Intermediates and methods useful in the semisynthesis of paclitaxel and analogs

The semisynthesis of paclitaxel and its analogs using new intermediates which are derivatives of 10-deacetyl-baccatin III, as well as to a method for preparing these derivatives. These novel derivatives have alkyl carbonate or alkyl carbonyl substituents in the 7 position.

TECHNICAL FIELD 
The present invention relates to semisynthes paclitaxel and its analogs 
using new intermediates which are derivatives of 10-deacetyl-baccatine 
III, as well as to a method for preparing these derivatives. These novel 
derivatives have carbonate substituents in the 7 position, such as 
t-butoxy-carbonate. 
BACKGROUND ART 
Paclitaxel, a well known potent antitumor compound having a broad spectrum 
of antitumor activities, has the following structure of formula (I): 
##STR1## 
Commercial pharmaceutical products containing this compound are available, 
e.g., for treating ovarian and breast cancer in women. For these reasons, 
greater and greater supplies of this compound are required each year. 
Paclitaxel and baccatine III are extracted with difficulty and in general 
in low yields from the trunk barks of different Taxus species. Thus, 
alternative sources of this compound are necessary. 
Several synthetic methods have been reported both in scientific and patent 
literature. U.S. Pat. No. RE-34,227 (a reissue of U.S. Pat. No. 4,924,011) 
discloses the semisynthesis of paclitaxel using a 10-deacetyl-baccatine 
III derivative which is protected in the 7 position with a tri-alkyl-silyl 
group which is specifically shown as a tri-ethyl-silyl ("TES") group and 
which is also protected in the 10 position with an acetyl group. This 
baccatine III derivative is allowed to react with a 
(2R,3S)-N-benzoyl-2-O-(1-ethoxyethyl)-3-phenylisoserine compound before 
removal of the protecting groups to obtain the paclitaxel. 
In PCT application WO-93/06094, paclitaxel was prepared by reacting a side 
chain precursor of a .beta.-lactam compound with 7-O-TES-baccatine III 
derivative to provide a 7-TES-baccatin III reaction product. After a mild 
acidic post-reaction treatment, paclitaxel was obtained. 
In U.S. Pat. No. 5,476,954, the synthesis of paclitaxel was conducted 
starting from a protected 10-deacetyl-baccatine III derivative that 
contained a 2,2,2-tri-chloroethoxy-carbonyl ("TROC") protective group in 
both the 7 and 10 positions of the derivative. 
It is well known that the key step in the semisynthesis of paclitaxel is to 
selectively protect the 7 position with a leaving group that can be easily 
removed. This is because the hydroxy group in that position of the taxane 
structure is much more reactive than those in position 10 or 13, and the 
paclitaxel product to be synthesized needs to have a hydroxy group in that 
position. Until now, however, the most useful protecting group was 
considered to be TES. The derivatization yield of 10-deacetyl-baccatine 
III with TES is typically about 85% when 20 moles of the reagent are used. 
The acetylation step, using 5 equivalents of acetylchloride, provides 
about 85% of 7-TES-baccatine III. as per the teachings of PCT application 
WO-93/06094 and its U.S. equivalent documents such as U.S. Pat. No. 
5,574,156. 
In view of the importance of paclitaxel, however, new and improved methods 
for its production are desirable. The present invention provides such 
improved syntheses of paclitaxel and its analogs primarily using new 
derivatives of 10-deacetyl-baccatin III as intermediates. 
SUMMARY OF THE INVENTION 
The present invention relates to an intermediate for use in the 
semisynthesis of paclitaxel, comprising a compound of formula (II): 
##STR2## 
with n being an integer of 0 to 3, R.sub.1 is a hydroxy-protecting group 
or a hydrogen atom; and 
R.sub.2 is a hydroxy-protecting group or a hydrogen atom. 
Preferably, R.sub.1 is A, an acetyl group or a tri-alkylsylil group wherein 
each alkyl group contains 1 to 3 carbon atoms, R.sub.2 is a 
(2R,3S)-3-phenylisoserine derivative having the structure: 
##STR3## 
where R.sub.3 is a hydroxy-protecting group, such as A; a methoxy methyl, 
1-ethoxyethyl, benzyloxymethyl, (.beta.-trialkyl-silylethoxy)methyl where 
each alkyl group contains 1 to 3 carbon atoms, tetrahydropyranyl or 
2,2,2-trichloroethoxycarbonyl group; or a hydrogen atom. 
The invention further relates to a process for producing paclitaxel by the 
steps of forming the intermediate compound of formula (II) and removing 
the A and R.sub.3 groups to form paclitaxel. In this method, when n is 0, 
R.sub.1 is an acetyl group, and R.sub.2 is a (2R,3S)-3-phenylisoserine 
derivative having the structure: 
##STR4## 
where R.sub.3 is a hydrogen atom, the method further comprises forming the 
intermediate compound by reacting 10-deacetylbaccatine III with 
t-butoxy-pyrocarbonate to obtain 7-t-butoxy-carbonyl-10-deacetyl baccatine 
III; acetylating 7-t-butoxy-10-deacetyl baccatine III to obtain 
7-t-butoxy-carbonyl-baccatine III and esterifying the hydroxy group in 
position 13 of the 7-t-butoxy-carbonyl-baccatine III with an oxazolidine 
derivative of formula (III): 
##STR5## 
wherein R.sub.4 is an aryl group or a straight or branched chain alkyl or 
alkenyl group having 1-5 carbon atoms; and R.sub.5 is R.sub.4 or a 
t-butoxy group, and each of R.sub.7 and R.sub.8 is a halogenated methyl 
group. Advantageously, an excess of the 7-t-butoxycarbonyl-baccatine III 
compound is used relative to the oxazolidine derivative. 
Another intermediate for use in the semisynthesis of paclitaxel according 
to the present invention comprises the oxazolidine derivative of formula 
(III), where R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are as defined above. 
Preferably, R.sub.4 is phenyl, R.sub.5 is phenyl or a t-butoxy group, and 
each of R.sub.6 and R.sub.7 is a ClCH.sub.2 --, BrCH.sub.2 -- or F.sub.3 
C-- group. 
DETAILED DESCRIPTION OF THE INVENTION 
The semisynthesis of paclitaxel of the general formula (I) given above 
through the use of the new intermediates of formula (II). These new 
intermediates are key intermediate which also can be used for the 
semisynthesis of docetaxel and other analogs of paclitaxel. The process 
for their preparation is also described. 
It has been found, surprisingly, that protecting the hydroxy group at 
position 7 of 10-deacetylbaccatine III or similar taxane derivatives with 
the same basic structure, with certain carbonate compounds provides 
enhancements in the preparation of paclitaxel from such derivatives. 
A preferred protective group is t-butoxypyrocarbonate (BOC), although other 
carbonate groups having the structure 
##STR6## 
can be used. In this structure, n is an integer of 0 to 3, with n being 0 
preferred. This protective group can be substituted in position 7 and, if 
desired, as well as in position 10. 
As position 10 is not as reactive as position 7, a number of other 
protective groups can be used in position 10. In particular, the group 
--OR.sub.1 can be used, where R.sub.1 is a hydroxy-protecting group or a 
hydrogen atom. Any of a wide variety of hydroxy-protecting groups can be 
used, including the carbonate groups described above for A, the G.sub.1 
groups of the compounds of formula III of U.S. Pat. Nos. 5,578,739 or 
5,621,121, the R.sub.2 groups of the compounds of formula III of U.S. Pat. 
No. 5,476,954, or the R.sub.3 substituents of the compounds of formula IV 
of U.S. Pat. No. Re. 34,277. 
It is possible to obtain almost quantitative yields of the 
7-BOC-10-deacetylbaccatine III derivative from 10-deacetylbaccatine III. 
The use of BOC as a protecting group for alcohols was not previously 
reported before in literature, and particularly on taxane structures. This 
group is easily and selectively removed in very mild acidic conditions 
using a catalytic amount of mineral or organic acids, preferably formic or 
F3C--COOH. 
The synthesis of 7-BOC-10-deacetylbaccatine III or its analog is performed 
in chlorinated solvents, preferably in methylene chloride using 
dimethylformamide as a cosolvent. 1 Mole of 10-deacetyl-baccatine III or 
the chosen taxane analog is reacted with 1.2 to 2.5 equivalents 
t-terbutoxy-pyrocarbonate in the presence of 1.2 equivalents of 
ethyldiisopropylamine and a catalytic amount of 4-dimethylaminopyridine. 
Under these conditions, it is possible to obtain in almost quantitative 
yields the 7-BOC-derivative. This compound can be converted into 
7-BOC-10-acetyl derivative using acetyl chloride, bromide or diketene as 
shown in the examples. 
These derivatives can be converted to biologically active compounds by 
esterifying the hydroxy group at the 13 position with an oxazolidine 
derivative of formula (III): 
##STR7## 
wherein R.sub.4 is an aryl group or a straight or branched chain alkyl or 
alkenyl group having 1-5 carbon atoms; and R.sub.5 is R.sub.4 or a 
t-butoxy group, and each of R.sub.6 and R.sub.7 is a halogenated methyl 
group. 
The reaction is performed in aprotic solvents, preferably benzene, toluene, 
xylene, chlorobenzene or ethylbenzene in the presence of 
dicycloecxylcarbodiimmide (DCC) and a catalytic amount of dialkylamino 
pyridine, preferably 4-dimethylaminopyridine at temperatures ranging from 
about 50.degree. C. to 100.degree. C., and preferably 70.degree. C. 
Preferably, to obtain the desired compounds, 4 Moles of condensing agent 
and 1.5 Mole of the oxazolidine derivative are used for 1 Mole of 
protected taxane. After elimination of the reaction byproducts and the 
solvent the ester at position 13 is isolated in crude form. This compound 
is treated in methanol with a catalytic amount of anhydrous HCl at room 
temperature or at temperatures ranging from about 5.degree. C. to 
10.degree. C., and preferably at 0.degree. C., with concentrated formic 
acid (98%) until complete deprotection of the BOC group at the 7 position 
and the protective group R.sub.3 of the side chain at position 13 is 
achieved. After treatment of the reaction mixture with brine, the taxane 
derivative is extracted with a solvent that is non-miscible with water, 
and preferably with ethylacetate. After distillation of the extraction 
solvent, the taxane derivatives are directly crystallized with suitable 
solvents or submitted to chromatographic process using silica-gel and as 
eluting solvents, a mixture preferably constituted by exane/ethylacetate 
in a suitable range. 
Alternatively, paclitaxel and its analogs can be prepared by esterifying 
the protected baccatine with a phenylisoserine chain esterified at 2 
position with BOC. The reaction conditions are those above described for 
the oxazolidine derivatives. 
The hydroxy group at position 13 can be esterified in a number of other 
ways as disclosed, e.g., in U.S. Pat. Nos. 5,578,739, 5,574,156, 
5,621,121, 5,476,954, 5,470,866, 4,857,653, 4,814,470, and Re. 34,277, and 
in European Patent Application 0,525,589-A1. To the extent necessary to 
understand the present invention, all patents cited in the detailed 
description of this specification are expressly incorporated by reference 
herein.

EXAMPLES 
The examples below are reported, without implied limitation, to show how 
the invention can be put in practice. 
EXAMPLE 1 
Synthesis of 7-BOC-10-deacetylbaccatine III 
A 500 mg sample of 10-deacetylbaccatine III (0.92 mMol) was suspended in 
CH.sub.2 Cl.sub.2 (5 mL) and ethyldiisopropylammine (1.10 mMol, 1.2 
Equiv.), t-butoxypirocarbonate (240 mg, 1.10 mMol, 1.2 Equiv.) and DMAP 
(4-dimethylaminopyridine, 20 mg) were added. 
The reaction was stirred 48 h at room temperature and then additioned with 
the same quantity of reagents and allowed to stay under stirring per other 
48 h. The reaction was worked up by dilution with CH.sub.2 Cl.sub.2 
washing with HCl and brine. After drying, 580 mg of 
7-Boc-10-deacetylbaccatine III were obtained having the following 
characteristics: mp 148.degree. C. and 162.degree. C.; 1H-NMR 200 Mhz, 
CDCl.sub.3, TMS as internal standard; Bz .delta. 8.10, br d, J 8; Bz 
.delta. 7.70, br t J 8; Bz .delta. 7.55, br t J 8; H2, 5.64 d J 7; H10, 
5.54, s; H7, 5.36, dd, J 11.0, 8.0; H5, 4.95, d J 8; H13, 4.91, br t, 
J7.5; H2Oa, 4.32 d, J 8.0; H2Ob 4.26, d, J 8.0; H3, 4.09 d, J 8.8; Ac. 
2.29 s; H18 2.09 s; H19 1.83 s; Boc 1.46 s; H16 1.34 s; H17 1.20 s; IR 
(KBr) 3480 (OH), 1740 (br, C.dbd.O), 1603, 1371, 1275, 1259, 1158, 1092, 
712. 
EXAMPLE 2 
Synthesis of 7-BOC-10-deacetylbaccatine III 
A 500 mg sample of 10-deacetylbaccatine III (0.92 mMol) was solubilized in 
1 ml of dimethylformamide and diluted with 4 ml of CH.sub.2 Cl.sub.2. The 
reagents and the reaction conditions are the same of Example I. 
EXAMPLE 3 
Synthesis of 7-BOC-baccatine III 
644 mg (1 mMol) of 7-Boc-10-deacetylbaccatine III prepared according to 
example 1 or 2 were dissolved in 5 mL of pyridine and at 0.degree. C. 
under stirring 1.2 g of acetylchloride were added (15 mMol) in 15 h. When 
the reaction is finished the solution is diluted with CH.sub.2 Cl.sub.2 
under stirring and washed with 60 mL of H.sub.2 O. The organic phase is 
washed several times with H.sub.2 O and diluted HCl until the elimination 
of pyridine. The solvent dried on Na.sub.2 SO.sub.4 is evaporated under 
vacuum and the residue crystallized from hexane/acetone. 660 mg of 
7-Boc-baccatine III were obtained having the following characteristics: mp 
190-97.degree. C. 1H-NMR 200 Mhz, CDCl.sub.3, TMS as internal standard; Bz 
8.10 br d, J 8; Bz 7.70 br t, J 8; Bz .delta. 7.55, br t J 8; H2, 5.64 d, 
J 7; H10, 5.52 s; H7, 5.44 dd, J 10.3, 7.0; H5, 4.98, d, J 7.9; H13, 4.50 
br t; H2Oa, 4.32 d, J 8.0; H2Ob 4.22 d, J 8.0; H3, 4.02 d, J 6.7; Ac. 2.30 
s; H18 2.19 s; Ac. 2.16 s; H19 1.80 s; Boc 1.48 s; H16 1.17 s; H17 1.07 s. 
EXAMPLE 4 
Synthesis of paclitaxel 
1.65 gr of (4S,5R)-2,2-di(chloro methyl)-4-phenyl-N-Benzoyl-5-oxazolidine 
acid were allowed to react in toluene with 0.69 gr of 7-Boc-baccatine III 
in the presence of 1.1 Equival. of DCC and 60 mg of 
4-dimethylaminopyridine. The reaction mixture was maintained at 60.degree. 
C. for 12 h under stirring in Argon atmosphere. 
At the end of the reaction (TLC) the reaction mixture was filtered form 
insoluble byproducts and the solvent washed with H.sub.2 O and distilled 
under vacuum. The residue is solubilized in 10 mL of conc. formic acid at 
0.degree. C. and kept in this condition for 2 h. The reaction mixture was 
diluted with 100 mL of H.sub.2 O and cloudy solution extracted three times 
with 50 mL CH.sub.2 Cl.sub.2. The organic phase was washed with a solution 
of NaHCO.sub.3 and then with H.sub.2 O. The organic phase after drying on 
Na.sub.2 SO.sub.4 is concentrated under vacuum. 
The residue was crystallized from ethanol/water and 0.81 gr of paclitaxel 
having the well known characteristics which have been reported in the 
literature was obtained.