Note: Descriptions are shown in the official language in which they were submitted.
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C-2 HYDROXYL PROTECTED-N-ACYL(2R,3S)-3-PHENYLISOSERINE
ACTIVATED ESTERS AND METHODS FOR PRODUCTION THEREOF
FIELD OF THE INVENTION
This invention generally relates to the synthesis of paclitaxel from
precursor compounds. More particularly, though, this invention concerns the
semi-synthesis of paclitaxel using a protected baccatin III backbone which is
esterified with suitably protected side chain activated esters to produce an
intermediate that may be converted to paclitaxel.
BACKGROUND OF THE INVENTION
The chemical compound referred to in the literature as taxol, and
more recently "paclitaxel", has received increasing attention in the
scientific
and medical community due to its demonstration of anti-tumor activity.
Paclitaxel has been approved for the chemotherapeutic treatment of several
different varieties of tumors, and the clinical trials indicate that
paclitaxel
promises a broad range of potent anti-leukemic and tumor-inhibiting activity.
As is known, paclitaxel is a naturally occurring taxane diterpenoid having
the formula and numbering system as follows:
0
Ph NH O H
O
Ph O
OH OH OCOPh OAc
(Formula 1 )
9 19
(Numbering System)
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While the paclitaxel molecule is found in several species of yew
(genus Taxus, family Taxaceae), the concentration of this compound is very
low. Moreover, these evergreens are slow-growing. Thus, a danger exists
that the increasing use of paclitaxel as an effective anti-cancer agent will
deplete natural resources in the form of the yew trees. Indeed, while the
bark of the yew trees typically exhibit the highest concentration of
paclitaxel,
the production of 1 kilogram of paclitaxel requires approximately 16,000
pounds of bark. Thus, the long term prospects for the availability of
paclitaxel through isolation is discouraging.
The paclitaxel compound, of course, is built upon the baccatin III
backbone, and there are a variety of other taxane compounds, such as
baccatin III, cephalommanine, 10-deacetylbaccatin III, etc., some of which
are more readily extracted in higher yields from the yew tree. Indeed, a
relatively high concentration of 10-deacetylbaccatin III can be extracted from
the leaves of the yew as a renewable resource. Typically, however, these
other taxane compounds present in the yew tree do not exhibit the degree of
anti-tumor activity shown by the paclitaxel compound.
Since the paclitaxel compound appears so promising as a
chemotherapeutic agent, organic chemists have spent substantial time and
resources in attempting to synthesize the paclitaxel molecule. A more
promising route to the creation of significant quantities of the paclitaxel
compound has been proposed by the semi-synthesis of paclitaxel by the
attachment of the A-ring side chain to the C-13 position of the naturally
occurring baccatin III backbone derived from the various taxanes present in
the yew. See, Denis et al, "Highly Efficient, Practical Approach to Natural
Taxol", Journal of the American Chemical Society, page 5917 (1988). In that
article, the partial synthesis of paclitaxel from 10-deacetylbaccatin III is
descri bed.
The most straightforward implementation of partial synthesis of
paclitaxel requires convenient access to chiral, non-racemic side chains and
derivatives, an abundant natural source of baccatin III or closely related
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diterpenoid substances, and an effective means of joining the two. Of
particular interest then is the condensation of baccatin III or 10-
deacetylbaccatin III with the paclitaxel A-ring side chain. However, the
esterification of these two units is difficult because of the hindered C-13
hydroxyl of baccatin III located within the concave region of the
hemispherical taxane skeleton. For example, Greene and Gueritte-
Voegelein reported only a 50% conversion after 100 hours in one partial
synthesis of paclitaxel. J. Am. Chem. Soc., 1988, 110, 5917.
In U.S. Patent No. 4,929,011 issued May 8, 1990 to Denis et al
entitled "Process for Preparing Taxol", the semi-synthesis of paclitaxel from
the condensation of a (2R,3S) side chain acid of the general formula:
0
Ph NH
~ /C02H
Ph
oP, (Formula 2)
wherein P, is a hydroxyl protecting group with a taxane derivative of the
general formula of:
'''' H
H O ''''' ~ _ H
OH pCOPh oAc (Formula 3)
wherein P2 is a hydroxyl protecting group is described wherein the
condensation product is subsequently processed to remove the P, and P2
protecting groups. In Denis et al, the (2R, 3S) 3-phenylisoserine derivative,
with the exception of the P, protecting group, is the A-ring side chain for
the
paclitaxel molecule. The P2 protecting group on the baccatin III backbone is,
for example, a trimethylsilyl or a trialkylsilyl radical.
Ac ; ~~ OP2
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An alternative semi-synthesis of paclitaxel is described in U.S. Patent
No. 5,770,745 to Swindell et al. Swindell et al. discloses semi-synthesis of
paclitaxel from a baccatin III backbone by the condensation with a side chain
having the general formula:
0
R10 'NH
~ /C02H
P h~
OP1
(Formula 4)
wherein R, is alkyl, olefinic or aromatic or PhCH2 and P, is a hydroxyl
protecting group.
The side chain in Swindell et al is distinct from the side chain
attachment used in Denis et al, above, in that the nitrogen is protected as a
carbamate. Preferably, the A-ring side chain is benzyloxycarbonyl (CBZ)
protected. After esterification, the CBZ protecting group is removed and
replaced by PhCO to lead to paclitaxel. This process generated higher
yields than that described in Denis et al. In Swindell et al., the preferred
masking groups were selected to be trichloroethoxymethyl or
trichloroethoxycarbonyl. Benzyloxymethyl (BOM) was, however, disclosed
as a possible side chain hydroxyl protecting group for the 3-phenylisoserine
side chain, but, according to the processes described therein, the BOM
protecting group could not be removed from the more encumbered C-2
hydroxyl in the attached 3-phenylisoserine side chain. The use of the BOM
protected side chain was not extensively investigated, for that reason.
Subsequently, it has been shown in U.S. Patent No. 5,675,025 to Sisti
et al., issued October 7, 1997, that the BOM group could be removed from
the more encumbered C-2 hydroxyl in the attached 3-phenylisoserine side
chain.
U.S. Patent No. 4,924,012, issued May 8, 1990 to Colin et al
discloses a process for preparing derivatives of baccatin III and of 10-
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deacetylbaccatin III, by condensation of an acid with a derivative of a
baccatin III or of 10-deacetylbaccatin III, with the subsequent removal of
protecting groups by acid hydrolysis. Several syntheses of TAXOTERE~
(Registered to Rhone-Poulenc Sante) and related compounds have been
reported in the Journal of Organic Chemistry: 1986, 51, 46; 1990, 55, 1957;
1991, 56, 1681; 1991, 56, 6939; 1992, 57, 4320; 1992, 57, 6387; and 993,
58, 255; also, U.S. Patent No. 5,015,744 issued May 14, 1991 to Holton
describes such a synthesis. Additional techniques for the synthesis of
paclitaxel and paclitaxel analogues are discussed in U.S. Patent No.
5,688,977 to Sisti et al., U.S. Patent No. 5,750,737 to Sisti et al., U.S.
Patent
No. 5,684,175 to Sisti et al. and U.S. Patent No. 5,750,736 to Sisti.
Despite the advances made in the semi-synthesis of the paclitaxel
molecule in the above-described processes, there remains a need for more
efficient protocols for the synthesis of paclitaxel in order to increase
efficiencies in yields and production rates. There remains such a need for
semi-synthesis that may be implemented into commercial processes. There
is a further need for efficient protocols for the synthesis of paclitaxel
analogs,
intermediates and various A-ring side chain structures.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide new C-2 hydroxyl
protected-N-Acyl (2R,3S)-3-phenylisoserine activated esters and production
processes therefore, that are useful in the semi-synthesis of paclitaxel.
Another object of the present invention is to provide a new and useful
process for the production of an hydrogenatable benzyl-type protected side
chain which may be readily attached to a protected baccatin III backbone
during the semi-synthesis of paclitaxel.
Still a further object of the present invention is to provide N-CBZ
protected C-2 hydroxyl-benzyl protected (2R,3S)-3-phenylisoserine activated
esters and production methods therefor.
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Still a further object of the present invention is to develop efficient
cost effective processes for the production of N-CBZ protected C-2 hydroxyl-
benzyl protected (2R,3S)-3-phenylisoserine activated esters.
According to the present invention, then, a chemical compound is
provided having the formula:
0
R1 'NH O
Ph O Z
OP1
wherein P, is a hydroxyl protecting group, R, may be an alkyl group, an
olefinic group, an aromatic group, an O-alkyl group, an O-olefinic group, or
an O-aromatic group, and Z may be a substituted phenyl moiety or an N-
imido moiety. R, is preferably Ph, PhCH2, O-Ph or O-CH2Ph, and P, is
preferably benzyl, benzyloxymethyl or benzoyl.
Z may be a phenyl moiety having the formula:
R~ Rz
Ra
such that the chemical compound has the formula:
0
R1 'NH O
Ph R4
OP1
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where each of R2 to Rs is H or an electron withdrawing group. Exemplary
electron withdrawing groups include N02 or halogens, such as F. It is
preferred that at least one of R2 to Rs is a halogen or N02.
A preferred embodiment is where P, is benzyloxymethyl; R2, Rs, Rs,
and Rs are H; R4 is N02; and R, is OCH2Ph. Also preferred is where P, is
benzyloxymethyl; R2, Rs, Ra, Rs and Rs are F; and R, is OCH2Ph. It is further
preferred where P, is benzyloxymethyl; R2 and Ra are N02; Rs, Rs, and Rs
are H; and R, is OCH2Ph.
Z may alternatively be a heterocyclic N-imido moiety, preferably
having 5 to 7 atoms in the ring, and alternatively substituted with at least
one
electron withdrawing group, such as a nitro or a halogen group. The N-imido
moiety may be substituted with a plurality of the same or different electron
withdrawing groups. In particular, Z may be succinimido, phthalimido, 5-
norbornene-2,3-dicarboxyimido, and maleimido moieties and substituted
derivatives thereof.
The present invention is also directed to a process for producing an
ester derivative useful in the production of paclitaxel, paclitaxel analogues
and their intermediates. The process comprises the step of reacting a first
compound of the general formula:
0
R1 'NH
~ /C02Rio
Ph
OP1
wherein R, is an alkyl group, an olefinic group, an aromatic group, an O-alkyl
group, an O-olefinic group, or an O-aromatic group; P, is a hydroxyl
protecting group; and R,o is H or C02X, where X is an alkyl group, an olefinic
group or an aromatic group, with a second compound of the general formula:
H O-Z
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wherein Z is selected from the group consisting of a substituted phenyl
moiety and an N-imido moiety, to give a third compound of the general
formula:
0
R~ ~NH O
Ph O Z
OP1
wherein R, is an alkyl group, an olefinic group, an aromatic group, an O-alkyl
group, an O-olefinic group, or an O-aromatic group; P, is a hydroxyl
protecting group; and Z is selected from the group consisting of a substituted
phenyl moiety and an N-imido moiety. When R,o is H, the step of reacting
may be conducted in the presence of THF and a carbodiimide, such as
dicyclohexylcarbodiimide. When R,o is COzX, X may particularly be
-CH2CH(CH3)2, and the step of reacting may be conducted in the presence
of N-methyl morpholine and THF.
The first compound may be formed by reacting a compound having
the formula:
o
R1 ~NH
~ /C02H
Ph
OPi
with a compound having the formula CI-C02X to give the first compound.
The second compound may be one having the formula:
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H R4
wherein each of R2 to Rs is selected from the group consisting of H and an
electron withdrawing group. In particular, each of R2 to Rs may be H,
halogen or N02 with at least one of R2 to Rs being halogen or N02. R, may
particularly be Ph, PhCH2, O-Ph or O-CH2Ph, and P, may be benzyl,
benzyloxymethyl or benzoyl. Alternatively, the second compound may be
one where Z may be a heterocyclic N-imido moiety, such as one having 5 to
7 atoms in the ring. In particular, Z may be succinimido, phthalimido, 5-
norbornene-2,3-dicarboxyimido, or maleimido moieties and substituted
derivatives thereof.
These and other objects of the present invention will become more
readily appreciated and understood from a consideration of the following
detailed description of the exemplary embodiments.
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
The present disclosure is broadly directed to a chemical process for
the efficient production of paclitaxel, intermediates and precursors therefor.
More specifically, the present invention concerns the semi-synthesis of
paclitaxel by esterifying suitably protected 3-phenylisoserine activated
esters
having protecting groups at C-2 to the C-13 hydroxyl of 7-O-protected
baccatin III. More particularly, the present invention preferably utilizes CBZ
protection at the C-7 site of the baccatin III. The general process described
herein involves the production of C-7 CBZ baccatin III, the production of a
suitably protected 3-phenylisoserine activated ester having a suitable
protecting group at C-2, the condensation of the two compounds, and the
subsequent deprotection, acylation, deprotection of the condensation
product to form paclitaxel.
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A. Production of C7-CBZ Protected Baccatin III
According to the present invention, two alternative routes are
described which appear in copending Application Serial No. 08/922,684,
now U.S. Patent No. X,XXX,XXX to Sisti et al., for producing C7-CBZ
protected baccatin III. On one hand, baccatin III can be protected at the C-7
site to yield C-7 CBZ baccatin III. On the other hand, 10-deacetylbaccatin III
(10-DAB) can be directly converted to C-7 CBZ baccatin III without going
through a baccatin III intermediate. Production from baccatin III is
advantageous for its yield and simplicity. The method using 10-
deacetylbaccatin III has an advantage since 10-deacetylbaccatin III is much
more naturally abundant, and thus less expensive, than baccatin III;
however, this alternative method has a reduced yield.
Route 1 Usinct baccatin III)_
C-7 CBZ baccatin III has the formula:
Ac ; ~0 OC02CH2Ph
''''' H
''' _
'''' H ~~0
H O ='~~i'
OH
- OAc
OCOPh
and can be synthesized from baccatin III according to the following reaction:
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H
HO
OAc
OCOPh Ac0 0
OC02CH2Ph
\'~~~ H
H 0 ~~~\' H '~/0
O vH
OAc
OCOPh
Reaction I
Baccatin III is dissolved in THF (tetrahydrofuran) to form a first solution,
which is cooled under a nitrogen atmosphere to a reduced temperature of
less than -20°C. n-Butyl lithium (1.6 M in hexane) is then added
dropwise to
the first solution to form a second solution, which is stirred for
approximately
five minutes at the reduced temperature. This creates the C-7 lithium
alkoxide of baccatin III. Benzyl chloroformate (CBZ-CI) is added dropwise to
the second solution to form a third solution which is then stirred and allowed
to warm to 0°C over approximately one (1) hour. The third solution is
quenched with cold saturated ammonium chloride to eliminate any excess n-
butyl lithium and CBZ-CI, and the mixture is concentrated under vacuum to
yield a first residue. This first residue is next taken up in ethyl acetate
and
washed once with water to remove unwanted salts. Next, the organic layer
is washed with brine. The organic layer is then dried and concentrated
under vacuum to yield a second residue. The second residue is
recrystallized or column chromatographed with ethyl acetate: hexane to give
C-7 CBZ baccatin III as a white solid.
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Instead of using n-butyl lithium, it should be appreciated that other
alkali bases may be used, especially potassium hydride and sodium hydride,
to form the C-7 metal alkoxide of baccatin III, to the extent understood by
the
ordinarily skilled artisan.
Route 2 (Using 10-deacet~rlbaccatin IIII
Alternatively, C-7 CBZ baccatin III can be synthesized directly from
10-deacetylbaccatin III as follows:
H
HO
OH
OAc
OCOPh Ac0 0
OC02CH2Ph
''''' H
'' _
'''' H ,~0
H O ='~vi'
OH
OAc
OCOPh
Reaction II
Here, 10-DAB III is dissolved in THF to form a first solution which is cooled
to a reduced temperature of less than -20°C, and preferably to -
40°C, under
a nitrogen atmosphere. At least two equivalents of n-butyl lithium (1.6 M in
hexane) are then added dropwise to the first solution to form a second
solution which is then stirred for approximately five minutes at the reduced
temperature. Preferably, acetyl chloride (one equivalent) is added to the
second solution to form a third solution which is stirred at the reduced
temperature for approximately thirty minutes. Alternatively, acetic anhydride
(one equivalent) may possibly be used in place of the acetyl chloride to
acylate the 10-DAB III. In either case, benzyl chloroformate (one equivalent)
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is next added, and this fourth solution is stirred for an additional thirty
minutes at the reduced temperature and then warmed to 0°C over thirty
minutes. The fourth solution is then quenched with cold saturated
ammonium chloride at the reduced temperature to remove any excess n-
butyl lithium, acetyl chloride and CBZ-CI; this mixture is then warmed to
room temperature. The solvent is removed under vacuum to yield an initial
residue, which is taken up in ethyl acetate and washed with water to remove
unwanted salts. The organic layer is then washed with brine, dried and
concentrated under vacuum to yield a final residue. The final residue is
chromatographed (silica gel hexanes:ethyl acetate) to yield C-7 CBZ
baccatin III. It is important to note that this method represents a direct
synthesis of C-7 CBZ baccatin III from 10-DAB III, as the intermediate
formed in this reaction is a C-7 lithium alkoxide of baccatin III, that is,
the
intermediate is not baccatin III itself.
While both Routes 1 and 2 specifically are directed to the production
of derivatives of baccatin III, it should be apparent to the ordinarily
skilled
person that baccatin III analogs can be produced from the Route 2 process
simply by substituting the appropriate acid chloride to the second solution in
Route 2. This would result in the formation of analogues with different alkyl
groups, for example, at C-10.
It should now be appreciated that both Route 1 and Route 2 to the
production of C-7 CBZ baccatin III can be expressed as a generalized
method. This method starts with a step of dissolving a starting compound
selected from the group consisting of baccatin III and 10-deacetylbaccatin III
in a first solvent to form a first solution. The first solution is then cooled
to a
temperature of -20°C or less. Thereafter, an alkyl lithium base is
added to
the first solution thereby to form an intermediate compound having a lithium
alkoxide at the C-7 position thereof. Next, as would be required for the 10-
DAB III starting compound,~the method includes selectively acylating, at the
C-10 position; any of the first intermediate compound present in the first
solution where the intermediate compound does not already have an acetyl
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group at the C-10 position thereby to produce a second solution of C-7
lithium alkoxide of baccatin III. Of course, where the starting compound is
baccatin III, the C-10 position already has an acetyl group. In any event, the
method includes a step of thereafter adding CBZ-CI to the second solution to
form a third solution of C-7 CBZ baccatin III.
B. Production of N-Acvl C-2 Hydroxyl Protected (2R.3S)-3-
Phen~rlisoserine A-Rina Side Chain Activated Esters
The second precursor necessary for the semi-synthesis of paclitaxel
according to the present invention is the N-acyl C-2 hydroxyl protected
(2R,3S) phenylisoserine side chain activated ester having the general
formula:
o
R1 NH
~ /C02R2
P h~
OP1
wherein R, is an alkyl group, an olefinic group, an aromatic group, Ph,
PhCH2, an O-alkyl group, an O-olefinic group, an O-aromatic group, O-Ph, or
O-CH2Ph. P, is a hydroxyl protecting group and R2 can be an N-imido group
or a phenyl ring substituted with one or more electron withdrawing groups.
Imido groups contemplated by the present invention include such groups as
succinimido, phthalimido, 5-norbornene-2,3-dicarboxyimido, or derivatives
thereof such as a maleimido group or succinimido group substituted at the 3
and/or 4 positions, or other heterocyclic imido groups, preferably having 5 to
7 atoms in the ring, alternatively substituted with chloro, fluoro, nitro or
other
groups.
The preferred hydroxyl protecting group is a benzyloxymethyl (BOM)
protecting group. Benzyl has also been demonstrated to be suitable as has
benzoyl, and other protecting groups are believed suitable as well. The
preferred N-Acyl group is benzyloxycarbonyl (CBZ). Other protecting groups
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and acyl groups (having alkyl, olefinic, and aromatic substituents and
variations thereon) may be substituted, to the extent understood by the
ordinarily skilled artisan.
The starting compound to produce the desired side chain is (2R,3S)-
3-phenylisoserine ethyl ester to produce the preferred N-CBZ protected
(2R,3S)-3-phenylisoserine ethyl ester according to the reaction:
0
NH2 Ph~O NH
CBZ-CI -
/C02Et ~ ~C02Et
Ph' ~_ Na2C03 PhI
OH Et20:H20 OH
Reaction III
Here, (2R,3S)-3-phenylisoserine ethyl ester was alternatively dissolved in
either equal parts diethyl ether:water or equal parts methyl t-butyl
ether:water and the solution was cooled to 0°C. The sodium carbonate
was
then added to the solution and benzylchloroformate was added dropwise
over an interval of about five minutes and the resulting mixture stirred at
0°C
for approximately one hour. After the one hour stirring, the solution was then
poured into water and extracted with methylene chloride or ethyl acetate, as
desired. The organic layer is separated, dried and reduced under vacuum to
residue. The residue was then recrystallized from ethyl acetate:hexane to
result in N-CBZ (2R,3S)-3-phenylisoserine ethyl ester having the formula:
0
Ph~O NH
~ /C02Et
P h~
o H (Formula 5)
The N-CBZ (2R,3S)-3-phenylisoserine ethyl ester was next protected
by the hydrogenatable benzyl-type protecting group, in several ways. For
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example, one route to the desired hydrogenatable benzyl protected side
chain is as follows:
0 0
P h~0 N H P h~0 N H
- BOM-CI Co Et
/~ /C02Et ~ ~ z
Ph~ n-BuLi Ph
OH THF, -78°C OBOM
Reaction IV
Here, the CBZ (2R,3S)-3-phenylisoserine ethyl ester is dissolved in
anhydrous THF under a nitrogen atmosphere and cooled to a reduced
temperature such as -40°C or -78°C, for example, in a dry
ice/acetone bath
followed by the dropwise addition of an alkylithium agent, such as n-butyl
lithium, although it is desirable that the alkylithium agent be a straight
chain
alkyl. In any event, the reaction is best done at a temperature no greater
than 0°C. The resulting mixture was stirred for about ten minutes.
Benzyloxymethyl chloride (BOM-CI) was then added dropwise over an
interval of about five minutes and the mixture stirred for approximately two
to
five hours at the reduced temperature. Thereafter, the solution was warmed
to 0°C and quenched with water. The resulting mixture is reduced under
vacuum to residue, and this residue is thereafter taken up in ethyl acetate
and washed with water and brine. The organic layer may then be dried and
reduced under vacuum and the residue recrystallized from ethyl
acetate:hexane or chromatographed with ethyl acetate:hexane to give the
compound:
0
P h~0 N H
~ /C02Et
P h~
0 BO M Formula 6)
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Another route in the production of the compound according to formula
6 is accomplished by dissolving the compound N-CBZ (2R,3S)-3-
phenylisoserine ethyl ester in anhydrous methylene chloride. Thereafter, a
tertiary amine base, such as diisopropylethylamine, is added along with
BOM-CI and the mix is refluxed for twenty-four hours. While this reaction
route will produce N-CBZ, C-2 (hydroxyl] protected (2R,3S)-3-
phenylisoserine ethyl ester, the reaction proceeds much slower than the
preferred route, discussed above.
In either instance, the resulting protected (2R,3S)-3-phenylisoserine
ethyl ester compound of formula 6 may simply be converted to the N-CBZ C-
2 O-BOM-protected (2R,3S) phenylisoserine intermediate by the reaction:
0 0
P h~0 N H P h~0 N H
- LiOH
~ /C02Et -~ ~ /C02H
Ph~ Ph
EtOH:H20 -
OBOM OBOM
Reaction V
Here, the protected (2R,3S)-3-phenylisoserine ethyl ester is dissolved
in ethanol/water (ratio 8:1 ). Lithium hydroxide (or other suitable alkali
hydroxide) is added to the solution and the resulting mixture stirred for
approximately three hours in order to saponify the compound. The mixture is
then acidified (1 N HCI) and extracted with ethyl acetate. The resulting
organic layer is separated, dried and reduced under vacuum. The residue
acid is then isolated for use without further purification. This produces the
desired side chain having the general formula:
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0
Ph~O NH
~ /C02H
P h~
0 BO M Formula 7)
Benzyl itself is another example of a hydrogenatable benzyl
protecting group that may be used instead of BOM. The compound of the
formula:
0
Ph~O NH
~ /C02Et
Ph
oan Formula 8)
was therefore produced as above with the substitution of benzyl bromide for
BOM-CI in Reaction IV according to the reaction
0 0
P h~0 N H P h~0 N H
- BnBr, THF - C02Et
/~ /C02Et
Ph~ n-BuLi Ph _
OH OBn
Reaction VI
Here, the CBZ protected (2R,3S)-3-phenylisoserine ethyl ester is dissolved
in anhydrous THF under a nitrogen atmosphere and cooled to a reduced
temperature such as -40°C or -78°C, for example, in a dry
ice/acetone bath
followed by the dropwise addition of an alkylithium agent, such as n-butyl
lithium, although it is desirable that the alkylithium agent be a straight
chain
alkyl. The resulting mixture was stirred for about ten minutes. Benzyl
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bromide (BnBr) was then added dropwise over an interval of about five
minutes and the mixture stirred for approximately two to five hours at the
reduced temperature. Thereafter, the solution was warmed to 0°C and
quenched with water. The resulting mixture is reduced under vacuum to
residue, and this residue is thereafter taken up in ethyl acetate and washed
with water and brine. The organic layer may then be dried and reduced
under vacuum and the residue recrystallized from ethyl acetate:hexane or
chromatographed with ethyl acetate:hexane to give the compound of
Formula 8.
Alternatively, the compound of Formula 8 may be obtained according
to the reaction:
o o
P h~0 N H P h~0 N H
- NaH, DMF
~C02Et -~ ~C02H
Ph - BnBr Ph -
OH OBn
Reaction VII
Here, to a stirred solution of NaH in anhydrous DMF under N2 was added the
compound of Formula 5 dissolved in DMF over five minutes. The mixture
was then stirred at 0°C for one half hour, after which time benzyl
bromide
(1.1 equivalents) was added dropwise over five minutes and the reaction
stirred for two hours. The mixture was then quenched with H20. Thereafter,
a selected one of diethyl ether and methyl t-butyl ether was added. The
organic layer was then washed with four portions of H20, brine, and then
dried and reduced under vacuum to produce the compound of Formula 8.
Formula 8 may then be readily converted into:
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0
Ph~O NH
~ /C02H
P h~
oBn (Formula 9)
by the process of Reaction V, above.
N-CBZ C-2-OBOM protected (2R,3S)-3-phenylisoserine (Formula 7)
may be converted into its corresponding activated esters by one of two
routes, although it should be appreciated that other esterification methods
known in scientific literature may be used to produce the activated ester, to
the extent understood by the ordinarily skilled artisan. Further, it should be
appreciated that the methods are applicable to C-2 and C-3N variations of
Formula 7, to the extent understood by the ordinarily skilled artisan.
In the first route N-CBZ-C-2-OBOM protected (2R,3S)-3-
phenylisoserine is mixed with 1.2 equivalents of dicyclohexylcarbodiimide or
other suitable carbodiimide and 1.2 equivalents of either p-nitrophenol,
pentaflurophenol, 2,4-dinitrophenol (or other substituted phenols, to the
extent understood by the ordinarily skilled artisan) or N-hydroxy succinimide
(or other N-hydroxy imides, to the extent understood by the ordinarily skilled
artisan) in THF and stirred for several hours at room temperature. The
preferred substituted phenol is p-nitrophenol. The preferred N-hydroxy
imide is N-hydroxy succinimide. It should be appreciated that the substituted
phenols and N-hydroxy imides contemplated by the present invention are
either readily available or may be synthesized from readily available starting
materials according to procedures known in the art.
The resulting mixture is diluted with ethyl acetate, cooled to 0°C
for
several hours, stirred for an additional several minutes and filtered. The
filtrate is then washed with 1 N HCI, water, 20% aqueous NaHCOs, water,
brine, dried over sodium sulfate and reduced in vacuo to a residue. The
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21
residue may then be column chromatographed and/or recrystallized from
ethyl acetate:heptane.
Exemplary reactions of the first route are as follows:
0
Ph~O- -NH
~ /C02H p
P h~
OBOM Ph~O NH 0
DCC - II
~0 ~ ~ N02
'
~h
P
THF
OBOM
HO ~ ~ N02
(Formula 10)
Reaction VIII
0
Ph~O- 'NH
~ /COzH p
Ph~ O2N
OBOM Ph~O NH p
DCC _ II
~0 ~ ~ N02
02N P ' ~h
THF
OBOM
HO ~ ~ NOp
(Formula 11 )
Reaction IX
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0
Ph~O ~NH
~ /COzH 0
P h~
F F
OBOM Ph~O~NH 0
DCC o ~ ~ F
F F Ph
THF
OBOM F F
HO ~ ~ F
F F
(Formula 12)
Reaction X
0
Ph~O- 'NH
~ /C02H
Ph~ 0
0
OBOM ~
Ph~O- 'NH 0
DCC o-N
0 Ph
THF
OBOM
HO-N 0
0
(Formula 13)
Reaction XI
In the second route, a mixed anhydride of N-CBZ-C-2-OBOM 3-
phenylisoserine may be reacted with either p-nitrophenol, pentaflurophenol,
2,4-dinitrophenol (or other substituted phenols, to the extent understood by
the ordinarily skilled artisan) or N-hydroxy succinimide (or other N-hydroxy
imides, to the extent understood by the ordinarily skilled artisan) to afford
the
corresponding activated esters. It is contemplated that mixed anhydrides
having alkyl, olefinic, aromatic or other appropriate radicals might be used,
to the extent understood by the ordinarily skilled artisan.
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23
To a solution of N-CBZ-C-2-OBOM-3-phenylisoserine in THF cooled
to -15° to -20°C under nitrogen was added 1.5 equivalents of i-
butyl
chloroformate followed by 1.5 equivalents of N-methyl morpholine. The
resulting mixture was stirred for several minutes followed by addition of 1.5
equivalents of either p-nitrophenol, pentaflurophenol, 2,4-dinitrophenol (or
other substituted phenols, to the extent understood by the ordinarily skilled
artisan) or N-hydroxy succinimide (or other N-hydroxy imides, to the extent
understood by the ordinarily skilled artisan). The mixture was then stirred
for
several minutes between -15° to 0°C then one hour at between
0°C to 25°C.
After which time the mixture was diluted with ethyl acetate, washed with
water and brine, dried over sodium sulfate and reduced in vacuo to a
residue. The residue was then purified by column chromatography and/or
recrystallization from heptane:ethyl acetate.
0
Ph~0 ~ H 0
CH~
'C02H '+ CIO
P - ~h
w OBOM CH3
N-Methyl Morpholine
THF
-15 to 25° C
0
Ph~0- -NH 0 0
~ CHI
Ph~~O- _0
OBOM CH~,
+ F F
02N + +
HO ~ ~ 0~ + 0
HO F
HO ~ ~ 0
_ HO-IV~
F ~ F
0
[Formula 10] [Formula 11 ] [Formula 12] [Formula 13]
Reaction XII
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Reactions VIII, IX, X, XI and XII may be generalized by the following
reaction XIII:
0 0
R1 NH R1 NH O
~ /C02F
Ph~ Ph O-Z
OP1 OP1
HO-Z
Reaction XIII
where P, is a hydroxyl protecting group such as BOM or benzyl; R, is an
alkyl group, an olefinic group, an aromatic group (such as Ph, PhCH2), an O-
alkyl group, an O-olefinic group, or an O-aromatic group (such as O-Ph or O-
CH2Ph); R,o is H or C02X, where X is an alkyl group, an olefinic group, or an
aromatic group; and Z is either a substituted phenyl moiety such as:
wherein each of R2 to Rs is selected from the group consisting of H and an
electron withdrawing group (such as a halogen or N02), or Z is an N-imido
moiety, including but not limited to succinimido, phthalimido, 5-norbornene-
2,3-dicarboxyimido, maleimido or derivatives thereof such as a maleimido
group or succinimido group substituted at the 3 and/or 4 positions, or other
heterocyclic imido groups, preferably having 5 to 7 atoms in the ring,
alternatively substituted with chloro, fluoro, nitro or other groups.
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C. Condensation of C-7 CBZ Baccatin IIL.with the Side Chain
Activated Esters
The side chain activated esters (Formulas 10, 11, 12 or 13, or other
variations to the extent understood by the ordinarily skilled artisan) as well
as the C-7 CBZ baccatin III may now be condensed. This condensation may
proceed in the presence of an appropriate lithium base (e.g., lithium
hexamethyl disalizane or n-BuLi) in THF at 0°C according to the
reaction:
0
H
Ph 0 NH
~C02R~ + HC
Ph OAc
OBOM OBz
0
Ph 0 NH 0
Ph 0
- OH
OBOM OAc
OBz
(Formula 14)
Reaction XV
wherein R, is p-nitrophenyl (Formula 10), 2,4-dinitrophenyl (Formula 11 ),
pentaflurophenyl (Formula 12), or other substituted phenyl groups, or N-
succinimido (Formula 13), or other N-imido groups.
Here, C-7 CBZ baccatin III (1.0 equivalent) and the activated ester
(Formula 10, 11, 12, 13 or others as discussed, 1.5 equivalents) are
dissolved in anhydrous THF under nitrogen and brought to 0°C. It should
be
THF
0 °C
__ LiN(SiMe3)2
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26
noted that other temperatures, including ambient temperature, have been
shown to be suitable as well. To this is then added a suitable lithium base--
in this case lithium hexamethyl disalizane, but n-butyl lithium can also be
employed. This presumably generates the C-13 lithium alkoxide of C-7 CBZ
baccatin III in analogous fashion to the C-13 lithium alkoxide of C-7 TES
baccatin III as described by Holton (U.S. Patent No. 5,229,526 and U.S.
Patent No. 5,274,124). The mixture is then stirred for a period of time,
preferably several hours, although time periods as short as thirty minutes
have been employed. The mixture is then diluted with a 1:1 mixture of ethyl
acetate and 1 N HCI, the organic phase collected and washed with water and
brine, dried over sodium sulfate and reduced in vacuo to a residue. The
residue could then be purified by column chromatography (ethyl
acetate/heptane) or recrystallization (diethyl ether or methyl t-butyl ether
or
ethyl acetate/heptane) to afford the coupled product of Formula 14.
0
P h~0 N H 0 H
Ph 0
_ OH
OBOM OAc
OBz
C-7 TES baccatin III can also be used in place of C-7-CBZ baccatin III
to yield Formula 15.
Ac0 ~ OTES
0
P h~0 N H 0 ~ '\\\' H
_ ''' _
'''' _ H '\~~~0
Ph 0'
_ OH
OBOM OAc
OBz
(Formula 15)
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27
The synthesis of C-7 TES baccatin III has been described (see Denis
et al in "A Highly Efficient, Practical Approach to Natural Taxol," J. Am.
Chem. Soc., 1988, p. 5917 and Kant et al in "A Chemoselective Approach to
Functionalize the C-10 Position of 10-deacetyl Baccatin III. Synthesis and
Biological Properties of Novel Taxol Analogs", Tetrahedron Letters, Vol. 35,
No. 31, 1994, p. 5543). Other C-7 protected baccatin III compounds may
also be used, to the extent understood by the ordinarily skilled artisan.
The conversion of Formula 14 to paclitaxel has been previously
described (Sisti et al, S.N. 08/719,488 now U.S. Patent No. 5,750,737) and
may be accomplished as follows:
D. Deprotections and Acylations to Form Paclitaxel from Formula 14
The compound according to Formula 14 may now be converted into
paclitaxel by removing the nitrogen and C-7 CBZ groups, putting the benzoyl
group onto the nitrogen, and finally removing the C-2' benzyl-type protecting
group. Removal of the CBZ groups, and subsequent addition of the benzoyl
group to the nitrogen are accomplished as follows (BOM is shown as the
protecting group at the C-2' hydroxyl site, although benzyl could also be
used):
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Ac ; ~~ OC02CH2Ph
0
P h~0 N H 0 ~ ''''' H
'''
'''' - H '\\~~0
P h 0 ''
OH
= OAc
Ph~0~0 OCOPh
Ac0 0 0 H
0
P h N H 0 ~ ''''' H
_ '''
P h _ 0 ''''' - H '\\~~/0
_ O VH
= OAc
Ph~0~0 OCOPh
(Formula 16)
Reaction XVI
Here, the coupled product of Formula 14 is dissolved in isopropanol
to which the Pearl man's catalyst is added. The resulting mixture is
hydrogenated at 40 psi for twenty-four hours, although alternatively, the
mixture can be stirred under one atmosphere of hydrogen for twenty-four
hours. Alternatively, the mixture can be hydrogenated at 1 atm of hydrogen
in the presence of at least one equivalent of tri-fluroacetic acid resulting
in
the TFA salt of the resultant amine. Thereafter, the mixture is filtered
through diatomaceous earth and reduced under vacuum to residue.
Preferably, the residue is taken up in toluene and anhydrous potassium
carbonate added. Alternatively, the residue may be taken up in ethyl acetate
or toluene and a tertiary amine base, such as triethylamine, is added. In
either case, benzoyl chloride is then added dropwise, and the mixture stirred
for two hours. The resulting mixture is then washed with water and finally
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brine. The resulting organic phase is then separated, dried, and
concentrated under vacuum to yield C-2'-BOM paclitaxel (Formula 16).
Finally, the C-2'-BOM is removed according to the following reaction:
0
Ph NH 0 H
Ph 0
-_ 0 A c
Ph~0~0 OCOPh
0
Ph NH 0 H
Ph _ C
OH
OH OAc
OCOPh
Reaction XVII
The BOM protected paclitaxel is dissolved in isopropanol to which
Pearl man's catalyst is added. This mixture is hydrogenated for twenty-four
hours under 40 psi hydrogen or twenty-four hours under one atmosphere of
hydrogen in the presence of tri-fluroacetic acid to yield paclitaxel.
The conversion of Formula 15 to paclitaxel has been previously
described (Sisti et al, U.S. Patent No. 5,675,025, Oct. 7, 1997) and may be
accomplished as follows:
E. Deorotections and Acvlation to Form Paclitaxel from Formula 15
The compound according to Formula 15 may now be converted into
paclitaxel by removing the CBZ protecting group and acylating the side
chain, removing the TES protecting group and removing the hydrogenatable
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benzyl protecting group. Here, several convenient routes have been found
although in general, it is necessary to deprotect the C-7 site by removing the
TES protecting group prior to deprotecting the C-2' site with the
hydrogenatable benzyl protecting group. If the TES protecting group is not
removed first, it is believed difficult at best to remove the hydrogenatable
protecting group in a later processing step.
In any event, the preferred route of producing paclitaxel is to first
remove the CBZ protecting group according to the reaction:
Ac0 O
O
Ph~O NH O ~ '''' H
''' \
Ph _ O ''''' _ H _ '0
_ 1
OBOM OH OCOPh OAc
1.12.
OTES
O
Ph ~NH O ~ ''''H
_ ''' '
Ph _ O ''''' _ H _ 'O
OH OAc
OBOM OCOPh
1. Pearlmans Cat., 1 Atm H2, iPrOH
2. Benzoyl Chloride, EtOAc, TEA
Reaction XVIII
Here, the coupled product of Formula 15 is dissolved in isopropanol to which
the Pearl man's catalyst is added. The resulting mixture is stirred under one
atmosphere of hydrogen for twenty-four hours. Thereafter, the mixture is
filtered through diatomaceous earth and reduced under vacuum to residue.
The residue may then be taken up in ethyl acetate or toluene and a tertiary
amine base, such as triethylamine is added. Benzoyl chloride was added
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dropwise, and the mixture stirred for two hours. The resulting mixture was
then washed with dilute aqueous solution of NaHC03, water, and finally
brine. The resulting organic phase was then separated, dried and reduced
under vacuum to yield the CBZ deprotected/acylated compound:
Ac0 ,~ OTES
O
Ph ~NH O
'''' H
_ '''
Ph _ O ''''' _ H _ ~O
_ 1
OBOM OH OCOPh OAc
(Formula 17)
Next, the compound of Formula 17 is deprotected at C-7 according to
the reaction:
0
Ph NH O ~ '''' H
_ '''
Ph _ O ''''' _ H _ ~C
_ 1
OBOM OH OCOPh OAc
H F 40%
CH3CN
O
Ph NH O
Ph O
OBOM OCOPh
Reaction XIX
Here, the compound of Formula 17 was dissolved in acetonitrile (CHsCN) at
0°C. Hydrofluoric acid (40% aqueous) was then added and the mixture
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stirred for ten hours while being held at 0°C. Thereafter, the mixture
is
diluted with ethyl acetate, saturated aqueous solution of NaHC03, water and
finally brine. The organic phase was separated, dried and reduced under
vacuum to produce a deprotected product at the C-7 position according to
the formula:
OH
O
Ph ~NH O ~ '''' H
'
Ph _ O ''''' H \~O
OBOM OH OCOPh OAc
(Formula 18)
Finally, the compound of Formula 18 is deprotected at C-2' to remove
the hydrogenatable benzyl-type (BOM) protecting group and to liberate the
C-2' hydroxy group thereby resulting in the desired paclitaxel. This is
accomplished according to the reaction:
0
Ph NH O
Ph O
OBOM OH OCOPh OAc
Pearlmans Cat.
H2 40 psi, iPrOH
24 h.
PACLITAXEL
Alternatively, the compound of Formula 15 may first be dissolved in
CHsCN at 0°C and hydrofluoric acid (40% aqueous) added to
deprotect the
compound at the C-7 site by removing the TES protecting group. This
results in a compound according to the Formula 19:
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Ac0 O
O
Ph~O NH O
'''~ H
_ '''
Ph _ O ''''' _ H _ ~O
1
OBOM OH OCOPh OAc
(Formula 19)
Next, the CBZ protecting group may be removed in a manner similar to that
described above. Here, the compound of Formula 19 is dissolved in
isopropanol and Pearlman's catalyst was added along with trifluoroacetic
acid (TFA) (one equivalent). The mixture was held at 40 psi of hydrogen at
room temperature for approximately four days. This removes the CBZ
protecting group and forms the C-2' BOM protected paclitaxel compound as
a TFA salt. The mixture was filtered through diatomaceous earth and
reduced under vacuum. Next, a base plus an acylating agent was added to
the residue. Specifically, the TFA salt of the C-2' BOM protected compound
was dissolved in pyridine and either benzoyl chloride or benzoic anhydride
was added. The resulting product is:
0
0
Ph NH O
'''~ H
_ '''
Ph _ O ''''' _ H _ ~O
_ 1
OBOM OH OCOPh OAc
(Formula 16)
The compound of Formula 16 is dissolved in isopropyl alcohol and
placed in a Parr bottle and Pearl man's catalyst was added. The mixture was
hydrogenated for twenty-four hours at 40 psi of hydrogen. Thereafter, the
mixture was filtered through diatomaceous earth and the eluent reduced
under vacuum. The residue may then be column chromatographed
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according to any desired technique or recrystallized from ethyl
acetate:hexane for the final paclitaxel product.
Accordingly, the present invention has been described with some
degree of particularity directed to the exemplary embodiments of the present
invention. It should be appreciated, though, that the present invention is
defined by the following claims construed in light of the prior art so that
modifications or changes may be made to the exemplary embodiments of the
present invention without departing from the inventive concepts contained
herein.
