BACKGROUND ART Paclitaxel, a well known potent antitumor compound having a broad spectrum of antitumor activities, has the following structure of formula (1): 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. patent RE-34,227 (a reissue of U. S. patent 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-phenyl-isoserine 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 ß-lactam compound with 7-0-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. patent 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.

Patent 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 (11): R, is a hydroxy-protecting group or a hydrogen atom; and R2 is a hydroxy-protecting group or a hydrogen atom.

Examples of hydroxy-protecting groups include C14 carboxylic acid acyl groups, for example acetyl, trialkylsilyl groups wherein each alkyl group contains 1 to 3 carbon atoms and the group A defined above, e. g. a t-butoxycarbonyl group of formula Preferably, therefore R, is a C, 4 carboxylic acid acyl group, for example acetyl, a trialkylsilyl group wherein each alkyl group contains 1 to 3 carbon atoms or the group A defined above, e. g. a t-butoxycarbonyl group R2 may be any of these groups, but because the 13-hydroxy group is less reactive, protection is not essential, so R2 can conveniently be hydrogen.

During the semisynthesis of paclitaxel, an N-benzol (2R, 3S)-3- phenylisoserine group is introduced atthe 13-position of an appropriately protected derivative of baccatine III. The resulting protected derivatives of paclitaxel have the general formula (Ila) R, is a hydroxy-protecting group or a hydrogen atom; and R2a is a (2R, 3S)-3-phenylisoserine derivative having the structure: where R3 is a hydroxy-protecting group, such as A (as defined above), a methoxy methyl, 1-ethoxyethyl, benzyloxymethyl, 3-trialkylsilylethoxy) 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 (Ila) and removing the A and R3 groups to form paclitaxel.

For the preparation of compounds of formula 11 and Ila where R is acetyl the process of the invention preferably comprises forming the intermediate compound II by reacting 10-deacetylbaccatine III with a reagent capable of introducing a t- butoxycarbonyl group, for example t-butoxy-pyrocarbonate to obtain 7-t-butoxycarbonyl-10-deacetyl baccatine III. The thus obtained 7-t-butoxycarbonyl-10-deacetyl baccatine III may then be acetylated to obtain 7-t-butoxycarbonyl-baccatine 111.

10-Deacetylbaccatine III is highly insoluble in most common solvents and accordingly the choice of solvent is important in order to ensure that the reaction in which the t-butoxycarbonyl group is introduced proceeds at an acceptable rate and in high yields. Thus, for example, 10-deacetyl is highly insoluble in methylene dichloride. If methylene dichloride is used as a solvent for the reaction of 10- deacetylbaccatine with t-butoxypyrocarbonate (see, e. g. Example 1 below), the reaction proceeds at a reasonably fast rate on a small scale, but when operated on a large scale, the reaction can be unacceptably slow.

On the other hand, if a polar aprotic nitrogen-containing solvent such as pyridine is used for carrying out the reaction (see Example 8 below) the reaction proceeds more rapidly, but the yield can be lower as a result of formation of 7,10- di (t-butoxycarbonyl) baccatin as a by-product.

Although there may be a minor penalty in terms of lower yield, the use of pyridine as a solvent has advantages on the industrial scale in view of the higher rate of the reaction. Also it is possible to carry out the next step in the process, i. e. the introduction of an acetyl group in the 10-position, without separating or purifying the desired 7-t-butoxycarbonyl 10-deacetylbaccatin. I. e. the reaction in which the t-butoxycarbonyl group is introduced and the acetylation reaction can be carried out in one pot.

Further, if the starting material is in the amorphous (anhydrous) form the reaction proceeds more rapidly than if the starting material is in the form of the crystalline hemihydrate.

The hydroxy group in position 13 of the 7-t-butoxy-carbonyl baccatine III may then be converted to a (2R, 3S)-3-phenylisoserine group having the structure wherein R3 is hydrogen by reaching the 7-t-butoxy-carbonyl-baccatine III with an oxazolidine derivative of formula (III): wherein R4 is an aryl group or a straight or branched chain alkyl or alkenyl group having 1-5 carbon atoms; and R5 is R4 or a t-butoxy group, and each of R7 and R8 is a halogenated methyl group. Advantageously, an excess of the 7-t-butoxy-carbonyl-baccatine III compound is used relative to the oxazolidine derivative.

The oxazolidine derivatives of formula (III) where R4, R5, R6 and R7 are as defined above from a further aspect of the present invention. Preferably, R4 is phenyl, R5 is phenyl or a t-butoxy group, and each of R6 and R7 is a CICH2-, BrCH2-or F3C-group.

The oxazolidine derivatives of Formula III may be prepared by reacting a compound of Formula IV with a ketone of formula R6R7 CO (V) In formula IV and V R4, R5, R6 and R7 are as defined above, and R8 is a residue of an alcohol RU OH in whic R8 is, e. g. a C14 alkyl group, e. g. methyl.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel methods for the semisynthesis of paclitaxel of the general formula (I) given above through the use of the new intermediates of formulae (II) and (Ila). These new intermediates are versatile intermediates 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-butoxy-pyrocarbonate (BOC) : 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, can be used, where R, 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 G1 groups of the compounds of formula III of U. S.

Patents 5,578,739 or 5,621,121, the R2 groups of the compounds of formula III of U. S. patent 5,476,954, or the R3 substituents of the compounds of formula IV of U. S. patent 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 alcools has not previously been reported before in literature, and particularly in respect of 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 may be performed in chlorinated solvents, preferably in methylene chloride using dimethylformamide as a co-solvent. 1 Mole of 10-deacetyl-baccatine III or the chosen taxane analog may be reacted with 1.2 to 2.5 equivalents t-terbutoxy-pyrocarbonate in the presence of 1.2 equivalents of ethyidiisopropylamine 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 then be converted to biologically active compounds by esterifying the hydroxy group at the 13 position with an oxazolidine derivative of formula (III): wherein R4 is an aryl group or a straight or branched chain alkyl or alkenyl group having 1-5 carbon atoms; and R5 is R4 or a t-butoxy group, and each of R6 and R7 is a halogenated methyl group.

The reaction is generally performed in aprotic solvents, preferably benzene, toluene, xylene, chlorobenzene or ethylbenzene, preferably in the presence of a condensing agent such as dicyclohexylcarbodiimmide (DCC) and a catalytic amount of a base such as dialkylamino pyridine, preferably 4-dimethylaminopyridine at temperatures ranging from about 50°C to 100°C, and preferably 70°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 13-ester may be isolated in crude form. This compound may then be treated in methanol with a catalytic amount of anhydrous HCI at room temperature or at temperatures ranging from about 5°C to 10°C, and preferably at 0°C, with concentrated formic acid (98%) until complete deprotection of the BOC group at the 7 position and of the protective group R3 of the side chain at position 13 is achieved. After treatment of the reaction mixture with brine, the taxane derivative may be extracted with a solvent that is non-miscible with water, and preferably with ethylacetate. After distillation of the extraction solvent, the taxane derivatives may directly be crystallized with suitable solvents or subjected to chromatographic process using silica-gel and as eluting solvents, a mixture preferably constituted by hexane/ethylacetate in a suitable ratio.

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 described above for the reaction using a oxazolidine derivative may be used.

The hydroxy group at position 13 can be esterified in a number of other ways as disclosed, e. g., in U. S. patents 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 Ill.

A 500 mg sample of 10-deacetylbaccatine III (0.92 mMol) was suspended in CH2CI2 (5 mL) and ethyldiisopropylammine (1.10 mMol, 1.2 Equiv.), t-butoxypyrocarbonate (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 CH2CI2 washing with HCI and brine.

After drying, 580 mg of 7-Boc-10-deacetylbaccatine III were obtained having the following characteristics: mp 148°C and 162°C; 1H-NMR 200 Mhz, CDCI3, TMS as interna standard; Bz 8.10, br d, J 8; Bz 7.70, br t J 8; Bz 7.55, br t J 8; H2,5.64d 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; H20a, 4.32 d, J 8.0; H20b 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; I R (KBr) 3480 (OH), 1740 (br, C = O), 712.

Example 2: Synthesis of 7-BOC-10-deacetylbaccatine lil A 500 mg sample of 10-deacetylbaccatine III (0.92 mMol) was solubilized in 1 ml of dimethylformamide and diluted with 4 mi of CH2CI2. The reagents and the reaction conditions are the same of Example I.

Example 3: Synthesis of 7-BOC-baccatine Ill.

644 mg (1 mMol) of 7-Boc-10-deacetylbaccatine Iil prepared according to example 1 or 2 were dissolve in 5 mL of pyridine and at 0°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 CH2CI2 under stirring and washed with 60 mL of H2O.

The organic phase is washed several times with H20 and diluted HCI until the elimination of pyridine. The solvent dried on Na2SO4 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°C. 1 H-NMR 200 Mhz, CDC13, TMS as internal standard; Bz 8.10 br d, J 8; Bz 7.70 br t, J 8; Bz 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; H20a, 4.32 d, J 8.0; H20b 4.22 d, J 8.0; H3,4.02 d, J 6.7; Ac. H 18 2. 19 s; Ac. H 19 1. 80 s; Boc 1. 48 s; H16 1.17 s; H17 1.07s.

Example 4: Synthesis of paclitaxel.

1.65 gr of (4S, 5R)-2,2-di (chloromethyl)-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°C for 12h 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 H2O and distille under vacuum.

The residue is solubilized in 10 mL of conc. formic acid at 0°C and kept in this condition for 2h. The reaction mixture was diluted with 100 mL of H20 and cloudy solution extracted three times with 50 mL CH2C12. The organic phase was washed with a solution of NaHC03 and then with H20. The organic phase after drying on Na2SO4 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.

Example 5: Reaction of 10-deacetylbaccatin III with Boc-pyrocarbonate A 500 mg sample of 10-deacetylbaccatin III (0.92 mMol) was suspended in CH2CI2 (5 mL) and ethyldiisopropylamine (190 NL, 1.10 mMol, 1.2 mol. Equiv.), BOC-pyrocarbonate (240 mg, 1.10 mMol, 1.2 mol. Equiv) and DMAP (4- dimethylaminopyridine, 20 mg) were added. The reaction was stirred 48 h at room temp, and then further BOC-pyrocarbonate (240 mg, 1.10 mMol, overall 2.4 mol equiv.) and ethyldiisopropylamine (190 vil, 1.10 mMol, overall 2.4 mol, equiv.) were added. After stirring for an additional 120 hours, the reaction was worked up by dilution with CH2C12, washing with HCI and brine. After drying the residue <BR> <BR> <BR> <BR> was purified by column chromatography (ca. 5 g silica gel). Elution with hexane- EtOAc 6: 4 gave 327 mg BOC DAB (yield: 55% conversion: 92%) Elution with ETOAc gave 195 mg recovered DAB.

Example 6-Reaction of 10-deacetylbaccatin III With BOC-ON To a solution of 10-deacetylbaccatin lil (400 mg, 0.73 mmol) in pyridine (3 mL), BOC-ON [=2-(tert-butoxywarbonyloxiymino) 2-phenylacetonitrile]- (543 mg, 2.19 mmol, 3 mol. equiv.) and 4-dimethylaminopyridine (90 mg, 0.73 mmol, 1 mol. equiv) were added.

The reaction was followed by TLC (hexane-EtOAc 4: 6, Rf starting material: 0.1; Rf 7-BOC derivative 0.50; Rf 7,10-diBOC derivative: 0.56). After stirring at room temp. for 10 days, the reaction was worked up by dilution with water and extraction with chloroform. After washing with sat. citric acid, sat. NaHCO3 and brine, the solution was dried (MgS04) and evaporated, to afford a semi-solid residue (1.07 g). The latter, when analyzed by'H NMR spectroscopy (200 MHz), turned out to contain the 7-BOC and the 7,10-diBOC derivatives in a 85: 15 ratio.

Purification by column chromatography (hexane-EtOAc 6: 4) afforded 265 mg of 7- BOC-10-deacetylbaccatin III (yield: 56%).

Analysis-7 BOC-10-deactylbaccatin III White powder, mp 162 °C; IR (KBr): 3480,1740,1603,1371,1275,1259,1158,1092,712 Cl-MS: 645 (M+H), C34H440, 2 'H NMR (200 MHz, CDCI3): 8.10 (br d, J=8.0 Hz, Bz); 7.70 (br t, J = 8.0 Hz, Bz).

7.55 (br t, J = 8.0 Hz, Bz), 5.64 (d, J = 7.0 Hz, H-2), 5.54 (s, H-10), 5.36 (dd, J = 11.0,8.0 Hz, H-7), 4.95 (d, J = 8.0 Hz, H-5), 4.91 (br t, J = 7.5 Hz, H-13), 4.32 (d, J= 8.0 Hz, H-20a), 4.26 (d, J = 8.0 Hz, H-20b), 4.09 (d, J = 8.0 Hz, H-3), 2.29 (s, OAc), 2.09 (br s, H-18), 1.83 (s, H-19), 1.46 (s, BOC), 1.34 (s, H-16), 1.20 (s, H-17).

Analysis-7-BOC-baccatin III White powder, mp 197 °C; IR (KBr): 980.

M. W.: 686, C36H46013 rH NMR (200 MHz, Ceci3): 8.10 (br d, J= 8.0 Hz, Bz); 7.70 (br t, J = 8.0 Hz, Bz).

7.55 (br t, J = 8.0 Hz, Bz), 6.52 (s, H-10), 5.64 (d, J = 7.0 Hz, H-2), 5.41 (dd, J = 11.0,8.0 Hz, H-7), 4.98 (d, J = 8.0 Hz, H-5), 4.90 (br t, J = 7.5 Hz, H-2), 4.32 (d, J = 8.0 Hz, H-20a), 4.22 (d, J = 8.0 Hz, H-20b), 4.02 (d, J = 7.0 Hz, H-3), 2.30 (s, OAc), 2.19 (br s, H-18), 2.16 (s, OAc), 1.80 (s, H-19), 1.48 (s, BOC), 1.17 (s, H-16), 1.07 (s, H-17).

Example 7-Preparation of Dichlorooxazolidine Derivative Illa (Methyl Ester of Compound of Formula III with R4 = R5 = phenyl; R6 = R7 = Cl) 500 mg of N-benzoyl-phenyl-isoserine methylester in 30ml of tetrahydrofuran/benzene 1: 1 mixture was allowed to react with 1 g of dichloroacetone and 50 mg of PTSA (pyridinium p-toluenesulfonate) in the presence 0 of a molecular sieve (3A). The reaction mixture was heated and refluxed for 2 days. At the end of the reaction, the residue was washed with hexane in order to eliminate the excess of dichloroacetone.

The residue in 20mut of MeOH was mixed with 220 mg of K2CO3 in 20ml of H20. After 2 hours, methanol was evaporated under vacuum and the aqueous phase was acidified with a 5% solution of KHSO4 and then extracted with ethylacetate.

The obtained acid was used then used directly for the esterification of 7-Boc- 10 acetylbaccatine III (see Example 4).

Example 8-Reaction of 10-Deacetylbaccatin III with Boc-Pyrocarbonate BOC20 (800 mg, 37 mMol, 2 mol. equiv.) and DMAP (220 mg, 18.5 mMol, 1 mol. equiv.) were added to a solution of 10-deacetylbaccatin III (1.0 g, 18.5 mMol) in pyridine (15 mL),. The reaction was followed by TLC (hexane-EtOAc f: 6, Rf st. m = 0.10; Rf7-boc-derivative 0.50; Rf 7,10-diBOC derivative: 0.56).

After stirring (16 h at room temp.), the reaction mixture was cooled to 0°C, and AcCI (261, uL, 37 mMol, 2 mol. equiv.) was added. The reaction was followed by TLC (1,2-dichloroethane-EtOH 96: 4 x 4).

After stirring at 0°C for 5 h, two further equivalents of AcCI were added, and stirring is continued at 0° for a further 2 hours. The reaction mixture was then worked up by addition of water (ca 150 mL) to the reaction flask. After 30 min. water was decanted from the reaction flask, and the sticky precipitate on the walls of the was taken up in EtOAc (30 mL), washed with dil HCI (10 mL) and brine (10 mL). After drying (MgS04) and evaporation of the solvent, 1.18 g of a yellow powder are obtained. When analyzed by NMR, the product was a 82: 18 mixture of 7-BOC baccatin III and 7,10-diBOC baccatin Ill.