Method of resolution of hydroxy-cyclopentenones using a lipase and transacylation agents

Abstract: A process for irreversible regio- and stereoselective enzyme catalyzed acylation of alcohols using enol esters as acylating reagents is disclosed. The present invention permits the selective modification of hydroxyl group(s) of chiral and meso alcohols, including sugars, organometallics, and glycosides. The enol freed upon transesterification rapidly tautomerizes to the corresponding volatile aldehyde or ketone thereby preventing the reverse reaction from occurring.

Description: The present invention relates to an enzymatic resolution method for preparing chiral hydroxycyclopentenones by lipase catalyzed irreversible transesterification using enol esters as transacylation reagents. Hydrolytic enzymes such as lipases, esterases, and proteases have been used extensively as catalysts in enantioselective syntheses. Whitesides, G.M., Wong, C-H. Angew. Chem. Int. Ed. Engl . 24 (1985) 617; Jones, J.B. Tetrahedron 42 (1986) 3351; Roberts, S.M. Chem. Br. (1987) 127; Akiyama, A., Bednarski, M., Kim, M.J., Simon, E.S., Waldmann, H.I., Whitesides, G.M. Ibid. (1987) 645. Because of their relatively high stability in organic media, many hydrolytic enzymes also can be used in organic solvents for certain types of transformations which are difficult to do in water. The most common reactions are esterase and lipase-catalyzed stereoselective esterifications and transesterifications. Klibanov, A.M. CHEMTECH (1986) 354-9; Klibanov, A.M., Cambou, B. J. Am. Chem. Soc. 106 (1984) 2687-92. Chen, C-S., Wu, S-H., Girdaukas, G., Sih, C.J. J. Am. Chem. Soc. 109 (1987) 2812-17; Guo, Z.W., Sih, C.J. Ibid. 110 (1988) 1999-2001; Gil, G., Ferre, E., Meou, A., Petit, J.L., Triantaphylides, C. Tetrahedron Lett. 28 (1987) 1647; Yokozeki, K., Yamanaka, S., Takinami, K., Hirose, Y., Tanaka, A., Sonomoto, K., Fukui, S. Eur. J. Appl. Microbiol. Biotechnicol 14 (1982) 1; Tambo, G.M.R., Schar, H-P., Busquets, X.F., Ghisalba, O. Tetrahedron Lett. 27 (1986) 5705-10; Belan, A., Bolte, J., Fauve, A., Gourey, J.G., Veschambre, H. J. Org. Chem. 52, 256-60. Langrand, G., Baratti, J., Buono, G., Triantaphylides, C. Tetrahedron Lett. 27 (1986) 29-32. One disadvantage of enzyme catalyzed hydrolytic reactions is that they are very slow compared to simple hydrolyses. Langrand, G., Baratti, J., Buono, G., Triantaphylides, C. Tetrahedron Lett. 27 (1986) 29-32. In addition, the products produced by enzymatic hydrolyses very often have to be separated from other by-products (particularly alcohol generated from the acylating reagent). Due to the reversible nature of these reactions, and due to the same stereoselectivity of the enzyme catalysis in both directions, the optical purity of the product obtained decreases as the reverse reaction proceeds. This situation is illustrated in FIG. 1 where a racemic alcohol is to be resolved via an enzymatic esterification (R'' = H) or transesterification. As shown in FIG. 1, if the D-isomer is a better substrate than the L-isomer for the enzyme, accumulation of the D-ester and the unreactive L-alcohol will be observed. In the reverse reaction, however, the D-ester is a better substrate and will be converted to the D-alcohol. The enantiomeric excess of both the D-ester and the L-alcohol therefore will decrease progressively as the extent of the reverse reaction increases. This reverse reaction problem clearly has been illustrated in the kinetic resolution of menthol, Chen, C-S., Wu, S-H., Girdaukas, G., Sih, C.J. J. Am. Chem. Soc. 109 (1987) 2812-17; Guo, Z.W., Sih, C.J. Ibid. 110 (1988) 1999-2001, and can be seen in the enantioselective esterification or transesterification of meso compounds. SUMMARY OF THE INVENTION The method of the present invention blocks the progress of the reverse reaction. The present invention is a process for irreversible regio- and stereoselective enzyme catalyzed acylation of alcohols using enol esters as acylating reagents. The present invention permits the selective modification of hydroxyl group(s) of chiral hydroxycyclopentenones. The simplicity of this irreversible transesterification makes the operation useful for the preparation of chiral alcohols or esters that may be difficult to prepare by other means. Enzymatic Resolution of Hydroxycyclopentenones Utilizing Lipase Lipase catalyzed reactions were utilized in the resolution of hydroxycyclopentenones as illustrated in the following reaction schemes, illustrating specific embodiments of the present invention. Using the procedure described herein, resolution of hydroxy-cyclopentenones was achieved. Specifically, hydroxy-alkyl-carbonyl-alkyl, alkenyl or alkynyl cyclopentenones, alternatively including a heteroatom in the side chain, are suitable for practice of the described method. The compounds can be described illustratively by the formula: wherein X represents alkyl, alkenyl or alkynyl, of from 2 to 10 carbon atoms, alternatively including a heteroatom, namely S or O, at the 2, 3, 4, 5, 6, 7, 8 or 9 position; and R is CH₂OR, wherein R is hydrogen, alkyl of 1 to 6 carbon atoms, tetrahydropyranyl, ethoxyethyl, acyl or CO₂R₂ or (R₃)₃Si wherein R₂ is alkyl of 1 to 6 carbon atoms and R₃ is alkyl of 1 to 10 carbon atoms or aryl. Practical and presently preferred embodiments of the method are described in the following examples. Several lipases which are available commercially were utilized, namely: Candida cylindracea (CC), Pseudomonas Species (P.Sp.), porcine pancreatic lipase (PPL), all from Sigma Chemical, and Amano P, ANL, Aspergilus Niger, ChE, Cholesterol Esterase, all from Amano Co. The role of solvent with respect to both rate of reaction and extent of kinetic resolution was also studied. The conversions were monitored utilizing normal phase HPLC on silica gel (Zorbax sil) and the optical purities of products were analyzed using HPLC on chiral stationary phase (Chiracel OD column). A preferred lipase among the above mentioned is porcine pancreatic lipase or amano P. Preferred acylating agents are isopropenyl acetate, isopropenyl valerate, vinyl acetate, vinyl propionate and vinyl valerate and among these especially isopropenyl acetate or vinyl acetate. Furthermore the method is especially useful when porcine pancreas (PPL) is the lipase, vinylacetate as acylating compound and the compound for resolution is Methyl 7-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)-4Z-heptenoate (1S) Methyl 7-(3-hydroxy-5-oxo-1-cyclopenten-1-yl)-4-heptenoate (4) 3-(3-hydroxy-5-oxo-1-cyclopenten-1-yl)-propyne (5) Methyl 7-(3-hydroxy-5-oxo-1-cyclopenten-1-yl)-5Z-heptenoate (6) or Methyl 7-(3-hydroxy-5-oxo-1-cyclopenten-1-yl)-heptenoate (7) or mostly preferred methyl 7-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)-4Z-heptenoate. Furthermore the lipase may be immobilized on a support. Preferred solvents which may be used in the method of the present invention are toluene, t-butylmethyl ether, tetrahydrofuran, diethyl ether, hexane and most preferred CHCl₃, benzene. Two critical challenges were considered: a) enone (1S) which has the undesired (S)-configuration must be inverted with high stereospecificity to give the necessary (R)-enone (1R); b) over half of the product from the lipase reaction was 11-OAc-enone, (2R) which was predominantly (94%) the (R)-isomer. Methodology for enrichment of this material to usable optical purity (i.e. 98%) was desirable. With respect to the first point, it was found that enone (1S) could be converted to (1R) via Mitsunobu chemistry. In the present case, when (1S) of 99.9% (S) purity was submitted to Mitsunobu conditions (Bull. chem. Soc. Japan 44 3427, 1971) using formic acid as nucleophile and then immediate hydrolysis of the formate ester intermediate, a 91-94% chromatographed yield of inverted enone (1R) was obtained which was 99.3-99.6% (R) isomer. Therefore, only a maximum of 0.3 to 0.5% of racemization had occurred. This product was then converted to its triethyl silyl derivative, (3) in 94% yield after chromatography. Again, HPLC analysis on Chiracel OD column showed this material to be of identical optical purity to its precursor (99.3%R). This enzymatic resolution method, coupled with the Mitsunobu alcohol inversion technology described here enalbes the preparation of either antipodes of the optically pure alcohol desired. The following important features of the described process should be noted. No special apparatus or inert atmosphere are required for the lipase reactions since all reactions were carried out in organic solvents in the absence of water. A 0.1-0.2 kg scale kinetic resolution of racemic enone were easily performed in the laboratory. The four hydroxy enones shown above were also submitted to enzymatic resolution using PPL in vinyl acetate as described for compound (1). Greater than 92% ee (unoptimized) was achieved in all cases after 4 days reaction time. Chromatography is a necessary purification process for each hydroxy to acetate transformation since failure to remove acetate from the alcohol quantitatively will lead to effective racemization. All chemical and enzymatic reactions take place with a high degree of stereoselectivity and in high yield. Three batches of target Triethyl Silyl (3) were prepared: 40.25 g (99.3% R), 6.59 g (99.7% R) and 8.90 g (99.4% R). It was found the all of the lipases were selective in acylating the R-isomer of the starting enone compound with ( 1 ). Amano-P lipase the most active catalyst. Using Amano-P as the enzyme source, relative rates of reaction were examined in five solvent systems using isopropenyl acetate as the acylating agent. Use of t-butyl methyl ether (TMBE) and aromatic solvents gave the highest rates. In addition to the lipase PPL, candida cylindracea (CC) lipase and a Pseudomonas Sp. (Amano-P) were studied both as their free powders and also immobilized on Amberlite XAD-8 resin, SIGMA Chem Co. In all cases, reaction at room temperature or 50°C were extremely slow, usually <10% conversion after 5 days. However, with PPL immobilized on XAD-8 resin, a 25% conversion was achieved after 5 days at room temperature. Optimum conditions were established when excess vinyl acetate was employed in place of isopropenyl acetate and no solvent was used. Under these conditions reactions could be run at room temperature, were much faster and proceeded smoothly in 3-5 days. Moreover, the side products observed when isopropenyl acetate was employed as the acylating agent (due to interference by the methyl ester) were eliminated. The viability of the process was demonstrated on a 100 g scale. A two-step process in which the acetate is removed by a purely chemical means and the recovered alcohol then resubmitted to the lipase acylation conditions was found to be most advantageous. Referring to Scheme 2, when ( 2R ) was treated with 2 equiv. of guanidine in CH₃OH, very rapid, clean conversion to the desired compound ( 1R ) was observed in less than 5 minutes at 0°C. In fact, when only 0.25 equiv. of guanidine were employed, the reaction profile and rate were identical to the stoichiometric case and compound ( 1R ) (93% R) was recovered in 75-77% yield after chromatography. When this material was resubmitted to the action of PPL in vinyl acetate for 2 days, a 90% yield of compound ( 2R ) was obtained with an enantiomeric excess of 99.6% (this represents a 98% conversion of available ( R ) alcohol ( 1R )). Deacylation via guanidine in methanol as described above provided the target (R)-enone ( 1R )). HPLC showed no racemization again during acetate removal. These results allow for complete conversion of both antipodes of (SC-37321) into chiral enone and all recovered non-enriched intermediates can be recycled to high optical purity (see Scheme 2). Experimental Section Column chromatography separations were performed by using Merck SiO₂ 60 silica gel with ethyl acetate/hexane mixtures as eluants. TLC analyses were performed on Merck SiO₂ 60 F254 precoated glass plates and were visualized by charring with phosphomolybdic acid in ethanol. Melting points (differential scanning calorimetry) were obtained on a Dupont 9900 Thermal Analyzer. NMR were recorded at room temperature in CDCl₃ using a General Electric QE-300 or Varian XL-400 spectrometer with TMS as internal standard. HPLC analyses were performed on Chiralcel OD, OA, or OC columns employing a chiral stationary phase (Daicel Chemical Industries) on a Waters Associates Model 590 solvent deliver system with Waters Intellingent Sample Processor (WISP). Optical rotations were measured with a Perkin-Elmer 241 polarimeter. IR spectra were recorded (as solutions in chloroform) on a Perkin-Elmer 681 spectrometer. CD spectra were recorded on a JASCO J-20 ORD/CD spectropolarimeter. UV spectra recorded (in CH₃OH) on a Beckman DU-7HS UV-Vis spectrophotometer. Diethyl azodicarboxylate, triphenylphosphine, formic acid, guanidine carbonate, triethylsilyl chloride, sodium spheres were purchased from Aldrich and used without purification. Lipases: candida cylindracea (CC), porcine pancreatic lipase (PPL), pseudomonas species (PSp.) were purchased from Sigma Chemical Co. Amano P lipase was purchased from Amano Co. Isopropenyl acetate and vinyl acetate were purchased from Aldrich and fractionally distilled prior to use. All solvents were purchased from Burdich and Johnson and were reagent grade. CH₃OH was distilled from Mg; DMF was distilled at reduced pressure from magnesium sulfate, benzene and toluene were azeotropically distilled, chloroform was distilled from P₂O₅ and t-butyl methyl ether was distilled from benzophenone ketyl (all under inert atmosphere). EXAMPLE 1 Preparation of Methyl 7-[3R-(acetyloxy)-5-oxo-1-cyclopenten-1-yl]-4Z-heptenoate (2R) and Methyl 7-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)-4Z-heptenoate (1S) via enzymatic resolution A mixture of 100.0 g of (0.42 mole) ±methyl 7-(3-hydroxy-5-oxo-1-cyclopenten-1-yl)-4Z-heptenoate (1), 100 g of porcine pancreatic lipase (14M units) and 2.5 L of distilled vinyl acetate were vigorously stirred at room temperature for 4 days. An additional 50-g portion of porcine pancreatic lipase was added and the mixture was stirred for one more day. The course of the reaction was monitored via HPLC on Chiralcel OD using 93:7 hexane:isopropanol as eluant until the enantiomeric excess of unreacted 3(S) alcohol was >99.8%. The crude mixture was then treated with 50 g of diatomaceous earth and filtered through a bed of diatomaceous earth. The filter cake was washed with 1.5 L of methylene chloride. The combined filtrates were concentrated under reduced pressure to give approximately 119 g of product mixture which contains predominantly 3(R)-acetate and 3(S) alcohol having R f values of 0.25 and 0.49 respectively on TLC using 80% ethyl acetate/hexane. The product was chromatographed on silica gel using a solvent gradient of 50% to 100% ethyl acetate/hexane to give 50.5 g (43%) of (R) enriched methyl 7-[(3R-acetyloxy)-5-oxo-1-cyclopenten-1-yl]-4Z-heptenoate (2R) ¹H NMR (CDCl₃): δ 7.10 ( m,1H, C₂H), 5.67 (m, 1H, C₃H), 5.30 (m, 2H, olefinic H), 3.68 (S, 3H, OCH₃), 2.86 (dd, 1H, C 4β H) J=6.5, 18.5 Hz, 2.36 (dd, 1H, C 4α H) J=2.1, 18.5 Hz, 2.4-2.25 (m, 8H, CH₂); ¹³C NMR (CDCl₃) δ 204.3, 173.1, 170.3, 151.8, 148.8, 129.4, 128.6, 71.3, 51.3, 41.3, 33.7, 24.7, 24.3, 22.6, 20.7 ppm; IR (CHCl₃) 3030, 3010, 1735, 1720, 1440, 1370, 1230 cm⁻¹; [α] 20 / D +45.4° (-643.3° at 365 nM) (c 1.080 g/dL, CHCl₃); UV ( CH₃OH) V max = 220 nM; Anal. calc'd. for C₁₅H₂₀O₅ = C, 64.27; H, 7.19 Found: C, 64.24; H, 7.32 and 35.1 g (35%) of methyl 7-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)-4Z-heptenoate ( 1R ) ¹H NMR (CDCl₃) δ 7.10 (m, 1H, C₂H), 5.24 (m, 2H, olefinic), 4.93 (m, C₃H, 1H), 4.05 (b, 1H, OH), 3.68 (S, 3H, OCH₃), 2.80(dd, 1H, C 4β H) J = 6.0, 18.5 Hz, 2.4-2.2 (m, C 4α H + CH₂'S,9H); ¹³C NMR δ 207.1, 174.1, 157.6, 146.9, 130.1, 128.8, 68.5, 51.9, 45.1, 34.2, 25.4, 24.6, 23.0 ppm; IR (CHCl₃) 3610, 3480 (broad), 3030, 3010, 1715 (shoulder at ∼1730), 1440, 1230 cm⁻¹; [α] 20 / D -13.3° (c 0.867 g/dL, CH₃Cl₃) (+1202° at 365 nM); UV (CH₃OH) λ max = 221 nM; CD [ϑ]²⁵ (nM) -11900 (320), +64909 (224) (CH₃OH); Anal. calc'd. for C₁₃H₁₈O₄: C, 65.52; H, 7.61; Found: C, 64.78; H, 7.74. HPLC (Chiralcel OD using 93:7 hexane in isopropanol as eluant) indicated the purified acetate (2R) was of 92% ee in R-isomer and that recovered alcohol (IS) was 99.4% ee in S-isomer. EXAMPLE 2 Mitsunobu Inversion of Methyl 7-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)-4Z-heptenoate (1S) To a mixture of 7.14 g (30.0 mmol) of the title alcohol, 15.70 g (60.0 mmol) of triphenylphosphine in THF (100 mL) under argon was added formic acid via syringe. The solution was cooled to ∼10°C in an ice bath. The reaction mixture was maintained at ≦15°C while 10.44 g (9.49 mL, 60.0 mmol) of diethyl azodicarboxylate was added dropwise via syringe. The pale yellow solution was warmed to room temperature and stirred at room temperature overnight. TLC (80% ethyl acetate/ hexane on silica gel) showed complete consumption of starting alcohol. The solvents were removed at reduced pressure to give a viscous oil. This was dissolved in 200 mL of t-butyl methyl ether (TBME) and to this was slowly added 400 mL of hexane and stirred at room temperature for 20 min. The mixture was filtered. The filter pad was washed with two 100-mL portions of 1:1 TBME:hexane. The combined filtrates were concentrated at reduced pressure to give 12.80 g of an amber oil which was dissolved in 300 mL of absolute methanol and stirred mechanically. To this was added gradually 200 g Woelm Super I (neutral) alumina. The mixture was stirred at room temperature for 5 hours to hydrolyze the formate ester intermediate. The mixture was filtered through a glass fritted funnel and the filter cake was washed with three 100-mL portions of CH₃OH. The combined filtrates were concentrated at reduced pressure to give ∼12 g residue which was purified by flash chromatography on silica gel using gradient elution (30 to 75% ethyl acetate in hexane) to give 8.22 g of product which still contains 6-10% of 1,2-dicarbethoxyhydrazine (determined by ¹H NMR) which is removed in the subsequent step. An analytical sample was obtained by PTLC on 2000µ silica gel plates using two elutions of 65% ethyl acetate in hexane. The twice purified sample was identical to 3S-alcohol by normal phase HPLC, TLC, ¹H and ¹³C NMR, UV and IR spectroscopy. [α] 25 / D + 16.6° (c 1.024 g/dL, CHCl₃ (-1174° at 365 nM). CD [ϑ]²⁵ (nM) -11900 (320) (negative maximum), +64909 (224) (positive maximum) (CH₃OH). HPLC on Chiralcel OD using 93/7 hexane/ isopropanol as eluant indicated that the ratio of 3R to 3S alcohols was 99.4/0.6. EXAMPLE 3 A stock solution of 0.5M guanidine in CH₃OH was prepared by adding 1.78 g (77.4 mmol) of 3X hexane washed sodium spheres to an ice-cooled CH₃OH (154 mL) under argon atmosphere. When an the sodium had reacted, 14.22 g (79.0 mmol) of guanidine carbonate was added. This was stirred at room temperature for 25 min and the mixture allowed to stand to settle out precipitated salts. In a separate flask was placed 12.8 g (45.6 mmol) of a ∼93:7 mixture of R:S alcohols in 50 mL of absolute CH₃OH under argon. This was cooled to 0°C in an ice bath and to it was added via syringe 100 mL of 0.5M guanidine in CH₃OH prepared above, over ∼5 min. This mixture was stirred at ∼10°C for 5 min. TLC (80% ethyl acetate in hexane on silica gel) showed complete consumption of acetate. To the reaction mixture was then added 2.86 mL (3.0 g, 50.0 mmol) of glacial acetic acid to neutralize the guanidine. After stirring for 5 minutes, solvent was removed at reduced pressure to give a thick slurry. The residue was partitioned between 100 mL of water and 100 mL of 1:1 toluene: ethyl acetate. The aqueous layer was further extracted with two 50-mL portions of ethyl acetate. The combined organic layers were washed with two 50-mL portions of water, 50 mL of brine and dried over sodium sulfate. Removal of solvent at reduced pressure gave a deep amber oil which was purified by flash chromatography on silica gel with 50% ethyl acetate in hexane to give 8.06g of ( 1R ) (77%) after exhaustive removal of solvent. ¹H and ¹³C NMR were identical to previously isolated pure 3S-alcohol ( 1S ). HPLC on Chiralcel OD using 93/7 hexane/isopropanol as eluant indicated a 93:7 R:S mixture of alcohols which showed that no racemization had taken place during deacylation. EXAMPLE 4 Enzymatic Optical Enrichment of Methyl 7-(3R-hydroxy-5-oxo-cyclopenten-1-yl)-4Z-heptenoate (1R) A mixture of 7.50 g (31.5 mmol) of 93:7 R:S alcohols, 7.50 g (99, 750 units) of porcine pancreatic lipase in 180 mL of distilled vinyl acetate was stirred vigorously at room temperature for 45 hours. HPLC of an aliquot on Chiralcel OD using 93:7 hexane:isopropanol showed excellent conversion of R alcohol ( 1R ) to the corresponding acetate ( 2R ). In fact, 98% of available R-alcohol had been consumed to give R-acetate with greater than 98.8% ee. The mixture was filtered through diatomaceous earth and the filter cake washed with two 100-L portions of methylene chloride. The combined filtrates were concentrated under reduced pressure to give 8.90 g of residue which was purified by chromatography on silica with 20% ethyl acetate in hexane as eluant. By this technique, 7.53 g (85%) of 98.8% ee R-acetate ( 2R ) was obtained which was identical to previously isolated R-acetate by ¹H and ¹³C NMR, HPLC and TLC. EXAMPLE 5 Deacylation of Optically Enriched Methyl 7-(3R-acetyloxy-5-oxo-1-cyclopenten-1-yl)-4Z-heptenoate (2R) To a room temperature solution of 7.47 g (26.6 mmol) of 98.8% ee 3R-acetate ( 2R ) in 25 mL of absolute methanol under argon was added dropwise via syringe 5.2 mL (2.6 mmol) of stock 0.5M guanidine in methanol prepared above. The reaction was stirred at room temperature for 30 minutes. TLC on silica gel with 80% ethyl acetate in hexane showed complete conversion of acetate to free alcohol. The reaction was quenched by the addition of 314 µL (5.5 mmol) of glacial acetic acid. The solvent was removed at reduced pressure and the residue partitioned between 150 mL of 1:1 toluene/ethyl acetate and 50 mL of water. The aqueous layer was further extracted with 50 L of ethyl acetate. The combined organic layers were washed with two 25-mL portions of water, 25 mL of brine and dried over sodium sulfate to give 6.25 g of crude residue. This was purified by flash chromatography on silica gel with gradient elution of 50 to 75% ethyl acetate in hexane to give 4.89 g (77%) of R-alcohol ( 1R ). HCLC on Chiralcel OD using 93:7 hexane:isopropanol as eluant indicated a 98.8% ee for the desired product. This product was identical to previously prepared 3R-alcohol by HPLC, ¹H and ¹³C NMR and TLC. EXAMPLE 6 Preparation of Methyl 7-[5-oxo-3R-[(triethylsilyl)oxy]-1 -cyclopenten-1-yl]-4Z-heptenoate (3) To a 10°C solution of 34.60 g (0.136 mole) of 94% pure 3R-alcohol ( 1R ), 34.3 g (0.34 mole) of triethylamine, 4.76 g (0.07 mole) of imidazole in 100 mL of DMF under nitrogen, was added dropwise via syringe 24.0 g (26.7 mL, 0.16 mole) of triethylsilylchloride. The mixture was warmed to room temperature for 4 hours. TLC (silica gel with 1:1 ethyl acetate:hexane as eluant) showed complete conversion of alcohol (R f = .60). The mixture was poured into 300 mL of 1:1 toluene:hexane and this was washed with 300 mL of water followed by three 100-mL portions of water, then 50 mL of brine and dried over sodium sulfate. Removal of solvent at reduced pressure followed by in vacuo treatment at 2 x 10⁻² torr at 50° for 2 hours gave 44.76 g crude product which was purified by chromatography on silica gel using a step gradient of 10 to 20% ethyl acetate in hexane. 40.25 g (84%) of purified TES-enone was obtained in this manner . HPLC on Chiralcel OD using 93:7 hexane:isopropanol indicated an enantiomer ratio (R/S) of 99.3:0.7. ¹H NMR (CDCl3): δ 7.04 (m, 1H, C₂H), 5.34 (m, 2H, cis olefin), 4.90 (m, 1H, C₃H), 3.68 (S, 3H, OCH₃), 2.75 (dd, 1H, C 4β H) J = 6.0, 18.0 Hz, 2.29 (dd, 1H, C 4α H) obscured, 2.4-2.2 (m, 8H, CH₂), 1.0 (t, 9H, 3CH₃) J = 8.0 Hz, 0.67 (q, 6H, 3CH₂) J = 8Hz; ¹³C NMR (CDCl₃): δ 206.3, 173.7, 157.3, 146.8, 130.3, 129.0, 69.1, 51.9, 45.9, 34.4, 25.4, 24.8, 23.2, 7.1, 5.1 ppm; IR (CHCl₃):3020, 3010, 1735, 1710, 1440, 1355, 1235, 1080 cm⁻¹; UV (CH₃OH) λ max = 222 nM; [α] 20 / D + 12.3° (c 0.814 g/dL, , CHCl₃) (-1018.4° at 365 nM); CD [ϑ]²⁵ (nM) -12166 (315) (negative maximum); +66507 (224) (positive maximum) (CH₃OH); Anal. calc'd. for C₁₉H₃₂O₄Si: C, 64.75; H, 9.15; found: C, 64.67; H, 9.20. EXAMPLE 7 A mixture of 240 mg (1.0 mmol) of the title enone, 240 mg (3192 units) of porcine pancreatic lipase in 3 mL of distilled vinyl acetate was sealed and stirred at room temperature for a total of 9 days. Analysis on HPLC using Chiralcel OC at 50°C using 90:10 hexane: isopropanol as eluant indicated that remaining alcohol was of >99.8% S-isomer. Resolution of the corresponding acetate was not possible under a variety of conditions. The reaction mixture was filtered through a bed of diatomaceous earth and the filter cake rinsed with methylene chloride. The combined filtrates were concentrated at reduced pressure to give 69 mg of crude residue which was purified by PTLC on silica gel (2000µ) using 65% ethyl acetate in hexane as eluant (R f (ROH) = .30 and R f (ROAc) = 0.61). In this manner was isolated 130 mg (46%) of methyl 7-(3R-acetyloxy-5-oxo-1-cyclopenten-1-yl)-heptanoate:¹H NMR (CDCl3) δ 7.10 (m, C₂H, 1H), 5.66 (dm, C₃H, 1H), 3.68 (s, OCH₃, 3H), 2.87 (dd, C 4β H, 1H) J = 6, 19.0 Hz, 2.38 (dd, C 4α H, 1H) J = 2.0, 19 Hz, 2.31 (t, CH₂,2H) J = 7.5 Hz, 2.21 (bt, 2H) J = 7.5 Hz, 2.10 (s, OAc, 3H), 1.62 (m, 2H), 1.35 (m, 4H); ¹³C NMR (CDCl₃), δ 204.5, 173.8, 170.3, 151.4, 149.6, 70.2, 51.2, 41.3, 33.7, 28.7, 28.5, 26.9, 24.5, 24.3, 20.6 ppm; IR (CHCl₃) 3020, 3010, 1715 (shoulder at 1735), 1435,1370, 1240, 1025 cm⁻¹; UV (CH₃OH) λ max = 221 nM; [α] 20 / D +47.6° (c 0.871 g/dL, CHCl₃) (-649.3° at 365 nM); CD [ϑ]²⁵ (nM) -7556 (315) (negative maximum), +49533 (224) (positive maximum); Anal. calc'd. for C₁₅H₂₂O₅: C, 63.80; H, 7.86; found: C, 63.32; H, 7.91 and then 99 mg (41%) of methyl 7-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)-heptanoate, mp = 60.1° (DSC), whose ¹H and ¹³C NMR, IR, and UV spectra were identical to racemic alcohol. [α] 20 / D -9.8° (c 1.072 g/dL, CHCl₃) (+1216.5° at 365 nM); CD []²⁵ (nM) -7556 (315) (negative maximum), +49533 (224) (positive maximum) (CH₃OH); anal. calc'd. for C₁₃H₂₀O₄: C, 64.98; H, 8.39; found: C, 64.78; H, 8.52. EXAMPLE 8 A mixture of 116 mg (0.49 mmol) of title compound, 116 mo (1543 units) of porcine pancreatic lipase (PPL) in 3 mL of distilled vinyl acetate was sealed and stirred at room temperature for 7 days. Aliquots were removed every 24 hours for HPLC analysis on Chiralcel OC at 50°C using 90/10 hexane/isopropanol as eluant. After 4 days, HPLC showed that remaining S-alcohol was of 96% ee in S-isomer. The product acetate could not be resolved under these conditions. The crude reaction mixture was filtered through diatomaceous earth and the filter pad was washed with methylene chloride. The combined filtrates were concentrated at reduced pressure to give 128 mg crude residue. Purification by PTLC on 2000µ silica plates (R f (ROAc) = 0.55 and R f (ROH) = 0.28) using 65% ethyl acetate/hexane as eluant gave 58 mg (43%) of methyl 7-(3R-acetyloxy-5-oxo-1-cyclopenten-1-yl)-4-heptynoate ¹H NMR (CDCl₃) δ 7.25 (m, C₂H, 1H), 5.80 (dm, C₃H, 1H), 3.70 (s, OCH₃, 3H), 2.88 (dd, C 4β H, 1H) J = 6.5, 19 Hz, 2.55-2.3 (m, 4CH₂ + C 4α H, 9H), 2.10 (S, OAc, 3H); ¹³C NMR (CDCl₃) δ 204.7, 172.4, 170.5, 152.8, 147.8, 79.4, 79.4, 70.4, 41.7, 41.4, 33.7, 24.1, 20.9, 16.8, 14.6 ppm; IR (CHCl₃) 3020, 3010, 1735, 1717, 1437, 1370, 1240, 1027 cm⁻¹ [α] 20 / D ; + 41° (c 0.976 g/dL, CHCl₃) (-606.4° at 365 nM); CD [ϑ]²⁵ (nM) -6022 (315) (negative maximum), +4668 (220 nM) (positive maximum) and 51 mg (45%) of methyl 7-(3S-hydroxy-5-oxo-1-cyclopentenyl)-4-heptynoate whose TLC, ¹H and ¹³C NMR, IR were identical to racemic alcohol; [α] -16.7° (c 0.927 g/dL CHCl₃) (+984.4° at 365 nM); CD [ϑ]²⁵ (nM) + 7690 (312), -55275 (225) (CH₃OH). EXAMPLE 9 A mixture of 166 mg (1.22 mmol) of the title alcohol, 166 mg (2208 units) of porcine pancreatic lipase in 4 mL of distilled vinyl acetate was sealed and stirred at room temperature for a total of 7 days. Aliquots were removed periodically for assay on Chiralcel OA at 50°C using 96/4 hexane/isopropanol as eluant. After 7 days, HPLC indicated that remaining S-alcohol was of 98% ee and that the product acetate R/S isomers were again unresolved. The reaction mixture was filtered through diatomaceous earth and the filter cake washed with methylene chloride. The combined filtrates were concentrated at reduced pressure to give 205 mg of crude residue which was purified by PTLC on 2000µ silica gel plates using 65% ethyl acetate in hexane as eluant (R f (ROAc) = 0.60 and R f (ROH) = 0.37). In this manner was isolated 76 mg (35%) of 3-(3R-acetyloxy-5-oxo-1-cyclopenten-1-yl)propyne: ¹H NMR (CDCl₃) δ 7.44 (q, C₂H, 1H) J = 2.1 Hz, 5.75 (m, C₃H, 1H), 3.13 (q, CH₂, 2H) J = 2.1 Hz 2.93 (dd, C 4β H, 1H) J = 2.2, 19.0 Hz, 2.21 (t, C≡C- H , 1H) J = 5.3 Hz, 2.11 (s, OAc, 3H); ¹³C NMR (CDCl₃) δ 202.6, 170.2, 153.5, 144.7, 78.8, 71.2, 69.8, 41.6, 20.7, 15.2 ppm; IR (CHCl₃): 3300, 3020, 3010, 1740, 1720, 1640, 1410, 1370, 1240, 1025 cm⁻¹; UV (CH₃OH) λ max = 219 nM; [α] 20 / D 52.2° (c 0.928 g/dL CHCl₃) (-713° at 365 nM); CD [ϑ]²⁵ (nM) -3968 (319), +27494 (216) (CH₃OH), no elemental analysis, and 65 mg (48%) of 3-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)propyne whose ¹H NMR, IR, and UV were identical to racemic alcohol; [α] 20 / D -8.7° (c 0.863 g/dL, CHCl₃) (+1693° at 365 nM; CD [ϑ]²⁵ (nM) + 9923 (318 nM), -50781 (224) (CH₃OH). EXAMPLE 10 A mixture of 83 mg (0.35 mmol) of the title alcohol, 83 mg (1104 units) of porcine pancreatic lipase in 3.0 mL of distilled vinyl acetate was sealed and stirred at room temperature for a total of 7 days. Aliquots were removed every 24 hours for HPLC analysis on Chiralcel OD using 97/3 hexane/isopropanol as eluant. After 7 days, HPLC indicated that remaining alcohol had a 96/4 S/R in isomer ratio and that this ratio was still improving. Nevertheless, the mixture was filtered through bed of diatomaceous earth and the filter pad was rinsed with methylene chloride. The combined filtrates were concentrated at reduced pressure to give 86 mg crude residue. PTLC on 2000µ silica gel plates using 65% ethyl acetate in hexane as eluant gave 45 mg (R f = 0.61, 46%) of methyl 7- (3R-acetyloxy-5-oxo-1-cyclopentene-1-yl)-5Z-heptenoate: ¹H NMR (CDCl₃) δ 7.12 (m, C₂H, 1H), 5.72 (dm, C₃H, 1H), 5.47 (m, olefinic H, 2H), 3.68 (s, OCH₃, 3H), 2.96 (m, bis allylic CH₂), 2.89 (dd, C 4β H, 1H) J = 6.5, 19.0 Hz, 2.48 (dd, C 4α H, 1H) J = 6.5, 19.0 Hz, 2.48 (dd, C 4a H, 1H) J = 2.1, 19.0 Hz, 2.31 (t, CH₂CO₂, 2H) J = 10.1 Hz, 2.10 (m, 2H), 2.10 (s, OAc, 3H), 1.70 (quint, isolated CH₂, 2H) J = 7.5 Hz; IR (CHCl₃) 3020, 3010, 1715 (shoulder at ∼1735), 1435, 1370, 1240, 1025 cm⁻¹; [α] 20 / D +47.1° (c 0.935 g/dL, CHCl₃) (-649.2° at 365 nM); UV (CH₃OH) λ max = 218 nM; CD [ϑ]²⁵ (nM) -7556 (315), +49533 (224) (CH₃OH); anal. calc'd. for C₁₅H₂₀O₅: C, 63.81; H, 7.14; found: C, 63.65; H, 7.04 and then 33 mg (R f = 0.34, 40%) of methyl 7-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)-5Z-heptenoate whose ¹H NMR, IR, TLC were identical to racemic alcohol; [α] 20 / D -19.9° (c 0.627 g/dL, CHCl₃) (+1031° at 365 nM); CD [ϑ]²⁵ (nM) + 24731 (314), -175176 (225) (CH₃OH), no elemental analysis. EXAMPLE 11 Immobilization of PSL Using XAD-8 Resin A solution of PSL (100 mg) in 10 ml of 0.05 M phosphate buffer solution (pH = 7.0) was mixed with 10 g of polystyrene beads (XAD-8 from Sigma). The suspension was stirred overnight at 8°°C. Most of the water was removed by pipet, the residue was dried over a cacuum pump (rt, 24 hours) to give the immobilized enzyme which was directly used for transesterification.

Claims: A method for resolution of a compound of the formula: wherein X represents alkyl, alkenyl or alkynyl of from 2 to 10 carbon atoms, optionally including a S or O atom at the 2, 3, 4, 5, 6, 7, 8 or 9 postion, and R is -CH₂OR₁ wherein R₁ is hydrogen, alkyl of 1 to 6 carbon atoms, tetrahydropyranyl, ethoxyethyl, acyl, or -CO₂R₂, or (R₂)₃Si wherein R₃ is independently alkyl of 1-10 carbon atoms or aryl, wherein R₂ is alkyl of 1 to 6 carbon atoms, said method comprising: mixing said compound with a lipase in the presence of an acylating reagent to thereby obtain the corresponding S alcohol and R acetat; and treating said S alcohol with diethylazocarboxylate, triphenyl phosphine and formic acid followed by hydrolysis of the intermediate formate ester with neutral alumina and methanol to obtain the corresponding R alcohol with essentially complete inversion. The method of Claim 1 wherein the acylating reagent is selected from the group consisting of isopropenyl acetate, isopropenyl valerate, vinyl acetate, vinyl propionate and vinyl valerate. The method of Claim 2 wherein the lipase is selected from the group consisting of Pseudomonas species (PSL, Type XIII), porcine pancrease (PPL, Type II), candida cylindracea (CCL, Type VII), amano P, ANL, aspergillus niger, or ChE cholesterol esterase. The method of Claim 1 wherein the lipase is porcine pancreas (PPL, Type II) or amano P. The method of Claim 4 wherein the acylating reagent is isopropenyl acetate or vinyl acetate. The method of Claim 1 wherein the lipase is porcine pancreas (PPL), the acylating reagent is vinyl acetate and said compound is: Methyl 7-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)-4Z-heptenoate (1S) Methyl 7-(3-hydroxy-5-oxo-1-cyclopenten-1-yl)-4-heptenoate (4) 3-(3-hydroxy-5-oxo-1-cyclopenten-1-yl) propyne (5) Methyl 7-(3-hydroxy-5-oxo-1-cyclopenten-1-yl)-5Z-heptenoate (6) or Methyl 7-(3-hydroxy-5-oxo-1-cyclopenten-1-yl)-heptenoate (7). The method of Claim 6 wherein said compound is methyl-7-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)-4Z-heptenoate. The method of Claim 6 wherein the lipase is immobilized on a support. A method for resolution of a compound of the formula: wherein X represents alkyl, alkenyl or alkynyl of from 2 to 10 carbon atoms, optionally including a S or O atom at the 2, 3, 4, 5, 6, 7, 8 or 9 position and R is -CH₂OR₁ wherein R₁ is hydrogen, alkyl of 1 to 6 carbon atoms, tetrahydropyranyl, ethoxyethyl, acyl or -CO₂R₂ wherein R₂ is alkyl of 1 to 6 carbon atoms, said method comprising: mixing said compound with a lipase in the presence of an acylating reagent to thereby obtain the corresponding S alcohol and R acetate: treating said R acetate compound with guanidine and an alcohol to obtain the corresponding R alcohol; mixing said R alcohol with a lipase in the presence of an acylating reagent to thereby obtain the corresponding R acetate; and treating said R acetate with guanidine and an alcohol to thereby obtain the optically pure R alcohol compound. The method of Claim 9 wherein the acylating reagent is selected from the group consisting of isopropenyl acetate, isopropenyl valerate, vinyl acetate, vinyl propionate and vinyl valerate. The method of Claim 10 wherein the lipase is selected from the group consisting of Pseudomonias species (PSL, Type XIII), porcine pancreas (PPL, Type II), candida cylindracea (CCL, Type VII) and amano P. The method of Claim 9 wherein the lipase is porcine pancreas (PPL, Type II) or amano P. The method of Claim 12 wherein the acylating reagent is isopropenyl acetate or vinyl acetate. The method of Claim 9 wherein the lipase is porcine pancreas (PPL), the acylating reagent is vinyl acetate and said compound is: Methyl 7-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)-4Z-heptenoate (1S) Methyl 7-(3-hydroxy-5-oxo-1-cyclopenten-1-yl)-4-heptenoate (4) 3-(3-hydroxy-5-oxo-1-cyclopenten-1-yl) propyne (5) Methyl 7-(3-hydroxy-5-oxo-1-cyclopenten-1-yl)-5Z-heptenoate (6) or Methyl 7-(3-hydroxy-5-oxo-1-cyclopenten-1-yl)-heptenoate (7). The method of Claim 14 wherein said compound is methyl-7-(3S-hydroxy-5-oxo-1-cyclopenten-1-yl)-4Z-heptenoate. The method of Claim 6 wherein the lipase is immobilized on a support. The method of Claim 1 wherein the lipase is mixed with the acylating reagent in the presence of a solvent selected from the group consisting of CHCl₃, benzene, toluene, t-butylmethyl ether, tetrahydrofuran, diethyl ether or hexane. The method of Claim 9 wherein the lipase is mixed with the acylating reagent in the presence of a solvent selected from the group consisting of CHCl₃, benzene, toluene, t-butylmethyl ether, tetrahydrofuran, diethyl ether or hexane.

Inventors: BABIAK KEVIN ANTHONY, DYGOS JOHN H, HENNEN WILLIAM J, NG JOHN S, WANG YI-FONG, WONG CHI-HUEY, BABIAK, KEVIN ANTHONY, DYGOS, JOHN H., HENNEN, WILLIAM J., NG, JOHN S., WANG, YI-FONG, WONG, CHI-HUEY
Applicants: SEARLE & CO, G.D. SEARLE & CO.
Application year: 1989
Main classification: C12P41
IPC codes: C12P41, C12P17, C12P9, C12P19, C12P7, C12P13