~ 0
HO
s OAc
/ \ O
HO~~w ~g r.~
O
H~~ O 1°i° Hcl
HO OAc
/ \ ~ o
Ac0 O OSO2 ~ ~ CH3
O ~~~NH o s
\ / Dln. 1g ~,,
OH H - / O
HO -
OAc
/ \ O
Et3N, by
O 11
MeOH
0
P,cO
OH
\ / NH O s
\ / O m. ~ 3 ~,,
OH H = , O
Hc~ -
OAc
~~O
~~-~,~~0
18
11
SUBSTITUTE SHEET (RULE 26)
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WO 98/50378 PCT/CA98/00401
SCHEME 3
Ac0 OH OH
CHZBr Ac0 OH Q
9
AcOm",. t3 ",
HO H =' 'O AcOm~,. ~3 ~"
OAc H ~ ~
/ \ HO -
OAc
O / \ O
O 16
1
TPAP
NaOCI
O
Q
AcO~ ~ ~".
HO H = '/
OAc
/ \
O
17
12
SUBSTITUTE SHEET (RULE 26)
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WO 98/50378 PCT/CA98/00401
1 9-dihydro-13-acetylbaccatin III
7-O-p-methoxybenzyl-9-dihydro-13-acetylbaccatin III
3 7-O-p-methoxybenzyl-13-acetylbaccatin III
4 7-O-p-methoxybenzylbaccati.n III
5 7-O-p-methoxybenzyl-(2'-ethoxyethyl) taxol
6 7-O-p-methoxybenzyl taxol
7 7-O-tosyl-9-dihydro-13-acet:ylbaccatin III
8 7-O-tosyl-13-acetylt>accatin III
9 7-O-tosylbaccatin III
l0 10 7-O-tosyl-( 2'-ethoxyethyl) taxol
11 7-O-tosyl taxol
12 10-deacetyl-7-O-p-methoxybenzylbaccatin III or 7-O-p-methoxybenryl-10-
deacetylbaccatin III
13 10-deacetylbaccatin III
is I 5 Baccatin III
16 7-O-benzyl-9-dihydro-13-aceaylbaccatin III
17 7-O-benzyl-13-acetylbaccatin III
18 taxol or paclitaxel
13
SUBSTITUTE SHEET (RULE 26)
CA 02307548 2001-O1-25
MATERIAL
All the reagents were obtained from Aldrich Chemicals, USA.
EXAMPLE 1
s Preparation of taaol
Step 1(a): 7-O-p-methoxybenzyl-9-dihydro-13-acetylbaccatin III ( 2 )
20 mg 9-dihydro-13-acetylbaccatin III ( 1 ) and 100 mg n-tetrabutylammonium
iodide
were dissolved in 3 mL of dichloromethane (CH2C12) in a 25 mL round bottom
flask. 23 mg
to of sodium hydride (NaH) was added and the mixture was stirred at room
temperature for five
minutes. 0. I mL of p-methoxybenzyl chloride was added dropwise over 5
minutes, and the
temperature was raised to 45°C. 'The mixture was stirred for 24 hours,
following which 30
mL of distilled water was added to stop the reaction. The product was
extracted with
dichloromethane, and purified by preparative thin layer chromatography (TLC)
to yield 17 mg
15 7-O-p-methoxybenzyl-9-dihydro-I3-acetylbaccatin III, compound 2, as
identified by NMR
spectroscopy.
'H-NMR (200 MHz, CDCI3), S: 8.10 (d,Ar-H-2,H-6), 7.60 (t, Ar-H-4), 7.50 (t, Ar-
H-3, H-
5), 7.21 (d, 2Ar-H), 6.90 (d, 2Ar-:(-i), 6.30 (d, H-10), 6.17 (t, H-13), 5.80
(d,H-2), 4.92 (d,H-
2o S), 4.75 (dd, H-7), 4.50 (d, H-9), 4.31 (d,H-20a), 4.29 (d,H-20b), 4.15 (s,
-OCHZ), 3.80 (s,
OCH3), 3.01 (s, H-3), 2.59 (m,H-6), 2.26 (s, CH3C=O), 2.18 (s, CH3C=O), 2.10
(H-14a),
2.02 (s, CH3C=O), 1.97 (s, CH3), 1.85 (H-14b), i.77 (s, CH3), 1.75 (s, CH3),
1.26 (S, CH3)
ppm.
14
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Step 1(b): 7-O-methoxybenzyl-13-acetylbaccatin III ( 3 )
mg of the product of step 1 (a), compound 2, was added to 10.8 mg of 4-
methylmorpholine N-oxide (NMO) in a 25 mL round bottom flask, and the mixture
was
dissolved in 3 mL of dichloromethane. 4d molecular sieve was added to the
mixture which
s was stirred for 5 minutes. 3.5 mg o~f tetra-n-propylammonium perruthenate
(TPAP) was
added, and the mixture wa;~ stirred for about 6 hours at room temperature,
following which
the temperature was raised to 40 °C'.. The mixture was maintained at
that temperature
overnight until the reaction was completed. Once the reaction was completed,
the mixture
was poured into a short silica gel column. The column was eluted with 50 mL of
chloroform
n> (CHCi3} to give a chloroform fraction which was concentrated to dryness.
The residue was
purified by preparative TL(~ to yield 2 mg white needles which were identified
as 7-O-
methoxybenzyl-13-acetylbaccatin III, compound 3, by NMR spectroscopy.
'H-NMR (400 MHz, CDC'.13), b: 8.10 (d, Ar-H-2, H-6), 7.60 (t, Ar-H-4), 7.49
(t, Ar-H-3,
H-S), 7.22 (s, 4Ar-H), 6.2f, (s, H-10), 6.18 (t, H-13), 5.65 (d, H-2), 5.60
(dd, H-7), 5.27 (s,
OCH3), 4.95 (d, H-5), 4.33 (d, H-2l)a), 4.17 (d, H-20b), 3.95 (s, -OCHZ), 3.81
(d, H-3), 2.60
(m, H-6), 2.35 (s, CH;C=O), 2.22 (~dd, H-14a), 2.20 (s, CH3C=O), 2.15 (s,
CHI), 2.05 (s,
CH3C=O), 2.01 (H-14b), 1.96 (s, CH3), 1.24 (s, CH3), 1.18 (s, CH;) ppm.
2o Step 1(c): 7-O-p-methoxybenzylbaccatin III ( 4 )
SO mg of the produ~~t of step 1(b), compound 3, was dissolved in 5 mL of
tetrahydrofuran at -44°C, <md 5 mole equivalent of methyllithium were
added dropwise to
remove the C-13 acetyl group. Upon completion of the deacetylation reaction,
the mixture
was partitioned between 50 mL of a mixture of saturated ammonium chloride
buffer and
SUBSTITUTE SHEET (RULE 26)
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WO 98/50378 PCT/CA98/00401
dichloromethane ( 1:1 ). The organic layer was concentrated and purified by
flash column
chromatography on silica gel, eluting with a mixture of dichloromethane and
methanol (97:3)
to yield 22 mg of 7-O-p-methoxybenzylbaccatin III as white solid.
Step 1(d): 7-O-p-methoxybenzyl-(2'-ethoxyethyl) taxol ( S )
mg of the product of step 1 (c) (4) was combined with 6 mole equivalents of
(2R,
2S)-N-benzoyl-O-(1-ethoxyethyl)-3-phenylisoserine or 5 mole equivalent of(3R,
4S)-3-(1-
ethoxyethoxy)-4-(phenyl)-N-benzoyl-2-azetidinone and 3 mole equivalents of
lithium
hexamethyldisilazide (LiHNU7S) in 5 mL of tetrahydrofizran at -78 °C
for about 20 minutes in
to a 25 mL round bottom flask. The reaction was warmed to 0 °C for
about 6 hours, or until
the reaction was completed as confirmed by TLC analysis. Once the reaction was
completed,
the mixture was quenched with 30 mL of a pH 7 buffer and the product was
extracted with
dichloromethane, dried and purified by flash column chromatography on silica
gel, eluting
with dichloromethane and methanol (97:3 ) to yield 16 mg of 7-O-p-
methoxybenzyl-(2'-
15 ethoxyethyl) taxol.
Step 1(e): 7-O-p-methoxybenzyl taxol ( 6 )
10 mg of the product of step 1 (d) was dissolved in ethanol at room
temperature in a ~
mL round bottom flask. An excess of 1% hydrochloric acid was added, and the
reaction
2o continued for about 4 hours. The reaction product was poured into a 25 mL
separation
funnel and 50 mL of a mixture of pH 7 buffer and dichloromethane ( 1:1 ) was
added. The
organic layer was evaporated to dryness to obtain a residue.
16
SUBSTITUTE SHEET (RULE 26)
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WO 98/50378 PCTlCA98/00401
Step 1(f): taxol or paciitax:el ( I 8)
mL of dichloromethane was added to the residue obtained in step 1(e), and 0.25
mL
of water was mixed thereto. In this case, the ratio of water to
dichloromethane was 1:20.
1.0-1.5 mole equivalent of :?,3-dichl~oro-5,6-dicyanobenzoquinone (DDQ) was
added to the
s mixture and the temperature was maintained at room temperature or
5°C. Upon
completion, 10 mL of saturated aqueous sodium hydrogen carbonate (NaHC03) was
added
to the reaction, and the mixture was extracted with 10 mL of dichloromethane.
The extract
was washed with S mL of saturated aqueous sodium hydrogen carbonate and dried
over
sodium sulfate (Na2S04). The solvent was evaporated and the residue was
purified on a silica
to gel column to give 3.5 mg of taxol.
EXAMPLE 2
Preparation of taxol
Step 2(a): 7-O-tosyl-9-dihvydro-13-acetylbaccatin III ( 7 )
1.0 g of 9-dihydro-13-acetylbaccatin III (1) was placed in a 50 mL round
bottom flask
with 454 mg of p-toluenesulfonyl chloride and 589 mg of tetrabutylammonium
iodide. The
mixture was dissolved in 1 S mL of dichloromethane and stirred at room
temperature for S
2o minutes. 57 mg of sodium hydride was added slowly to the mixture, and the
mixture was
stirred at room temperature for 2 hours, following which 100 mL of water was
added. The
mixture was extracted with 70 mL of dichloromethane. The organic layer was
concentrated
to dryness, and the residue ,vas purified by flash column chromatography on
silica gel, eluting
17
SUBSTITUTE SHEET (RULE 26)
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WO 98/50378 PCT/CA98/00401
with a mixture of dichloromethane and methanol (97:3) to yield 1.1 g of 7-O-
tosyl-9-dihydro-
13-acetylbaccatin III.
Step 2(b): 7-O-tosyl-13-acetylbaccatin III ( 8 )
400 mg of the product of step 2(a), compound 7, was placed in a 50 mL round
s bottom flask and 126 mg of 4-methylmorpholine N-oxide was added to thereto.
The mixture
was dissolved in 5 mL of acetonitrile (CH3CN) and 126 mg of 4 6 molecular
sieve was added
The mixture was stirred at room temperature for about 10 minutes, following
which 18 mg of
TPAP was added. The mixture was stirred for 5 hours at room temperature and
poured
through a short silica gel column, eluting with dichloromethane. The fraction
was further
1o purif ed by flash column chromatography on silica gel, eluting with a
mixture of ethyl
acetoacetate (EtOAc) and hexane (7:3) to yield about 350 mg of white crystals
which were
identified as 7-O-tosyl-13-acetylbaccatin III, purity 98%.
'H-NMR (400 MHz, CDCI3), 8: 8.05 (dd, 2H, Ar-H), 7.74 (d, 2H, tosyl), 7.60
(ddd, Ar-
15 Hd), 7.47 (dd, 2H, Ar-H), 7.30 (d, 2H, tosyl), 6.69 (s, H,o), 6.16 (t,
H,3), 5.66 (dd, H2), 5.30
(dd, H~), 4.85 (dd, H-5), 4.30 (d, H-20a), 4.10 (d, H-20b), 3.00 (s, 3H,
tosyl), 3.92 (d, H-3),
2.60 (m, H-6a), 2.42 (s, -OCH3), 2.33 (s, -OCH3), 2.30 (dd, H-14a), 2.21 (s, -
OCH;), 2.13
(s, CH3), 2.10 (dd, H-14b), 1.77 (s, CH3), 1.20 (s, CH3), 1.17 (s, CH3) ppm.
2o Step 2(c): 7-O-tosylbaccatin III ( 9 )
100 mg of the product of step 2(b) (compound 8) was dissolved in 10 mL of
tetrahydrofuran in a 25 mL round bottom flask. The flask was then placed into
a container
which was maintained at -43 °C. The solution was stirred and 0.4 mL of
n-butyllithium in
hexane was added dropwise for about 3 minutes. The mixture was stirred for
about 30
18
SUBSTITUTE SHEET (RULE 26)
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WO 98/50378 PCT/CA98/00401
minutes with the temperature raised to about 0 °C, and was further
transferred into a 500 mL
separation funnel with 100 mL of water and extracted with 150 mL of
dichloromethane. The
product was purified by preparative TLC to yield about 62 mg of white crystals
which were
identified as 7-O-tosylbaccatin III.
Step 2(d): 7-O-tosyl-(2'-e;thoxyethyl) taxol ( 10 )
100 mg of compound 8 was placed in a 25 mL round bottom flask , and 6 mole
equivalents of (2R, 2S)-N-benzoyl-O-(1-ethoxyethyl)-3-phenylisoserine were
dissolved in 5
mL of tetrahydrofuran at -'78 °C. 3 mole equivalents of LiHNIDS was
added slowly. The
to mixture was stirred at -78 "C for about 20 minutes, following which it was
warmed to about
0 to 20 °C. The reaction progress was followed by TLC until completion.
Once completed,
the reaction was quenched with pH 7 buffer and extracted with 40 mL of
dichloromethane.
The organic layer was concentrated to dryness under vacuum, and the residue
was purified by
preparative TLC to yield about 85 rng of white crystals which were identified
as 7-O-tosyl-
(2'-ethaxyethyl) taxol.
Step 2(e): 7-O-tosyl taxol ( 11 )
50 mg of the product of step 2(d), compound 10, was placed in a 25 mL round
bottom flask and dissolved with 15 mL of ethanol at room temperature. An
excess of 1
2o hydrochloric acid was added and the mixture was maintained at room
temperature for about 5
hours. The reaction was then quenc;hed with 20 mL of water and 40 mL of
dichloromethane.
The organic layer was evaporated to dryness under vacuum to yield 7-O-tosyl
taxol.
Step 2(~: taxol or paclita~:el ( 18 )
19
SUBSTITUTE SHEET (RULE 26)
CA 02307548 2000-05-11
WO 98/50378 PCT/CA98/00401
The residue obtained in step 2(e) was dissolved in 10 mL of a 0.02 M solution
of
triethylamine in methanol and irradiated by UV light for about 6 hours or
until the reaction is
completed as shown by TLC. After irradiation, the triethylamine solvent was
evaporated
under vacuum. The residue was purified by flash column chromatography on
silica gel,
eluting with a mixture of clichloromethane and methanol (97:3) to yield about
25 mg of taxol
which was characterized as natural taxol.
'H-NMR (400 MHz, CDI~l3), b: 8.12, 7.73, 7.6I, 7.51, 7.41, 7.38, 6.97, 6.27,
6.23, 5.78,
5.67, 4.94, 4.79, 4.40, 4.3 3, 4.19, 3.79, 3.54, 2.51, 2.46, 2.38, 2.36, 2.28,
2.24, 1.89, 1.89,
l0 1.79, 1.68, 1.24, 1.14 pprr~.
EXAMPLE 3
Preparation of 10-deacetylbaccatin III
IS
Step 3(a): 7-O-p-methox~benzyl-10-deacetylbaccatin III ( 12 )
70 mg of the prodG.ct of step 1 (c), compound 4, was placed in a 25 mL round
bottom
flask with 5 mL of dichloromethane. 1.5 mole equivalent of sodium hydride was
added
followed by 3 mL of trieth;ylamine, and the mixture was magnetically stirred
for 2 to 3 hours.
2o The reaction progress was monitored by TLC. When the deacetylation was
completed, the
solution was poured into 25 mL of water and extracted with 30 mL
dichloromethane. The
organic layer was concentrated to dryness under vacuum. The residue was
purified by flash
column chromatography to yield 42 mg of 7-O-p-methoxybenzyl-10-
deacetylbaccatin III as
white solid.
SUBSTITUTE SHEET (RULE 26)
CA 02307548 2000-05-11
WO 98/50378 PCT/CA98/00401
Step 3(b): 10-deacetylbaccatin IIIf ( 13 )
30 mg of compound 12, i.e. 7-O-p-methoxybenzyl-10-deacetylbaccatin III, was
placed into a 25 mL round bottom flask with 5 mL of dichloromethane which
contained a
small amount of water, thc: ratio of water to dichloromethane being 1:20. 1.0
mole equivalent
of 2,3- dichloro-5,6-dicyanobenzoquinone (DDQ) was added and the mixture was
stirred at
room temperature for about 3 hours. Once the reaction was completed, 15 mL of
staturated
sodium hydrogen carbonate was added and the mixture was extracted with 20 mL
of
dichloromethane. The extract was dried over sodium sulfate. The
dichloromethane solution
1o was evaporated and the residue was purified with a silica gel column to
yield 16 mg of 10-
deacetylbaccatin III.
EXAMPLE 4
Preparation of baccatin III
25 mg of compound 4 (7-O-p-methoxybenzylbaccatin III) or 9 (7-O-tosylbaccatin
III)
containing a small amount of water was placed in a 25 mL round bottom flask
and 5 mL of
dichloromethane was added. In this case, the ratio of water to dichloromethane
was 1:18. 1.0
to 1.5 mole equivalent of I)DQ was added to the mixture which was being
stirred at about
room temperature or S °C. Upon completion of the reaction, 20 mL of
saturated aqueous
2o sodium hydrogen carbonate was added, and the mixture was extracted with 30
mL
dichloromethane, and dried over sodium sulfate. The solvent was evaporated and
the residue
was purified by flash column chromatography, eluting with a muxture of
dichloromethane and
methanol (96:4) to yield 1',~ mg of baccatin III, compound 15., as white
solid.
21
SUBSTITUTE SHEET (RULE 26)
CA 02307548 2001-O1-25
EXAMPLE S
Preparation of 7-O-benzyl-13-acetylbaccatin IEI intermediate
Step 4(a): 7-O-benzyl-9-dihydro-13-acetylbaccatin III ( 16 )
mg of 9-dihydro-13-acetylbaccatin III, compound 1, was dissolved in 3 mL of
tetrahydrofi,rran (THF), and 20 mg of n-tetrabutylammonium iodide was added in
a 5 mL
round bottom flask. The mixture was stirred for 5 minutes and 12 mg of sodium
hydride was
added. I 70 mg of ben2yl bromide was added dropwise and the mixture was
stirred at 40°C.
The stirring continued overnight. 40 mL of distilled water was added to stop
the reaction.
The product was extracted with chloroform. The chloroform solution was
evaporated and
the residue was purified by preparative TLC to yield 8 mg of 7-O-benzyl-9-
dihydro-13-
acetylbaccatin III.
Step 4(b): 7-O-benzyl-13-acetylbaccatin III ( 17 )
t.s In a similar manner as in step I(b), compound 17 can be made. In this
case, the
reagent methylmorphine N-oxide (NMO) is replaced by sodium chloroxide (NaOCI).
Taxol, 10-deacetylbaccatin III and baccatin III can be obtained from 7-O-
benryl-13-
acetylbaccatin III in similar manners as shown in the previous examples.
Although embodiments of the invention have been described above, it is not
limited thereto and it will be apparent to those skilled in the art that
numerous
modifications form part of the present invention insofar as they do not depart
from the
spirit, nature and scope of the claimed and described invention.
22
