Title of Invention	A STEREOSELECTIVE CHEMOENZYMATIC PROCESS FOR THE PREPARATION OF OPTICALLY ENRICHED PHENYLGLYCIDATES ALKYL(2S,3R) - PHENYLGLYCIDATE AND ALKYL(2R,3S)- PHENYLGLYCIDATE AS PRECURSORS OF TAXOL SIDE CHAIN
Abstract	The present invention relates to a stereoselective chemoenzymatic process for the preparation of optically enriched phenylglycidates i.e. alkyl(2S,3R)-phenylglycidate and alkyl(2R,3S)-phenylglycidate as precursors of taxol side chain. The process involves preparation of halohydrins from alkyl cinnamates by the action of a halogenating agent followed by their conversion to alkyl acylates , incubating alkyl acylates with dry powder of lipase from Aspergillus niger .separation of hydrolyzed halohydrins , acid followed by base catalyzed reaction for the cyclization of resolved esters.There are a number of reports on the preparation of these chiral intermediates by biochemical or chemoenzymatic methods besides asymmetric synthesis.No prior art discloses the application of lipase from Aspergillus niger for the preparation of (2S,3R)-phenylglycidate and alkyl(2R,3S)-phenylglycidate which is mentioned in the present invention.

Title of Invention

A STEREOSELECTIVE CHEMOENZYMATIC PROCESS FOR THE PREPARATION OF OPTICALLY ENRICHED PHENYLGLYCIDATES ALKYL(2S,3R) - PHENYLGLYCIDATE AND ALKYL(2R,3S)- PHENYLGLYCIDATE AS PRECURSORS OF TAXOL SIDE CHAIN

Abstract

Full Text	The present invention relates to a stereoselective chemoenzymatic process for the preparation of optically enriched phenylglycidates i.e. alkyl(2S,3R)- phenylglycidate and alkyl(2R,3S)- phenylglycidate as precursors of taxol side chain.The invention particularly discloses a novel process for the chemoenzymatic synthesis of two enantiomers of trans alkyl phenylglycidate i.e.alkyl (2R,3S)- phenylglycidate and aikyi(2R,3S)-phenylglycidate of formulae 7 and 8 respectively, process of their synthesis comprises cohalogenation reaction of alkyl cinnamate of formula 1 where R' represents C-1 to C-5 alkyl group to form trans 2-halo-3-hydroxy-3-phenylpropanoates of formula 2 where x represents Br or I, then converting the halohydrins of formula 2 to corresponding alkyl acylates of formula 3 where x and R' represents as above, subsequently incubating the acyl derivatives of alkyl 2-halo-3-hydroxy-3-phenylpropanoates of formula 3 with crude dry powder of lipase f,om Aspergillus niger in an aqueous buffer phase in presence of an organic solvent, thereafter separating the hydrolysed halohydrins i.e. alky!(2R,3R) 2-haio-3-hydroxy-3-phenylpropanoates of formula 4 and unhydrolysed ester i.e. alkyl (2S,3S) 2-halo-3-acyloxy-3-phenylpropanoates of formula 5 from the mixture by conventional method, if required then again incubating the optically enriched acyl derivatives of formula 5 with crude dry powder of lipase from Aspergillus niger in an aqueous buffer phase in presence of an organic solvent to further improve the enantiopurity, followed by reaction of the optically enriched products of formula 5 with an acid to furnish optically enriched alkyl (2S.3S) 2-halo-3-hydroxy-3-phenylpropanoate of formula 6 and finally treating the compounds of formulae 4 and 6 with an alkali in an organic or aqueous phase to furnish optically enriched alkyl(2S,3R)-phenylglycidate and alkyl(2R,3S)-phenylglycidate of formulae 7 and 8 respectively . Use of lipase from Aspergillus niger for the kinetic resolution of trans alkyl 2-halo-3-hydroxy-3-phenylpropanoates where x and R' represents as above is novel as also the step of acid followed by a base catalysed reaction for the cyclisation of resolved esters of trans alkyl 2-halo-3-hydroxy-3-phenylpropanoates of formula 4 and 6, not reported in the art of its synthesis. Optically enriched alkyl(2S,3R)-phenylglycidate and alkyl(2R3S)-phenyl-glycidatd of formulae 7 and 8 are the key intermediates used in the synthesis of N-benzoyl-(2R,3S)-3-phenylisoserine (Taxol side chain). Paclitaxel or Taxol which was isolated from the bark of the Pacific Yew (Taxus 5rewfo//a)(Wani et Taxol 10-Deacetyl baccatin-lll Isoserine various types of cancers( Holmes et al J. Natl. Cancer Inst. 83, 1797-1805,1991). Despite being one of the most promising anti-cancer drugs, its very low occurrence (40-165 mg/kg) in nature is the main hindrance in its production. Fortunately it has been found that 10-deacetyl baccatin-lll which is structurally closely related to Taxol and occurs in comparatively higher concentrations (approx. 1g/kg), can be easily isolated from fresh leaves of European Yew (Taxus baccate). It is also reported that Paclitaxel is 1000 times more potent compared to 10-deacetyl baccatin-lll and its higher activity is due to a C-13 side chain comprising N-benzoyl-(2f?,3S)-3-phenylisoserine moiety (Wani et al J Therefore synthesis of optically active (2S.3R) and (2R,3S)-phenylglycidates of formula 7 and 8 which are the key chiral precursors for synthesising N-benzoyl- (2R,3S)-3-phenylisoserine has become very vital for the development of a practical and efficient route to synthesise enantiomerically pure isoserine chain . There are number of reports on the preparation of these chiral intermediates by biochemical or chemoenzymatic methods besides asymmetric syntheses. Most ?f these reports are related to kinetic resolution of racemic 2-halo-3-hydroxy- phenylpropanoate through enzymatic hydrolysis ( H.Honig et al., Tetrahedron 46, 3841-3850, 1990; Peter G. M Wuts et al, Tetrahedron Asymmetry 11, 2117-2123, 2000), or by transesterification reaction ( Ching-Shih Chen et al., J. Org. Chem. 58, 1287-1289, 1993; Ching-Shih Chen et al, U .S. Pat. No. 6,020,174, to The Board of Governors for Higher Education, Rhodes Island; Marco Villa et al., U.S.Pat. No. 6,187,936 to Zambon Group S.p.A; Tanebe JP 06/078790) or via resolution of azetidinones (C.J Sih et al., J. Org. Chem. 58, 1068-1075, 1993; R.N. Patel et al, J. American Oil Chemists Society, 73, 1363-1375, 1996; R. A. Holton et al, WO 2001029245, EP 1222305 to Bristol-Myers Squibb; R.N. Patel et al., Biotechnology and Applied Biochemistry, 20, 23-33, 1994). From the literature review it is quite clear that no prior art is available on kinetic resolution of racemic 2-halo-3-hydroxy-3-phenylpropanoates where x represents bromo and iodo groups and R' represents C-1 to C-5 alky group. Therefore the use of bromo and iodohydrins via kinetic resolution route for the preparation of desired (2S,3R) and (2R,3S)-phenylglycidates i n essentially n ovel a nd h as n ot been reported in the literature or known in the art of their synthesis of taxol side chain precursors. The present invention therefore discloses the application of a lipase for the preparation of (2S,3R) and (2R,3S)-phenylglycidates via kinetic resolution of halohydrin intermediates x and R' are defined as above. Thus the main objective of the present invention is to synthesise optically active alkyl (2S,3R) and (2R,3S)-phenylglycidates of formulae 7 and 8 using chemoenzymatic approach through resolution of its racemic precursor trans alkyl 2-halo-3-hydroxy-3-phenylpropanoates of formula 2 where x represents bromine or iodine and R' represenrts C-1 to C-5 alkyl group. Advantage of using bromo and iodohydrin intermediates are that they are easily preparable in almost quantitative yields and are obtained in crystalline form. Moreover these compounds can be easily converted to corresponding epoxides (glycidates) by acid and base catalysed transformations in almost quantitative yields. Additionally use of lipase from Aspergillus niger makes the process of resolution of trans alkyl 2-halo-3-hydroxy-3-phenylpropanoates facile. Accordingly, the present invention relates to a stereoselective chemoenzymatic process for the preparation of optically enriched phenylglycidates i.e. alkyl(2S,3R)-phenylglycidate and alkyl(2R,3S)- phenylglycidate as precursors of taxol side chain. The steps involved are: a. Preparation of halohydrins of formula 2 where x represents bromine or iodine and R' represents C-1 to C-5 alkyl group, from corresponding alkyl cinnamates of formula 1 by the action of a halogenating agent. b. Chemical transformation of halohydrins of formula 2 to alkyl acylatesof formula 3 using an acyl anhydride in presence of a base. c. Incubating the trans alkyl 3-acyloxy-2-halo-3-phenylpropanoates of formula 3 with dry powder of the lipase from Aspergillus niger in an aqueous buffer phase in absence of an organic medium or in presence of an organic medium to facilitate the reaction. The bioresolution process is effected using the commercial enzyme lipase AMANO AS (Aspergillus niger) gifted by M/s AMANO, Japan and used directly without further purification or modification, d. Separating the hydrolysed alkyl(2R3R)-2-halo-3-hydroxy-3- phenylpropanoates of formula 4 and unhydrolysed alkyl(2S,3S)-3- acyloxy-2-halo-3-phenylpropanoates of formula 5 by conventional method of chromatography. e. Reacting the optically enriched hydrolysed alkyl(2ft,3/?)-2-halo-3-hydroxy- 3-phenylpropanoates of formula 4 with a base to furnish alkyl (2S,3R) phenylglycidates of formula 7. f. Reaction of unhydrolysed alkyl(2S,3S)-3-acyloxy-2-halo-3- phenylpropanoates of formula 5 with an inorganic or organic acid to furnish corresponding alkyl(2S,3S)-2-halo-3-hydroxy-3-phenylpropanoates of formula 6. Further reacting the optically enriched hydrolysed alkyl(2S,3S)-2-halo-3-hydroxy-3-phenylpropanoates of formula 6 with a base to furnish alkyl(2R,3S) phenylglycidates of formula 8. In a preferred embodiment of the process of preparation of halohydrins from alkyl :innamates of formula 1 to halohydrins of formula 2 in step 'a' of the process, the nalogenating agent is selected from N-halosuccinimide such as NDromosuccinimide, N-iodosuccinimide or sodium bromate, periodic acid, 1,3- Jibromo-5,5-dimethyl hydantoin, iodine, bromine and the like but more Dreferably sodium bromate for bromoh -Voxylation or periodic acid for ' odohydroxylation in an aqueous phase or in a mixed organic aqueous phase where organic solvent may be selected from water miscible solvents such as acetone, tetrahydrofuran, dioxane, acetonitrile and the like. The formation of halohydrins is carried out at a temperature in the range of 0-60°C more preferably at 30-40°C. In step 'b' of the transformation of compound of formula to acyiate of formula 3, the acylating agent is selected from acyl anhydrides such as acetic anhydride; propionic anhydride and butyric anhydride or corresponding acyl chlorides but most suitable is acetic anhydride in presence of bases like pyridine, N,N-dimethyl aminopyridine (DMAP) and more preferably DMAP. In step 'c' of the process of kinetic resolution of the compound of formula 3 is effected by incubating the compound of formula 3 in presence of crude lipase enzyme (AS AMANO) from Aspergillus niger either in a buffered aqueous phase alone or a buffered aqueous phase in presence of an organic medium as a cosolvent which facilitates the hydrolytic reaction. The pH of the buffer is suitably adjusted at 5-7.5, more suitably at 6-7.5 and most suitably at 7. The temperature of the reaction is selected at 10-40°C, but more suitably at 20-35°C and most suitably at 30°C. The preferred aqueous phase is water, phosphate buffer (0.1M to 0.2M) or an acetate buffer and the most preferred one is phosphate buffer (0.1M). The preferred cosolvents that are added in the ratio of 10-90% are hexane, toluene, dichloromethane, acetone, acetonitrile, dimethylformamide, dimethyl sulphoxide, methanol, ethanol and the like. The more preferable are toluene, acetone and acetonitrile and most preferable is acetonitrile. After the completion of the hydrolysis reaction in step 'd' of the process the separation of hydrolysed alcohol of formula 4 and unhydrolysed ester of formula 5 is effected by column chromatography on silica gel columns by conventional chromatographic methods. In step 'e' of the process of base catalysed conversion of the compound of the formula 4 (hydrolysed ester) to alkyl(2S,3R)-phenylglycidates of the formula 7, is effected by an inorganic or organic base such as sodium hydroxide, sodium carbonate and the like and organic bases are selected from triethylamine, piperidine,1,4-diazabicyclo[2,2,2]octane (DABCO), 1,8-diazabicyclo[5,4,0]undec- 7-ene (UBU) and the like, the more preferred base is DBU. In step "f" of the process of acid catalysed conversion of the compound alkyl (2S,3S)-3-acyloxy-2-halo-3-phenylpropanoates of the formula 5 (unhydrolysed ester) first to alkyl(2S,3S)-2-halo-3-hydroxy-3-phenylpropanoates of formula 6 is effected by mineral acid such as hydrochloric acid , sulfuric acid or an organic or lewis acid such as trifluoro acetic acid, boron trifiuoride (BFs) in an organic solvent such as diethyl ether, methanol, acetone and the like but the preferred acidic conditions are 1N, 2N and 5N HCI , most preferred is 2N HCI in methanol, finally transformation to alkyl(2R,3S)-phenylglycidates of the formula 8 is effected by a base as described in step 'e". The invention is described herein with reference to the examples given below. These examples should not be construed as to restrict the scope of this invention. Step 'a' Example (i) Synthesis of (i)-methyl 2-bromo-3-hydroxy-3-phenylpropanoate of formula 2 where x=Br and R'= CH3 Potassium bromate (4g, 24 mmol), was dissolved in 40 ml water and adjusted to pH 1-2 with 2M H2SO4. To the resultant solution was added methyl cinnamate (3g, 20 mmol) in 40ml acetonitrile. 1M sodium bisulphite solution (5.2g in 50ml) was added to the above mixture over a period of two hours with stirring and at 40°C. The reaction mixture was further stirred for 36 hrs till the reaction was complete (TLC monitored). The resulting solution was extracted with ethyl acetate (3x100ml), and combined organic layer was washed with aqueous sodium sulphite followed by drying over anhydrous sodium sulphate. The contents concentrated in vacuo to give a crude material, which was purified by crystallization (benzene: hexane, 1:1) to furnish compound of formula 2 m.pt 63°C (yield 70%). 1HNMR (CDCI3) 6: 7.37(5H, s, Ar-H), 5.08(1 H, d, J=8.25 Hz, CH-OH), 4.38(1 H, d, 7=8.24 Hz, CH-Br), 3.80(3H, s, COOCH3). Example (ii) Synthesis of (±)-ethyl 2-bromo-3-hydroxy-3-phenylpropanoate of formula 2 where x=Br and R'= C2H5. It was prepared from ethyl cinnamate (3.26g, 20 mmol) potassium bromate (4g, 24 mmo!) following the procedure given in example step 'a' (i), m.pt 76-77°C, yield 3.7g, (70%). 1HNMR(CDCI3) 5: 7.38(5H, s, Ar-H), 5.08(1 H, d, J =8.29 Hz, CH-OH), 4.54(1 H, d, J =8.28 Hz, CH-Br), 4.25(2H, q, J=7.11 Hz, CH2), 1.25(3H, t, J=7.12 Hz CH3). Example (Mi) Synthesis of (±)-methyl 3-hydroxy-2-iodo-3-phenylpropanoate of formula 2 where x=l and R'= CH3. To a stirred suspension of methyl cinnamate(3g, 20 mmol), HIO4.2H2O (5.2g, 24 mmol) 12 ml water and 40 ml of acetonitrile, 1M sodium bisulphite solution (5.2g in 50ml) was added to the above mixture over a period of three-four hours with stirring and at 30°C. The reaction mixture was further stirred for 36 hrs till the reaction was complete (TLC monitored). The resulting solution was extracted with ethyl acetate (3x50ml), and combined organic layer was washed with aqueous sodium sulphite followed by drying over anhydrous sodium sulphate. The contents concentrated in vacuo to give a crude material, which was purified by crystallization (benzene: hexane, 1:1) to furnish compound of formula 2 m.pt 63°C, yield 3g (65%). 1HNMR(CDCI3) 8: 7.35(5H, s, Ar-H), 5.05(1 H, d, J=8.42 Hz, CH-OH), 4.55(1 H, d, J =8.43 Hz, CH-Br), 3.75(3H, s, CH3). Example (iv) Synthesis of (±)-ethyl 3-hydroxy-2-iodo-3-phenylpropanoate of formula 2 where x=l and R'= C2H5. It was prepared from ethyl cinnamate.(3.26g, 20 mmol), HIO4.2H2O (5.2g, 24 mmol) following the procedure given in example step 'a' (iii), yield 4g (77%), m.pt 79°C. 1HNMR(CDCI3) 6: 7.38(5H, s, Ar-H), 5.05(1 H, d, J=8.29 Hz, CH-OH), 4.55(1 H, d, J=8.29 Hz, CH-Br), 4.25(2H, q, J=7.11 Hz, Chfc), 1.25(3H, t, J=7.12 Hz, CH2CH3). Step 'b' Example (i) Synthesis of (±)-methyl 3-acetoxy-2-bromo-3-phenylpropanoate of formula 3 where x=Br and R'= CH3. A solution of 2 (2.59g, 10mmol) and acetic anhydride (12mmol) and dimethyl N,N-dimethyl aminopyridine (DMAP) (in catalytic amount) in 10 ml of dry dichloromethane was kept overnight at room temp. The reaction mixture was poured i nto i ce-cold w ater a nd e xtracted w ith d ichloromethane (3x100ml). T he organic layer was washed, dried, and evaporated to furnish compound of formula 3, which was purified by column chromatography (silica gel, ethyl acetate:hexane;3:97), in 90-95% yield, m.pt. 56°C 1HNMR (CDCI3)6: 7.45(5H, s, Ar-H), 6.23(1 H, d, J=10.00 Hz, CH-OAc), 4.58(1 H, d, J=10.00 Hz, CH-Br), 3.83(3H, s, COOCH3), 2.03(3H, s, OCOCH3). Example (ii) Synthesis of (±)-ethyl 3-acetoxy-2-bromo-3-phenylpropanoate of formula 3 where x=Br and R'= C2H5. It was prepared from 2 (2.73g, 10mmol) and acetic anhydride (12mmol) and N,Ndimethyl aminopyridine (DMAP), in catalytic amount) following the procedure given in step 'b' example (i) in 90-95% yield. 1HNMR (CDCI3)6: 7.50(5H, s, Ar-H), 6.23(1 H, d, J=10.50 Hz, CH-OAc), 4.53(11-1, d, J=10.50 Hz, CH-Br), 4.33(2H, q, J=7.11 Hz Chb), 2.06(3H, s, OCOCH3), 1.33(3H,t, J=7.12HzCH2CH3). Example (iii) Synthesis of (i)-methyl 3-acetoxy-2-iodo 2»phenylpropanoate of formula 3 where x=l and R'= CH3. It was prepared from 2 (3.06g, 10mmol) and acetic anhydride (12mmol) and N,Ndimethyl aminopyridine (DMAP), in catalytic amount) following the procedure given in step 'b' example (i) in 90-95% yield, m.pt, 58°C. 1HNMR(CDCI3) 5: 7.40(5H, s, Ar-H), 6.15(1 H, d, J=10.75 Hz, CH-OAc), 4.62(1H, d, J=10.75 Hz, CH-Br), 3.17(3H, s, COOChb), 2.00(3H, s, CH3). Example (iv) Synthesis of (±)-ethyl 3-acetoxy-2-iodo-3-phenylpropanoate of formula 3 where x=l and R'= C2H5. It was prepared from 2 (3.19g, 10mmol) and acetic anhydride (12mmol) and N,Ndimethyl aminopyridine (DMAP), in catalytic amount) following the procedure given in step 'b' example (i) in 90-95% yield. 1HNMR(CDCI3) 6: 7.50(5H, s, Ar-H), 6.15(1H, d, J=10.50 Hz, CH-OAc), 4.62(1H, d, J=10.50 Hz, CH-Br), 4.25(2H, q, J=7.11 Hz COOCH2), 2.06(3H, s, OCOCH3), 1.25(3H, t, J=7.12 Hz CH2CH3). Step 'c} Example (i) Preparation of (-)-methyl (2ft,3R)-2-bromo-3-hydroxy-3-phenylpropanoate of formula 4 by kinetic resolution, where x-Br and R'= CH3. (±)-Methyl 3-acetoxy-2-bromo-3-phenylpropanoate (SOOmg) of formula 3 where x=Br and R'= CH3, was added to biphasic system of aqueous phosphate buffer (16ml, 0.1M. pH 7.0) and toluene (1.6ml). To the above solution was added crude dry powder of lipase Aspergillus niger (Amano AS, 400mg, 12-15 units/mg) with the continuous stirring and maintaining pH 7.0 by addition of 0.5N sodium hydroxide solution. During the course of the reaction temperature was maintained at 30°C. The progress of the reaction was monitored after every six hours by TLC and HPLC. After completion of the reaction (48 hrs., approx., 43% conversion), the reaction was terminated by centrifuging the mixture at 10,000 to 15,000g to remove enzyme and the suspended particles. The clear solution and the centrifuged mass was extracted separately with ethyl acetate (3 x 20ml). The organic layer was combined and washed with water. The combined solvent layer was then dried and evaporated under reduced pressure to furnish a mixture comprising hydrolysed alcohol and unhydrolysed ester which were separated by column chromatography over silica gel using hexane:ethyl acetate (97:3) as eluent to furnish (-)-methyl (2f?,3R)-2-bromo-3-hydroxy-3-phenylpropanoate of formula 4 (250mg, 85%) having enantiomeric purity (EE)92%, [a]D 25 -19.7° (c,1,CHCI3) and unhydroiysed ester (+)-methyl (2S,3S)-3-acetoxy-2-bromo-3- phenyipropanoate of formula 5 (378mg, 83%) having enantiomeric purity (EE)70% (chiral HPLC), [a]D 25 +36.4° (c,1,CHCI3). Example (ii) Preparation of (-)-ethyl (2/?,3/?)-2-bromo-3-hydroxy-3-phenylpropanoate of formula 4 by kinetic resolution, where x=Br and R'= C2H5. It was prepared from (±)-ethyl 3-acetoxy-2-bromo-3-phenylpropanoate (800mg) of formula 3, phosphate buffer (16ml, 0.1M. pH 7.0), toluene (1.6ml) and crude dry powder of lipase Aspergillus niger (Amano AS, 400mg, 12-15 units/mg) following the procedure given in step 'c' example (i). After 48 hrs. (45% conversion) the hydrolyzed alcohol was obtained (256mg, 82%), having enantiomeric purity (EE) 86%, [a]D 25 -14.8° (c,1,CHCI3) and unhydrolysed ester (+)-ethyl (2S,3S)-3-acetoxy-2-bromo-3-phenylpropanoate of formula 5 (375mg, 85%) having enantiomeric purity (EE)73%(chiral HPLC), [a]D 25 +37.5° (c,1,CHCI3). Example (iii) Preparation of (-)-methyl (2/?,3/?)-3-hydroxy-2-iodo-3-phenylpropanoate of formula 4 by kinetic resolution, where x=l and R'= CH3. It was prepared from (t)-Methyl 3-acetoxy-2-iodo-3-phenylpropanoate (800mg) of formula 3, phosphate buffer (16ml, 0.1M. pH 7.0), toluene (1.6ml) and crude dry powder of lipase Aspergillus niger (Amano AS, 400mg, 12-15 units/mg) following the procedure given in step 'c' example (i). After 36 hrs. (44% pnversion) the hydrolyzed alcohol was obtained (274mg, 88%), having enantiomeric purity (EE) 94%, [a]D 25 0.0° (c,1,CHCI3); -3.0° (c 1, MeOH) and unhydrolysed ester (+)-methyl-(2S,3S)-3-acetoxy-2-iodo-3-phenylpropanoate of formula 5 (415mg, 92%) having enantiomeric purity (EE)76%(chiral HPLC), [a]D +48.0°(c,1,CHCI3). Example (iv) Preparation of (-)-ethyl (2R,3R)-3-hydroxy-2-iodo-3-phenylpropanoate of formula 4 by kinetic resolution, where x=l and R'= C2H5. It was prepared from (±)-Ethyl 3-acetoxy-2-iodo-3-phenylpropanoate (800mg) of formula 3, phosphate buffer (16ml, 0.1M. pH 7.0), toluene (1.6ml) and crude dry powder of lipase Aspergillus niger (Amano AS, 400mg, 12-15 units/mg) following the procedure given in step 'c' example (i). After 40 hrs. (40% conversion) the hydrolyzed alcohol was obtained (250mg, 88%), having enantiomeric purity(EE)95%, [a]D 25 -9.3° (c,1 ,CHCI3) and unhydrolysed ester (+)-ethyl(2S,3S)- 3-acetoxy-2-iodo-3-phenylpropanoate of formula 5 (440mg, 91%) having enantiomeric purity (EE)60%(chiral HPLC), [a]D 25 +36.0° (c,1,CHCI3). Example (v) Preparation of (+)-methyl (2S,3S)-3-acetoxy-2-bromo-3-phenylpropanoate of formula 5 by double kinetic resolution, where x=Br and R'= CH3. It was prepared from optically enriched (+)-Methyl (2S,3S)-3-acetoxy-2-bromo-3- phenylpropanoate (EE, 70%, 350mg) of formula 5, phosphate buffer (7ml, 0.1M. pH 7.0), toluene (0.7ml) and crude dry powder of lipase Aspergillus niger (Amano AS, 100mg, 12-15 units/mg) following the procedure given in step 'c' example (j). After completion of the reaction (72 hrs.), the unhydrolyzed ester on separation (290mg) was found to have enantiomeric excess (EE)>99%(chiral HPLC), [a]D 25 +52.0°(c,1,CHCI3) Example (vi) Preparation of (+)-ethyl (2S,3S)-3-acetoxy-2-bromo-3-phenylpropanoate of formula 5 by double kinetic resolution, where x=Br and R'= C2H5. It was prepared from optically enriched ethyl (2S,3S)-3-acetoxy-2-bromo-3- phenylpropanoate (EE 73%, 350mg) of formula 3, phosphate buffer (7ml, 0.1M. pH 7.0), toluene (0.7ml) and crude dry powder of lipase Aspergillus niger (Amano AS, 100mg, 12-15 units/mg) following the procedure given in step 'c' example (i). After 65 hrs the unhydrolyzed ester on separation (290mg) was found to have enantiomeric excess (EE)98%(chiral HPLC), [a]D 25 +50.5° (c,1, CHCI3). Example (vii) Preparation of (+)-methyl (2S,3S)-3-acetoxy-2-iodo-3-phenylpropanoate of formula 5 by double kinetic resolution, where x=l and R'= CH3. It was prepared from optically enriched methyl (2S,3S)-3-acetoxy-2-iodo-3- phenylpropanoate (EE 76%, 350mg) of formula 3, phosphate buffer (7ml, 0.1M. pH 7.0), toluene (0.7ml) and crude dry powder of lipase Aspergillus niger (Amano AS, 100mg, 12-15 units/mg) following the procedure given in step 'c' example (i). The unhydrolyzed ester of formula 5 (SOOmg) was found to have enantiomeric excess (EE)94%(chiral HPLC), [a]D 25 +59.0° (c,1, CHCI3). Example (viii) Preparation of (+)-ethyl (2S,3S)-3-acetoxy-2-iodo-3-phenylpropanoate of formula 5 by double kinetic resolution, where x=l and R'= C2H5. It was prepared from optically enriched methyl (2S,3S)-3-acetoxy-2-iodo-3- phenylpropanoate (EE 60%, 350mg) of formula 3, phosphate buffer (7ml, 0.1M. pH 7.0), toluene (0.7ml) and crude dry powder of lipase Aspergillus niger Amano AS (100mg, 12-15 units/mg) The unhydrolyzed ester (315mg)(72hrs.) was found to have enantiomeric excess (EE)73%(chiral HPLC), [a]D 25 +43.5° (c,1, CHCI3). Example (ix) Preparation of (-)-methyl (2R,3R)-2-iodo-3-hydroxy-3-phenylpropanoate of formula 4 by kinetic resolution in presence of a cosolvent acetonitrile, where x=l and R'= CH3. (±)-Methyl 3-acetoxy-2-iodo-3-phenylpropanoate (200mg) of formula 3 was added to biphasic system of aqueous phosphate buffer (3.6ml, 0.1M. pH 7.0) and acetonitrile (0.4ml). To the above solution crude dry powder of lipase Aspergillus n/ger (Amano AS, 100mg, 12-15 units/mg) was added with the continuous stirring and maintaining pH 7.0 by addition of 0.5N sodium hydroxide solution. During the course of the reaction temperature was maintained at 30°C. The progress of the reaction was monitored after every six hours. After the completion of the reaction (9 hrs., approx., 43% conversion), the reaction was terminated by centrifuging the mixture at 10,000 to 15,000g to remove enzyme and the suspended particles. The clear solution and the centrifuged mass was extracted separately with ethyl acetate (3X30 ml). The organic layer was combined and washed with water. The combined solvent layer was then dried and evaporated under reduced pressure to furnish a mixture comprising hydrolysed alcohol and unhydrolysed ester which were separated by column chromatography over silica gel using hexane: ethyl acetate (97:3) as eluent to furnish methyl (2R,3R)-3-hydroxy-2-iodo-3- phenylpropanoate of formula 4 (65mg, 85%) having enantiomeric purity (EE)92% and unhydrolysed ester methyl (2S,3S)-3-acetoxy-2-iodo-3-phenylpropanoate of formula 5 ( 92 mg, 77%), enantiopurity (EE) 62% determined by chiral HPLC. Step 'e' Example (i) Preparation o f (+)-methyl (2S,3R)-phenylglycidate o f t he f ormula 7 where 0.2 ml of 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) was added to a solution of optically enriched halohydrin (200 mg) of formula 4 in methanol (4ml) at 20° C for 5 minutes. The solvent was removed under reduced pressure and the reaction mixture was diluted with 10 ml water and extracted with ethyl acetate and solvent removed to furnish compound of formula 7; recovery 98mg, [a]D 25 +155.0° (c,1, CHCI3) Step 'f Example (i) Preparation of (+)-methyl (2S,3S)-2-halo-3-hydroxy-3-phenylpropanoate of formula 6 where R'= CH3 Alkyl (2S,3S)-3-acetoxy-2-holo-3-phenylpropanoate of formula 5 (200mg) was dissolved in 4ml of methanol and 0.2ml of 2N HCI was added and the reaction mixture was stirred at room temperature for 24 hrs till the complete conversion of the compound of formula 5 to the compound of the formula 6. Excess of solvent was evaporated and the reaction mixture was diluted with 10 ml water and extracted with ethyl acetate, and solvent removed to furnish compound of formula 6, recovery 150mg. Example (ii) Preparation of (-)-methyl (2R,3S)-phenylglycidates of the formula 8 where R'= CH3 It was prepared from 6 following the procedure given in step 'f example (i) for compound 7. [a]D 25 -153.0° (c,1, CHCI3) We claim, 1. A stereoselective chemoenzymatic process for the preparation of optically enriched phenylglycidates i.e. alkyl(+2S,3R)- phenylglycidate and alkyI(-2R,3S)- phenylglycidate as precursors of taxol side chain,wherein the said process comprises a. halohydroxylation reaction of 3-phenylpropanoate having C-1 to C-5 alkyl group to form trans 2-halo-3- hydroxyl-3-phenylpropanoates at a temperature between 0-60°C, wherein the halogenating agent used is N- halosuccinimide such as herein described. b. converting trans 2-halo-3- hydroxyl-3-phenylpropanoates to (±)- alkyl-3-acetoxy-2- halo-3-phenylpropanoates by an acylating agent such as herein described. c. substantially incubating the acyl derivatives of alkyl-3-acetoxy-2-halo-3- phenylpropanoates with crude dry powder of lipase from Aspergillus niger in an aqueous buffer phase in presence of an organic solvent, d. thereafter separating the hydrolysed halohydrins i.e. alkyl(2R,3R)- 2-halo-3- hydroxy- 3-phenylpropanoates and unhydrolysed ester i.e. alkyl(2S,3S)- 2-halo-3- acyloxy-3- phenylpropanoates from the mixture by conventional method, e.if required, then again incubating the optically enriched alkyl(2S,3S)- 2-halo-3-acyIoxy-3-phenylpropanoates with crude dry powder of lipase of Aspergillus niger in an aqueous buffer phase in presence of an organic solvent to further improve the enantiopurity, f reacting optically enriched (2S,3S)- 2-halo-3- acyloxy-3-phenylpropanoates with an acid to furnish optically enriched alkyl(2S,3S)- 2-halo-3- hydroxy-3-phenylpropanoates, g. treating the compounds alkyl(2R,3R)- 2-halo-3- hydroxy-3-phenylpropanoates and alkyl(2S,3S)- 2-halo-3- hydroxy-3-phenylpropanoates with an alkali in an organic or aqueous phase to furnish optically enriched (+)-methyl (2S,3R)-phenylglycidate and (-)-methyl (2S,3R)-phenylglycidate . 2. A novel process as claimed in claim 1 step(a), N-halosuccinimide used is N-bromosuccinimide, N-iodosuccinimide or sodium bromate, periodic acid, 1,3-dibromo-5,5-dimethyl hydantoin, iodine and bromine. 3. A process as claimed in claim 1, wherein the halohydroxilation process of step(a) is effected in aqueous or in an organic or aqueous organic phase wherein the organic phase is selected from water miscible solvents such as acetone, tetrahydrofuran, dioxane, dimethylformamide and methanol. 4. A novel process as claimed in claim 1, wherein for the incubation of alkyl acrylates ,the aqueous phosphate buffer is phosphate tris buffer and acetic acid having pH in the range of 5-7.5 and temperature 10-40°C. 5. A process as claimed in claims 1 wherein organic solvent used are hexane, toluene, dicholoromethane ,acetone, acetonitrile, dimethylformamide, dimethylsulphoxide, methanol and ethanol at 10-90% concentration. 6. A process as claimed in step(f) of claim 1, wherein the acid used to furnish optically enriched alkyl(2S,3S)- 2-halo-3- hydroxy-3-phenylpropanoates is an organic or inorganic acid such as hydrochloric acid .sulfuric acid, trifluoro acetic acid and boron trifluoride. 8. A process as claimed in step(g) of claim 1, wherein alkali used to furnish optically enriched (+)-methyl (2S,3R)-phenylglycidate and (-)-methyl (2S,3R)-phenylglycidate is organic or inorganic base selected from the group consisting of sodium hydroxide, sodium carbonate and triethylamine. 10. A process for the preparation of alkyl(+2S,3R)- phenylglycidate and alkyl(-2S,3R)-phenylglycidate shown in the drawing accompanying the specification substantially as herein described with refererence to examples.

Full Text

The present invention relates to a stereoselective chemoenzymatic process for the preparation of optically enriched phenylglycidates i.e. alkyl(2S,3R)- phenylglycidate and alkyl(2R,3S)- phenylglycidate as precursors of taxol side chain.The invention particularly discloses a novel process for the chemoenzymatic synthesis of two enantiomers of trans alkyl phenylglycidate i.e.alkyl (2R,3S)- phenylglycidate and aikyi(2R,3S)-phenylglycidate of formulae 7 and 8 respectively, process of their synthesis comprises cohalogenation reaction of alkyl cinnamate of formula 1 where R' represents C-1 to C-5 alkyl group to form trans 2-halo-3-hydroxy-3-phenylpropanoates of formula 2 where x represents Br or I, then converting the halohydrins of formula 2 to corresponding alkyl acylates of formula 3 where x and R' represents as above, subsequently incubating the acyl derivatives of alkyl 2-halo-3-hydroxy-3-phenylpropanoates of formula 3 with crude dry powder of lipase f,om Aspergillus niger in an aqueous buffer phase in presence of an organic solvent, thereafter separating the hydrolysed halohydrins i.e. alky!(2R,3R) 2-haio-3-hydroxy-3-phenylpropanoates of formula 4 and unhydrolysed ester i.e. alkyl (2S,3S) 2-halo-3-acyloxy-3-phenylpropanoates of formula 5 from the mixture by conventional method, if required then again incubating the optically enriched acyl derivatives of formula 5 with crude dry powder of lipase from Aspergillus niger in an aqueous buffer phase in presence of an organic solvent to further improve the enantiopurity, followed by reaction of the optically enriched products of formula 5 with an acid to furnish optically enriched alkyl (2S.3S) 2-halo-3-hydroxy-3-phenylpropanoate of formula 6 and finally treating the compounds of formulae 4 and 6 with an alkali in an organic or aqueous phase to furnish optically enriched alkyl(2S,3R)-phenylglycidate and alkyl(2R,3S)-phenylglycidate of formulae 7 and 8 respectively .
Use of lipase from Aspergillus niger for the kinetic resolution of trans alkyl 2-halo-3-hydroxy-3-phenylpropanoates where x and R' represents as above is novel as also the step of acid followed by a base catalysed reaction for the cyclisation of resolved esters of trans alkyl 2-halo-3-hydroxy-3-phenylpropanoates of formula 4 and 6, not reported in the art of its synthesis. Optically enriched alkyl(2S,3R)-phenylglycidate and alkyl(2R3S)-phenyl-glycidatd of formulae 7 and 8 are the key intermediates used in the synthesis of N-benzoyl-(2R,3S)-3-phenylisoserine (Taxol side chain). Paclitaxel or Taxol

which was isolated from the bark of the Pacific Yew (Taxus 5rewfo//a)(Wani et
Taxol 10-Deacetyl baccatin-lll Isoserine
various types of cancers( Holmes et al J. Natl. Cancer Inst. 83, 1797-1805,1991).
Despite being one of the most promising anti-cancer drugs, its very low
occurrence (40-165 mg/kg) in nature is the main hindrance in its production.
Fortunately it has been found that 10-deacetyl baccatin-lll which is structurally
closely related to Taxol and occurs in comparatively higher concentrations
(approx. 1g/kg), can be easily isolated from fresh leaves of European Yew
(Taxus baccate). It is also reported that Paclitaxel is 1000 times more potent
compared to 10-deacetyl baccatin-lll and its higher activity is due to a C-13 side
chain comprising N-benzoyl-(2f?,3S)-3-phenylisoserine moiety (Wani et al J
Therefore synthesis of optically active (2S.3R) and (2R,3S)-phenylglycidates of
formula 7 and 8 which are the key chiral precursors for synthesising N-benzoyl-
(2R,3S)-3-phenylisoserine has become very vital for the development of a
practical and efficient route to synthesise enantiomerically pure isoserine chain .
There are number of reports on the preparation of these chiral intermediates by
biochemical or chemoenzymatic methods besides asymmetric syntheses. Most
?f these reports are related to kinetic resolution of racemic 2-halo-3-hydroxy-
phenylpropanoate through enzymatic hydrolysis ( H.Honig et al., Tetrahedron 46,
3841-3850, 1990; Peter G. M Wuts et al, Tetrahedron Asymmetry 11, 2117-2123, 2000), or by transesterification reaction ( Ching-Shih Chen et al., J. Org. Chem. 58, 1287-1289, 1993; Ching-Shih Chen et al, U .S. Pat. No. 6,020,174, to The Board of Governors for Higher Education, Rhodes Island; Marco Villa et al., U.S.Pat. No. 6,187,936 to Zambon Group S.p.A; Tanebe JP 06/078790) or via resolution of azetidinones (C.J Sih et al., J. Org. Chem. 58, 1068-1075, 1993; R.N. Patel et al, J. American Oil Chemists Society, 73, 1363-1375, 1996; R. A. Holton et al, WO 2001029245, EP 1222305 to Bristol-Myers Squibb; R.N. Patel et al., Biotechnology and Applied Biochemistry, 20, 23-33, 1994). From the literature review it is quite clear that no prior art is available on kinetic resolution of racemic 2-halo-3-hydroxy-3-phenylpropanoates where x represents bromo and iodo groups and R' represents C-1 to C-5 alky group. Therefore the use of bromo and iodohydrins via kinetic resolution route for the preparation of desired (2S,3R) and (2R,3S)-phenylglycidates i n essentially n ovel a nd h as n ot been reported in the literature or known in the art of their synthesis of taxol side chain precursors.
The present invention therefore discloses the application of a lipase for the preparation of (2S,3R) and (2R,3S)-phenylglycidates via kinetic resolution of halohydrin intermediates x and R' are defined as above.
Thus the main objective of the present invention is to synthesise optically active alkyl (2S,3R) and (2R,3S)-phenylglycidates of formulae 7 and 8 using chemoenzymatic approach through resolution of its racemic precursor trans alkyl 2-halo-3-hydroxy-3-phenylpropanoates of formula 2 where x represents bromine or iodine and R' represenrts C-1 to C-5 alkyl group. Advantage of using bromo and iodohydrin intermediates are that they are easily preparable in almost quantitative yields and are obtained in crystalline form. Moreover these compounds can be easily converted to corresponding epoxides (glycidates) by acid and base catalysed transformations in almost quantitative yields. Additionally use of lipase from Aspergillus niger makes the process of resolution of trans alkyl 2-halo-3-hydroxy-3-phenylpropanoates facile. Accordingly, the present invention relates to a stereoselective chemoenzymatic process for the preparation of optically enriched phenylglycidates i.e. alkyl(2S,3R)-phenylglycidate and alkyl(2R,3S)- phenylglycidate as precursors of taxol side chain. The steps involved are:

a. Preparation of halohydrins of formula 2 where x represents bromine or
iodine and R' represents C-1 to C-5 alkyl group, from corresponding alkyl
cinnamates of formula 1 by the action of a halogenating agent.
b. Chemical transformation of halohydrins of formula 2 to alkyl acylatesof
formula 3 using an acyl anhydride in presence of a base.
c. Incubating the trans alkyl 3-acyloxy-2-halo-3-phenylpropanoates of
formula 3 with dry powder of the lipase from Aspergillus niger in an
aqueous buffer phase in absence of an organic medium or in presence of
an organic medium to facilitate the reaction. The bioresolution process is
effected using the commercial enzyme lipase AMANO AS (Aspergillus
niger) gifted by M/s AMANO, Japan and used directly without further
purification or modification,
d. Separating the hydrolysed alkyl(2R3R)-2-halo-3-hydroxy-3-
phenylpropanoates of formula 4 and unhydrolysed alkyl(2S,3S)-3-
acyloxy-2-halo-3-phenylpropanoates of formula 5 by conventional method
of chromatography.
e. Reacting the optically enriched hydrolysed alkyl(2ft,3/?)-2-halo-3-hydroxy-
3-phenylpropanoates of formula 4 with a base to furnish alkyl (2S,3R)
phenylglycidates of formula 7.
f. Reaction of unhydrolysed alkyl(2S,3S)-3-acyloxy-2-halo-3-
phenylpropanoates of formula 5 with an inorganic or organic acid to
furnish corresponding alkyl(2S,3S)-2-halo-3-hydroxy-3-phenylpropanoates
of formula 6. Further reacting the optically enriched hydrolysed
alkyl(2S,3S)-2-halo-3-hydroxy-3-phenylpropanoates of formula 6 with a
base to furnish alkyl(2R,3S) phenylglycidates of formula 8.
In a preferred embodiment of the process of preparation of halohydrins from alkyl
:innamates of formula 1 to halohydrins of formula 2 in step 'a' of the process, the
nalogenating agent is selected from N-halosuccinimide such as NDromosuccinimide,
N-iodosuccinimide or sodium bromate, periodic acid, 1,3-
Jibromo-5,5-dimethyl hydantoin, iodine, bromine and the like but more
Dreferably sodium bromate for bromoh -Voxylation or periodic acid for '
odohydroxylation in an aqueous phase or in a mixed organic aqueous phase
where organic solvent may be selected from water miscible solvents such as
acetone, tetrahydrofuran, dioxane, acetonitrile and the like. The formation of
halohydrins is carried out at a temperature in the range of 0-60°C more
preferably at 30-40°C. In step 'b' of the transformation of compound of formula
to acyiate of formula 3, the acylating agent is selected from acyl anhydrides such
as acetic anhydride; propionic anhydride and butyric anhydride or corresponding
acyl chlorides but most suitable is acetic anhydride in presence of bases like
pyridine, N,N-dimethyl aminopyridine (DMAP) and more preferably DMAP.
In step 'c' of the process of kinetic resolution of the compound of formula 3 is
effected by incubating the compound of formula 3 in presence of crude lipase
enzyme (AS AMANO) from Aspergillus niger either in a buffered aqueous phase
alone or a buffered aqueous phase in presence of an organic medium as a
cosolvent which facilitates the hydrolytic reaction. The pH of the buffer is suitably
adjusted at 5-7.5, more suitably at 6-7.5 and most suitably at 7. The temperature
of the reaction is selected at 10-40°C, but more suitably at 20-35°C and most
suitably at 30°C. The preferred aqueous phase is water, phosphate buffer (0.1M
to 0.2M) or an acetate buffer and the most preferred one is phosphate buffer
(0.1M). The preferred cosolvents that are added in the ratio of 10-90% are
hexane, toluene, dichloromethane, acetone, acetonitrile, dimethylformamide,
dimethyl sulphoxide, methanol, ethanol and the like. The more preferable are
toluene, acetone and acetonitrile and most preferable is acetonitrile.
After the completion of the hydrolysis reaction in step 'd' of the process the
separation of hydrolysed alcohol of formula 4 and unhydrolysed ester of formula
5 is effected by column chromatography on silica gel columns by conventional
chromatographic methods.
In step 'e' of the process of base catalysed conversion of the compound of the
formula 4 (hydrolysed ester) to alkyl(2S,3R)-phenylglycidates of the formula 7, is
effected by an inorganic or organic base such as sodium hydroxide, sodium
carbonate and the like and organic bases are selected from triethylamine,
piperidine,1,4-diazabicyclo[2,2,2]octane (DABCO), 1,8-diazabicyclo[5,4,0]undec-
7-ene (UBU) and the like, the more preferred base is DBU.
In step "f" of the process of acid catalysed conversion of the compound alkyl
(2S,3S)-3-acyloxy-2-halo-3-phenylpropanoates of the formula 5 (unhydrolysed
ester) first to alkyl(2S,3S)-2-halo-3-hydroxy-3-phenylpropanoates of formula 6 is
effected by mineral acid such as hydrochloric acid , sulfuric acid or an organic or
lewis acid such as trifluoro acetic acid, boron trifiuoride (BFs) in an organic
solvent such as diethyl ether, methanol, acetone and the like but the preferred
acidic conditions are 1N, 2N and 5N HCI , most preferred is 2N HCI in methanol,
finally transformation to alkyl(2R,3S)-phenylglycidates of the formula 8 is effected
by a base as described in step 'e".
The invention is described herein with reference to the examples given below.
These examples should not be construed as to restrict the scope of this
invention.
Step 'a'
Example (i)
Synthesis of (i)-methyl 2-bromo-3-hydroxy-3-phenylpropanoate of formula
2 where x=Br and R'= CH3
Potassium bromate (4g, 24 mmol), was dissolved in 40 ml water and adjusted to
pH 1-2 with 2M H2SO4. To the resultant solution was added methyl cinnamate
(3g, 20 mmol) in 40ml acetonitrile. 1M sodium bisulphite solution (5.2g in 50ml)
was added to the above mixture over a period of two hours with stirring and at
40°C. The reaction mixture was further stirred for 36 hrs till the reaction was
complete (TLC monitored). The resulting solution was extracted with ethyl
acetate (3x100ml), and combined organic layer was washed with aqueous
sodium sulphite followed by drying over anhydrous sodium sulphate. The
contents concentrated in vacuo to give a crude material, which was purified by
crystallization (benzene: hexane, 1:1) to furnish compound of formula 2 m.pt
63°C (yield 70%).
1HNMR (CDCI3) 6: 7.37(5H, s, Ar-H), 5.08(1 H, d, J=8.25 Hz, CH-OH), 4.38(1 H, d,
7=8.24 Hz, CH-Br), 3.80(3H, s, COOCH3).
Example (ii)
Synthesis of (±)-ethyl 2-bromo-3-hydroxy-3-phenylpropanoate of formula 2
where x=Br and R'= C2H5.
It was prepared from ethyl cinnamate (3.26g, 20 mmol) potassium bromate (4g,
24 mmo!) following the procedure given in example step 'a' (i), m.pt 76-77°C,
yield 3.7g, (70%).
1HNMR(CDCI3) 5: 7.38(5H, s, Ar-H), 5.08(1 H, d, J =8.29 Hz, CH-OH), 4.54(1 H,
d, J =8.28 Hz, CH-Br), 4.25(2H, q, J=7.11 Hz, CH2), 1.25(3H, t, J=7.12 Hz CH3).
Example (Mi)
Synthesis of (±)-methyl 3-hydroxy-2-iodo-3-phenylpropanoate of formula 2
where x=l and R'= CH3.
To a stirred suspension of methyl cinnamate(3g, 20 mmol), HIO4.2H2O (5.2g, 24
mmol) 12 ml water and 40 ml of acetonitrile, 1M sodium bisulphite solution (5.2g
in 50ml) was added to the above mixture over a period of three-four hours with
stirring and at 30°C. The reaction mixture was further stirred for 36 hrs till the
reaction was complete (TLC monitored). The resulting solution was extracted
with ethyl acetate (3x50ml), and combined organic layer was washed with
aqueous sodium sulphite followed by drying over anhydrous sodium sulphate.
The contents concentrated in vacuo to give a crude material, which was purified
by crystallization (benzene: hexane, 1:1) to furnish compound of formula 2 m.pt
63°C, yield 3g (65%).
1HNMR(CDCI3) 8: 7.35(5H, s, Ar-H), 5.05(1 H, d, J=8.42 Hz, CH-OH), 4.55(1 H, d,
J =8.43 Hz, CH-Br), 3.75(3H, s, CH3).
Example (iv)
Synthesis of (±)-ethyl 3-hydroxy-2-iodo-3-phenylpropanoate of formula 2
where x=l and R'= C2H5.
It was prepared from ethyl cinnamate.(3.26g, 20 mmol), HIO4.2H2O (5.2g, 24
mmol) following the procedure given in example step 'a' (iii), yield 4g (77%), m.pt
79°C.
1HNMR(CDCI3) 6: 7.38(5H, s, Ar-H), 5.05(1 H, d, J=8.29 Hz, CH-OH), 4.55(1 H, d,
J=8.29 Hz, CH-Br), 4.25(2H, q, J=7.11 Hz, Chfc), 1.25(3H, t, J=7.12 Hz,
CH2CH3).
Step 'b'
Example (i)
Synthesis of (±)-methyl 3-acetoxy-2-bromo-3-phenylpropanoate of formula
3 where x=Br and R'= CH3.
A solution of 2 (2.59g, 10mmol) and acetic anhydride (12mmol) and dimethyl
N,N-dimethyl aminopyridine (DMAP) (in catalytic amount) in 10 ml of dry
dichloromethane was kept overnight at room temp. The reaction mixture was
poured i nto i ce-cold w ater a nd e xtracted w ith d ichloromethane (3x100ml). T he
organic layer was washed, dried, and evaporated to furnish compound of formula
3, which was purified by column chromatography (silica gel, ethyl
acetate:hexane;3:97), in 90-95% yield, m.pt. 56°C
1HNMR (CDCI3)6: 7.45(5H, s, Ar-H), 6.23(1 H, d, J=10.00 Hz, CH-OAc), 4.58(1 H,
d, J=10.00 Hz, CH-Br), 3.83(3H, s, COOCH3), 2.03(3H, s, OCOCH3).
Example (ii)
Synthesis of (±)-ethyl 3-acetoxy-2-bromo-3-phenylpropanoate of formula 3
where x=Br and R'= C2H5.
It was prepared from 2 (2.73g, 10mmol) and acetic anhydride (12mmol) and N,Ndimethyl
aminopyridine (DMAP), in catalytic amount) following the procedure
given in step 'b' example (i) in 90-95% yield.
1HNMR (CDCI3)6: 7.50(5H, s, Ar-H), 6.23(1 H, d, J=10.50 Hz, CH-OAc), 4.53(11-1,
d, J=10.50 Hz, CH-Br), 4.33(2H, q, J=7.11 Hz Chb), 2.06(3H, s, OCOCH3),
1.33(3H,t, J=7.12HzCH2CH3).
Example (iii)
Synthesis of (i)-methyl 3-acetoxy-2-iodo 2»phenylpropanoate of formula 3
where x=l and R'= CH3.
It was prepared from 2 (3.06g, 10mmol) and acetic anhydride (12mmol) and N,Ndimethyl
aminopyridine (DMAP), in catalytic amount) following the procedure
given in step 'b' example (i) in 90-95% yield, m.pt, 58°C.
1HNMR(CDCI3) 5: 7.40(5H, s, Ar-H), 6.15(1 H, d, J=10.75 Hz, CH-OAc), 4.62(1H,
d, J=10.75 Hz, CH-Br), 3.17(3H, s, COOChb), 2.00(3H, s, CH3).
Example (iv)
Synthesis of (±)-ethyl 3-acetoxy-2-iodo-3-phenylpropanoate of formula 3
where x=l and R'= C2H5.
It was prepared from 2 (3.19g, 10mmol) and acetic anhydride (12mmol) and N,Ndimethyl
aminopyridine (DMAP), in catalytic amount) following the procedure
given in step 'b' example (i) in 90-95% yield.
1HNMR(CDCI3) 6: 7.50(5H, s, Ar-H), 6.15(1H, d, J=10.50 Hz, CH-OAc), 4.62(1H,
d, J=10.50 Hz, CH-Br), 4.25(2H, q, J=7.11 Hz COOCH2), 2.06(3H, s, OCOCH3),
1.25(3H, t, J=7.12 Hz CH2CH3).
Step 'c}
Example (i)
Preparation of (-)-methyl (2ft,3R)-2-bromo-3-hydroxy-3-phenylpropanoate of
formula 4 by kinetic resolution, where x-Br and R'= CH3.
(±)-Methyl 3-acetoxy-2-bromo-3-phenylpropanoate (SOOmg) of formula 3 where
x=Br and R'= CH3, was added to biphasic system of aqueous phosphate buffer
(16ml, 0.1M. pH 7.0) and toluene (1.6ml). To the above solution was added
crude dry powder of lipase Aspergillus niger (Amano AS, 400mg, 12-15 units/mg)
with the continuous stirring and maintaining pH 7.0 by addition of 0.5N sodium
hydroxide solution. During the course of the reaction temperature was
maintained at 30°C. The progress of the reaction was monitored after every six
hours by TLC and HPLC. After completion of the reaction (48 hrs., approx., 43%
conversion), the reaction was terminated by centrifuging the mixture at 10,000 to
15,000g to remove enzyme and the suspended particles. The clear solution and
the centrifuged mass was extracted separately with ethyl acetate (3 x 20ml). The
organic layer was combined and washed with water. The combined solvent layer
was then dried and evaporated under reduced pressure to furnish a mixture
comprising hydrolysed alcohol and unhydrolysed ester which were separated by
column chromatography over silica gel using hexane:ethyl acetate (97:3) as
eluent to furnish (-)-methyl (2f?,3R)-2-bromo-3-hydroxy-3-phenylpropanoate of
formula 4 (250mg, 85%) having enantiomeric purity (EE)92%, [a]D
25 -19.7°
(c,1,CHCI3) and unhydroiysed ester (+)-methyl (2S,3S)-3-acetoxy-2-bromo-3-
phenyipropanoate of formula 5 (378mg, 83%) having enantiomeric purity
(EE)70% (chiral HPLC), [a]D
25 +36.4° (c,1,CHCI3).
Example (ii)
Preparation of (-)-ethyl (2/?,3/?)-2-bromo-3-hydroxy-3-phenylpropanoate of
formula 4 by kinetic resolution, where x=Br and R'= C2H5.
It was prepared from (±)-ethyl 3-acetoxy-2-bromo-3-phenylpropanoate (800mg)
of formula 3, phosphate buffer (16ml, 0.1M. pH 7.0), toluene (1.6ml) and crude
dry powder of lipase Aspergillus niger (Amano AS, 400mg, 12-15 units/mg)
following the procedure given in step 'c' example (i). After 48 hrs. (45%
conversion) the hydrolyzed alcohol was obtained (256mg, 82%), having
enantiomeric purity (EE) 86%, [a]D
25 -14.8° (c,1,CHCI3) and unhydrolysed ester
(+)-ethyl (2S,3S)-3-acetoxy-2-bromo-3-phenylpropanoate of formula 5 (375mg,
85%) having enantiomeric purity (EE)73%(chiral HPLC), [a]D
25 +37.5°
(c,1,CHCI3).
Example (iii)
Preparation of (-)-methyl (2/?,3/?)-3-hydroxy-2-iodo-3-phenylpropanoate of
formula 4 by kinetic resolution, where x=l and R'= CH3.
It was prepared from (t)-Methyl 3-acetoxy-2-iodo-3-phenylpropanoate (800mg)
of formula 3, phosphate buffer (16ml, 0.1M. pH 7.0), toluene (1.6ml) and crude
dry powder of lipase Aspergillus niger (Amano AS, 400mg, 12-15 units/mg)
following the procedure given in step 'c' example (i). After 36 hrs. (44%
pnversion) the hydrolyzed alcohol was obtained (274mg, 88%), having
enantiomeric purity (EE) 94%, [a]D
25 0.0° (c,1,CHCI3); -3.0° (c 1, MeOH) and
unhydrolysed ester (+)-methyl-(2S,3S)-3-acetoxy-2-iodo-3-phenylpropanoate of
formula 5 (415mg, 92%) having enantiomeric purity (EE)76%(chiral HPLC), [a]D
+48.0°(c,1,CHCI3).
Example (iv)
Preparation of (-)-ethyl (2R,3R)-3-hydroxy-2-iodo-3-phenylpropanoate of
formula 4 by kinetic resolution, where x=l and R'= C2H5.
It was prepared from (±)-Ethyl 3-acetoxy-2-iodo-3-phenylpropanoate (800mg) of
formula 3, phosphate buffer (16ml, 0.1M. pH 7.0), toluene (1.6ml) and crude dry
powder of lipase Aspergillus niger (Amano AS, 400mg, 12-15 units/mg) following
the procedure given in step 'c' example (i). After 40 hrs. (40% conversion) the
hydrolyzed alcohol was obtained (250mg, 88%), having enantiomeric
purity(EE)95%, [a]D
25 -9.3° (c,1 ,CHCI3) and unhydrolysed ester (+)-ethyl(2S,3S)-
3-acetoxy-2-iodo-3-phenylpropanoate of formula 5 (440mg, 91%) having
enantiomeric purity (EE)60%(chiral HPLC), [a]D
25 +36.0° (c,1,CHCI3).
Example (v)
Preparation of (+)-methyl (2S,3S)-3-acetoxy-2-bromo-3-phenylpropanoate of
formula 5 by double kinetic resolution, where x=Br and R'= CH3.
It was prepared from optically enriched (+)-Methyl (2S,3S)-3-acetoxy-2-bromo-3-
phenylpropanoate (EE, 70%, 350mg) of formula 5, phosphate buffer (7ml, 0.1M.
pH 7.0), toluene (0.7ml) and crude dry powder of lipase Aspergillus niger (Amano
AS, 100mg, 12-15 units/mg) following the procedure given in step 'c' example (j).
After completion of the reaction (72 hrs.), the unhydrolyzed ester on separation
(290mg) was found to have enantiomeric excess (EE)>99%(chiral HPLC), [a]D
25
+52.0°(c,1,CHCI3)
Example (vi)
Preparation of (+)-ethyl (2S,3S)-3-acetoxy-2-bromo-3-phenylpropanoate of
formula 5 by double kinetic resolution, where x=Br and R'= C2H5.
It was prepared from optically enriched ethyl (2S,3S)-3-acetoxy-2-bromo-3-
phenylpropanoate (EE 73%, 350mg) of formula 3, phosphate buffer (7ml, 0.1M.
pH 7.0), toluene (0.7ml) and crude dry powder of lipase Aspergillus niger (Amano
AS, 100mg, 12-15 units/mg) following the procedure given in step 'c' example (i).
After 65 hrs the unhydrolyzed ester on separation (290mg) was found to have
enantiomeric excess (EE)98%(chiral HPLC), [a]D
25 +50.5° (c,1, CHCI3).
Example (vii)
Preparation of (+)-methyl (2S,3S)-3-acetoxy-2-iodo-3-phenylpropanoate of
formula 5 by double kinetic resolution, where x=l and R'= CH3.
It was prepared from optically enriched methyl (2S,3S)-3-acetoxy-2-iodo-3-
phenylpropanoate (EE 76%, 350mg) of formula 3, phosphate buffer (7ml, 0.1M.
pH 7.0), toluene (0.7ml) and crude dry powder of lipase Aspergillus niger (Amano
AS, 100mg, 12-15 units/mg) following the procedure given in step 'c' example (i).
The unhydrolyzed ester of formula 5 (SOOmg) was found to have enantiomeric
excess (EE)94%(chiral HPLC), [a]D
25 +59.0° (c,1, CHCI3).
Example (viii)
Preparation of (+)-ethyl (2S,3S)-3-acetoxy-2-iodo-3-phenylpropanoate of
formula 5 by double kinetic resolution, where x=l and R'= C2H5.
It was prepared from optically enriched methyl (2S,3S)-3-acetoxy-2-iodo-3-
phenylpropanoate (EE 60%, 350mg) of formula 3, phosphate buffer (7ml, 0.1M.
pH 7.0), toluene (0.7ml) and crude dry powder of lipase Aspergillus niger Amano
AS (100mg, 12-15 units/mg) The unhydrolyzed ester (315mg)(72hrs.) was found
to have enantiomeric excess (EE)73%(chiral HPLC), [a]D
25 +43.5° (c,1, CHCI3).
Example (ix)
Preparation of (-)-methyl (2R,3R)-2-iodo-3-hydroxy-3-phenylpropanoate of
formula 4 by kinetic resolution in presence of a cosolvent acetonitrile,
where x=l and R'= CH3.
(±)-Methyl 3-acetoxy-2-iodo-3-phenylpropanoate (200mg) of formula 3 was
added to biphasic system of aqueous phosphate buffer (3.6ml, 0.1M. pH 7.0) and
acetonitrile (0.4ml). To the above solution crude dry powder of lipase Aspergillus
n/ger (Amano AS, 100mg, 12-15 units/mg) was added with the continuous stirring
and maintaining pH 7.0 by addition of 0.5N sodium hydroxide solution. During the
course of the reaction temperature was maintained at 30°C. The progress of the
reaction was monitored after every six hours. After the completion of the reaction
(9 hrs., approx., 43% conversion), the reaction was terminated by centrifuging
the mixture at 10,000 to 15,000g to remove enzyme and the suspended particles.
The clear solution and the centrifuged mass was extracted separately with ethyl
acetate (3X30 ml). The organic layer was combined and washed with water. The
combined solvent layer was then dried and evaporated under reduced pressure
to furnish a mixture comprising hydrolysed alcohol and unhydrolysed ester which
were separated by column chromatography over silica gel using hexane: ethyl
acetate (97:3) as eluent to furnish methyl (2R,3R)-3-hydroxy-2-iodo-3-
phenylpropanoate of formula 4 (65mg, 85%) having enantiomeric purity (EE)92%
and unhydrolysed ester methyl (2S,3S)-3-acetoxy-2-iodo-3-phenylpropanoate of
formula 5 ( 92 mg, 77%), enantiopurity (EE) 62% determined by chiral HPLC.
Step 'e'
Example (i)
Preparation o f (+)-methyl (2S,3R)-phenylglycidate o f t he f ormula 7 where
0.2 ml of 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) was added to a solution of
optically enriched halohydrin (200 mg) of formula 4 in methanol (4ml) at 20° C for
5 minutes. The solvent was removed under reduced pressure and the reaction
mixture was diluted with 10 ml water and extracted with ethyl acetate and solvent
removed to furnish compound of formula 7; recovery 98mg, [a]D
25 +155.0° (c,1,
CHCI3)
Step 'f
Example (i)
Preparation of (+)-methyl (2S,3S)-2-halo-3-hydroxy-3-phenylpropanoate of
formula 6 where R'= CH3
Alkyl (2S,3S)-3-acetoxy-2-holo-3-phenylpropanoate of formula 5 (200mg) was
dissolved in 4ml of methanol and 0.2ml of 2N HCI was added and the reaction
mixture was stirred at room temperature for 24 hrs till the complete conversion of
the compound of formula 5 to the compound of the formula 6. Excess of solvent
was evaporated and the reaction mixture was diluted with 10 ml water and
extracted with ethyl acetate, and solvent removed to furnish compound of
formula 6, recovery 150mg.
Example (ii)
Preparation of (-)-methyl (2R,3S)-phenylglycidates of the formula 8 where
R'= CH3
It was prepared from 6 following the procedure given in step 'f example (i) for
compound 7. [a]D
25 -153.0° (c,1, CHCI3)

We claim,
1. A stereoselective chemoenzymatic process for the preparation of optically enriched phenylglycidates i.e. alkyl(+2S,3R)- phenylglycidate and
alkyI(-2R,3S)- phenylglycidate as precursors of taxol side chain,wherein the said process comprises
a. halohydroxylation reaction of 3-phenylpropanoate having C-1 to C-5 alkyl group to
form trans 2-halo-3- hydroxyl-3-phenylpropanoates at a temperature between 0-60°C,
wherein the halogenating agent used is N- halosuccinimide such as herein described.
b. converting trans 2-halo-3- hydroxyl-3-phenylpropanoates to (±)- alkyl-3-acetoxy-2-
halo-3-phenylpropanoates by an acylating agent such as herein described.
c. substantially incubating the acyl derivatives of alkyl-3-acetoxy-2-halo-3-
phenylpropanoates with crude dry powder of lipase from Aspergillus niger in an aqueous
buffer phase in presence of an organic solvent,
d. thereafter separating the hydrolysed halohydrins i.e. alkyl(2R,3R)- 2-halo-3- hydroxy-
3-phenylpropanoates and unhydrolysed ester i.e. alkyl(2S,3S)- 2-halo-3- acyloxy-3-
phenylpropanoates from the mixture by conventional method,
e.if required, then again incubating the optically enriched alkyl(2S,3S)- 2-halo-3-acyIoxy-3-phenylpropanoates with crude dry powder of lipase of Aspergillus niger in an aqueous buffer phase in presence of an organic solvent to further improve the enantiopurity,
f reacting optically enriched (2S,3S)- 2-halo-3- acyloxy-3-phenylpropanoates with an acid to furnish optically enriched alkyl(2S,3S)- 2-halo-3- hydroxy-3-phenylpropanoates, g. treating the compounds alkyl(2R,3R)- 2-halo-3- hydroxy-3-phenylpropanoates and alkyl(2S,3S)- 2-halo-3- hydroxy-3-phenylpropanoates with an alkali in an organic or aqueous phase to furnish optically enriched (+)-methyl (2S,3R)-phenylglycidate and (-)-methyl (2S,3R)-phenylglycidate .
2. A novel process as claimed in claim 1 step(a), N-halosuccinimide used is N-bromosuccinimide, N-iodosuccinimide or sodium bromate, periodic acid, 1,3-dibromo-5,5-dimethyl hydantoin, iodine and bromine.

3. A process as claimed in claim 1, wherein the halohydroxilation process of step(a) is effected in aqueous or in an organic or aqueous organic phase wherein the organic phase is selected from water miscible solvents such as acetone, tetrahydrofuran, dioxane, dimethylformamide and methanol.
4. A novel process as claimed in claim 1, wherein for the incubation of alkyl acrylates ,the aqueous phosphate buffer is phosphate tris buffer and acetic acid having pH in the range of 5-7.5 and temperature 10-40°C.
5. A process as claimed in claims 1 wherein organic solvent used are hexane, toluene, dicholoromethane ,acetone, acetonitrile, dimethylformamide, dimethylsulphoxide, methanol and ethanol at 10-90% concentration.
6. A process as claimed in step(f) of claim 1, wherein the acid used to furnish optically
enriched alkyl(2S,3S)- 2-halo-3- hydroxy-3-phenylpropanoates is an organic or inorganic
acid such as hydrochloric acid .sulfuric acid, trifluoro acetic acid and boron trifluoride.
8. A process as claimed in step(g) of claim 1, wherein alkali used to furnish optically enriched (+)-methyl (2S,3R)-phenylglycidate and (-)-methyl (2S,3R)-phenylglycidate is organic or inorganic base selected from the group consisting of sodium hydroxide, sodium carbonate and triethylamine.
10. A process for the preparation of alkyl(+2S,3R)- phenylglycidate and alkyl(-2S,3R)-phenylglycidate shown in the drawing accompanying the specification substantially as herein described with refererence to examples.

Documents:

275-DEL-2003-Abstract-(21-01-2009).pdf

275-del-2003-abstract.pdf

275-DEL-2003-Claims-(21-01-2009).pdf

275-DEL-2003-Claims-(23-12-2008).pdf

275-del-2003-claims.pdf

275-DEL-2003-Correspondence-Others-(21-01-2009).pdf

275-DEL-2003-Correspondence-Others-(23-12-2008).pdf

275-del-2003-correspondence-others.pdf

275-del-2003-correspondence-po.pdf

275-DEL-2003-Description (Complete)-(23-12-2008).pdf

275-del-2003-description (complete).pdf

275-DEL-2003-Drawings-(21-01-2009).pdf

275-del-2003-drawings.pdf

275-DEL-2003-Form-1-(21-01-2009).pdf

275-del-2003-form-1.pdf

275-del-2003-form-18.pdf

275-DEL-2003-Form-2-(21-01-2009).pdf

275-DEL-2003-Form-2-(23-12-2008).pdf

275-del-2003-form-2.pdf

275-DEL-2003-Form-3-(23-12-2008).pdf

275-del-2003-form-3.pdf

275-DEL-2003-Petition-137-(23-12-2008).pdf

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Patent Number

233623

Indian Patent Application Number

275/DEL/2003

PG Journal Number

20/2009

Publication Date

15-May-2009

Grant Date

31-Mar-2009

Date of Filing

12-Mar-2003

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	NAVEEN ANAND	RRL,JAMMU,INDIA
2	MUNISH KAPOOR	RRL,JAMMU,INDIA.
3	SUBHASH CHANDRA TANEJA	RRL,JAMMU,INDIA.
4	SURRINDER KOUL	RRL,JAMMU,INDIA.
5	RATAN LAL SHARMA	RRL,JAMMU,INDIA.
6	GHULAM NABI QAZI	RRL,JAMMU,INDIA.

PCT International Classification Number

C07C 45/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA