STEREOSELECTIVE MICROBIAL REDUCTION PROCESS

Abstract: A stereoselective reduction of compound of formula II to compound of formula I, which comprises adding compound of formula II to a culture broth of the Zygosaccharomyces bailii ATCC 38924, incubating the resulting mixture, and isolating a hydroxy compound of formula I, is described. A stereoselective reduction of a compound of formula IV to compound of formula III, which comprises adding a compound of formula IV to a culture broth of Schizosaccharomyces octosporus ATCC 2479, incubating the resulting mixture, and isolating a hydroxy compound of formula III is also described. The resulting compounds of formula I and III are useful as intermediates in the preparation of 1-(4-fluorophenyl)-3(R) -[3(S)-hydroxy-3-(4-fluorophenyl)propyl)]-4(S)-(4-hydroxyphenyl)-2-azetidionone which is a serum cholesterol lowering agent.

Description: STEREOSELECTIVE MICROBIAL REDUCTION PROCESS BRIEF SUMMARY OF THE INVENTION The present invention relates to microbiological reduction of carbonyl groups which comprises cultivating the microorganism Zygosaccharomyces bailii American Type Culture Collection (ATCC) 38924, which can be obtained from the American Type Culture Collection, in a medium to which the ketone compound, 4-(4- Fluorobenzoyl)butyric acid (also referred to here as FBBA) can be added so that a compound having a hydroxy group of desired stereochemistry can be formed, accumulated and isolated from said medium. The resulting hydroxy compound, (S)-5-(4-fluorophenyl)hydroxyvaleric acid (FPHVA), is useful as an intermediate in the preparation of 1 -(4-fluorophenyl)-3(R) -[3(S)-hydroxy-3-(4-fluorophenyl)propyl)]-4(S)-(4-hydroxyphenyl)-2- azetidinone which is a serum cholesterol lowering agent. More specifically, the hydroxy and acid groups of the hydroxy intermediate, FPHVA, can be protected by protecting group chemistry such as that set forth in Protective Groups in Organic Chemistry. T.W. Greene, John Wiley &Sons (1981) which is hereby incoφorated by reference. The resulting compound is encompassed by formula in set forth at page 8, line 18 of PCT/U S94/10099 published 30 March 1995, which is hereby incoφorated by reference. PCT/US94/10099 shows the protected form of FPHVA as being converted to the serum cholesterol lowering agent, mentioned above. The invention also relates to microbiological reduction of carbonyl groups in FBBA-phenyloxazolidinone conjugate by selected microbes including Schizosaccharomyces octosporus ATCC #2479. Detailed Description of the Invention As noted above, this invention relates to a method for performing the following stereospecific reduction using a microorganism. microbial reductase The microbiological reduction is carried out by adding the ketone substrate FBBA (the compound of formula II above), to the culture broth containing the microorganism. The incubation may be conducted at temperatures in the range from between about 20°C and about 40°C, and is preferably conducted at 3θOC, while adjusting the initial pH value of the culture medium in the range from between about 4.0 and about 8.0, preferably 5.5. The initial concentration of compound π in the reaction may vary from between about 1.0g/L and about 20.0 g/L, and is preferably 10.0 g/L. The duration of the chiral reduction reaction may vary from about 18 to about 96 hours, and is preferably about 48-72 hours. At the end of the reduction reaction, FPHVA ( the hydroxy compound of formula I), may be extracted using organic solvents such as ethyl acetate, t-butyl methyl ether (TBME), methylene chloride and the like. Adsoφtion to resins, chromatography, and other physical methods known to the art may also be used to extract compound I. A large number of microorganisms were investigated to determine whether they reduce the ketone group of compound II. Many such microorganisms failed to provide the desired specificity or productivity. Of those that did provide the desired specificity or productivity for this reduction, Z. bailii ATCC No. 38924, afforded the highest yields of compound I with the desired enantiomeric purity, from the highest starting concentration of the ketone, compound II . This is very desirable, since carrying out this reaction at the highest starting concentration of compound π , affords the most economical means of obtaining the desired alcohol, compound I. In the examples below are given the following: Example 1. A means for identifying the stereoselective reduction of compound π by microorganisms including Z. bailii ATCC No. 38924. Example 2. A means for determining that the stereoselective reduction of compound π by cultures including Z. bailii ATCC No. 38924, can be carried out at greater concentrations of compound π than in the original identification example. Example 3. A means for carrying out the stereoselective reduction of compound II by Z. bailii ATCC No. 38924, as flask fermentations using greater concentrations of compound π than used in example 2. Example 4. A means for carrying out the stereoselective reduction of compound II by Z. bailii ATCC No. 38924 in flask fermentations, so as to obtain gram quantities of hydroxy compound I. Example 5. A means for carrying out the stereoselective reduction of compound II by Z. bailii ATCC 38924, using a 10-liter fermentor. The invention also relates to microbiological reduction of carbonyl groups in FBBA-phenyloxazolidinone conjugate (the compound of formula IV) by selected microbes including Schizosaccharomyces octosporus ATCC #2479, to obtain the compound of formula III using the appropriate conditions of temperature, initial pH of the starting medium, initial concentration of the compound of formula IV, and duration of the reaction. microbial reductase i m The resulting compound of formula HI may also be converted to cholesterol lowering agents. Example 1. The general method for identifying the stereoselective microbial reduction of 4-(4-Fluorobenzoyl)butyric acid (FBBA, compound π) for use as a synthetic precursor for the production of 1-(4-fluorophenyl)-3(R) -[3(S)-hydroxy-3-(4-fluorophenyl)propyl)]-4(S)-(4-hydroxyphenyl)-2- azetidinone is described below. Seed cultures of yeast, filamentous fungi, and bacteria were grown in 125 mL flasks containing 25 mL of YPD (1 % yeast extract, 2% peptone, 2% dextrose; pH 5.5), SIM6 (3.5% soy flour, 5% white potato dextrose,0.5% cerelose, 2mg/L cobalt chloride, 0.5% calcium carbonate; pH 6.0 ) and NYC (0.8% nutrient broth, 2% yeast extract, 2% cerelose; pH 7.0) media respectively, for 72 hours at 30°C with agitation (175-250 φm) prior to inoculation (4% v/v) into flask fermentations (25mL YPD/125 mL flask for yeast and filamentous fungi or 25mL NYC /125 mL flask for bacteria) which were incubated at 30°C with agitation (250 rpm). In all fermentations, medium pH was adjusted prior to inoculation but was not controlled during culture propagation and ketone reduction. Ketone reduction was initiated by adding compound π (1 g/L) dissolved in methanol (100 mg/mL) directly to cultures following 24 hours of growth. Samples of fermentation broth extracted with TBME (1 :2) following 24-48 hours incubation with substrate were analyzed by reverse-phase HPLC. Cultures demonstrating consistent reduction activity without significant substrate degradation following repeated fermentations using this procedure, were further analyzed by chiral HPLC to determine the configuration of the product alcohol. Cultures capable of reducing compound II at 1 g/L in high enantiomeric excess (ee > 95%) producing compound I the S enantiomer, are summarized in Table 1. Table 1. Microorganisms capable of reducing 4-(4- Fluorobenzyol) butyric acid (compound II) at 1 g/L to (S)-5-(4-Fluorophenyl)-5-hydroxy- valeric acid (compound I) with > 95% ee. Genus species ATCC Strain # Candida kefyr 748 14244 Candida guilliermondii 9058 20118 Candida parapsilosis 7330 16632 20008 22019 34078 Geotrichum candidum 34614 Hansenula subpelliculosa 20281 Kluyveromyces marxianus 8554 10022 12424 Saccharomyces cerevisiae 26108 34081 36900 Saccharomyces uvarum 10613 32634 Torulaspora delbrueckii 10662 Torulaspora fermentati 20100 Torulaspora hansenii 34022 Yarrowia lipolytica 8661 9773 Zygosaccharomyces bailii 36946 38924 Example 2. The general method for identifying microorganisms including Z bailii ATCC 38924 capable of reducing compound π at concentrations greater than those used in Example 1 is described below. Seed cultures of yeast were grown in 125 mL flasks containing 25 mL of YPD medium (1% yeast extract, 2% peptone, 2% dextrose; pH 5.5) for 72 hours at 30°C with agitation (250 rpm) prior to inoculation (4% v/v) into flask fermentations (25 mL YPD/125 mL flask) incubated at 30°C with agitation (250 φm). In all fermentations, medium pH was adjusted prior to inoculation but was not controlled during culture propagation and ketone reduction. Ketone reduction was initiated by adding compound π (1-4 g/L) dissolved in methanol (100 mg/mL) directly to cultures at inoculation, following 24 hours of growth or both as indicated in Table 2. Samples of fermentation broth extracted with TBME (1 :2) following 24-48 hours incubation with compound II were analyzed by HPLC to assess the yield of compound I as summarized in Table 2. Table 2. Cultures capable of reducing Compound II at 1-4 g/L in flask fermentations. Genus species ATCC # Condition % Yield, Compound I 1g/L at log 24 98 Candida kefyr 748 2 g/L at log 24 70 4 g/l at log 24 34 1g/Lat log 24 95 Candida kefyr 14244 2 g/L at log 24 80 4 g/L at log 24 28 1 g/L at log 24 90 Candida guilliermondii 9058 2 g/L at log24 9 4 g/L at log 24 0 1g/Latlog24 100 Candida parapsilosis 7330 1 g/L at logs 88 0+24 4 g/L at log 24 6 1 g/L at log 24 100 Candida parapsilosis 16632 1 g/L at logs 100 0+24 4 gL at log 24 18 1g/Latlog24 100 Candida parapsilosis 22019 1 g/L at logs 99 0+24 4 g/L at log 24 15 1g/Latlog24 86 Kluyveromyces 2 g/L at log 24 93 marxianus 8554 4 g/L at log 24 32 1g/Latlog24 98 Saccharomyces 1 g/L at logs 42 cerevisiae 26108 0+24 4 g/L at log 24 13 1 g/L at log 24. 92 Saccharomyces 1 g/L at logs 83 cerevisiae 34081 0+24 34 4 g/Lat log 24 1g/Latlog24 95 Saccharomyces 1 g/Lat logs 66 cerevisiae 36900 0+24 4 g/L at log 24 25 1g/L at log 24 94 Saccharomyces 1 g/L at logs 47 uvarum 32634 0+24 4 g/L at log 24 6 1g/L at log 24 68 Torulaspora 2 g/L at log 24 42 delbruθckii 10622 4 g/L at log 24 10 1g/L at log 24 95 Torulaspora 2 g/L at log 24 30 fermentati 20100 4 g/L at log 24 6 Zygosaccharomyces 4 g/L at log 24 84 bailii 36946 Zygosaccharomyces 4 g/L at log 24 100 bailii 38924 Example 3. The general method for investigating the fermentation parameters for the reduction of compound π by Z. bailii ATCC 38924 in flask fermentations is described below. Seed cultures of Z. bailii ATCC 38924 were grown in 125 mL or 300 mL flasks containing 25mL or 100 mL of YPD (1% yeast extract, 2% peptone, 2% dextrose; pH 5.5) or TNC (1% Tastone 154, 2% NZ amine, 3% cerelose; pH 5.5) for 24-72 hours at 30°C with agitation (250 φm) prior to inoculation (2-8 % v/v) into flask fermentations (25mL-100 mL YPD or TNC 1.25-300 mL flask) which were incubated at 30°C with agitation (250 rpm) where indicated in Table 3. In all conditions, medium pH was adjusted prior to inoculation but was not controlled during culture propagation and ketone reduction. Ketone reduction was initiated by adding compound π (4-10 g/L) dissolved in methanol (MeOH; 100 mg/mL) or dimethylsulfoxide (DMSO; 500 mg/mL with slight heat) directly to cultures following 24 hours of growth. Samples of fermentation broth were taken following 24-72 hours of incubation with compound II and extracted with TBME (1 :4). Extracts were diluted with ethanol (1:1) prior to analysis by reverse-phase HPLC to determine the yield of compound I as summarized in Table 3. Example 4. Gram quantities of compound I derived from the stereoselective reduction of compound π were prepared using Z. bailii ATCC 38924 in flask fermentations employing conditions affording the highest yield and selectivity as summarized in Table 3 parameter set 27. Specifically, seed cultures of Z bailii ATCC 38924 were grown in 300 mL flasks containing 100 mL of TNC medium (1% Tastone 154, 2% NZ amine, 3% cerelose; pH 5.5) for 72 hours at 30°C with agitation (250 φm). Medium pH was adjusted prior to inoculation but was not controlled during culture propagation or subsequently during ketone reduction. Seed inoculum (4% v/v) was transfered into fermentation flasks (300 mL) containing 100 mL of TNC medium and propagated at 30°C with agitation (250 φm) for 24 hours prior to the addition of 10 g/L of compound π dissolved in dimethylsulfoxide (DMSO; 500 mg/mL with slight heat). Following 24 hours of incubation with compound II, additional cerelose (3% w/v) was added to the fermentation. Samples of fermentation broth taken during incubation with compound π, were extracted with TBME (1:4), diluted with ethanol (1 :1) and analyzed by reverse-phase HPLC. Reduction of compound π to compound I was shown to be complete following 72 hours of incubation. Compound I was recovered from the fermentation broth (5 X 100 mL) by removing cells by centrifugation and extracting the supematant with TBME (2X 250 mL) and ethyl acetate(1X200 mL). The pooled extract was washed with distilled water and saturated sodium chloride solution (2X 250 mL). Anhydrous magnesium sulfate was added to the washed solvent extract to adsorb residual water and removed by filtration. The filtrate was concentrated by evaporation and subjected to purification by silica gel chromatography (2X 5-10 inch column bed). Material was eluted from the column with a solution of methylene chloride:ethyl acetate (60:40). Peak fractions containing only compound I were identified by thin layer chromatography, pooled and concentrated by evaporation. A total of 3.43 g of compound I was isolated from a 5 g bioconversion. Samples of this material were confirmed to be the desired product compound I, by reverse phase and chiral HPLC, NMR, mass spectrum and elemental analyses. Example 5. The general method for the bioconversion of compound π to compound I using Z. bailii ATCC 38924 in 10 liter fermentors is described below. First stage seed cultures were grown in TNC medium (1% Tastone 154, 2% NZ amine, 3% cerelose; pH 5.5) using 100mL/300 mL flasks for 24 hours at 30° C with agitation (250 φm) prior to serial transfer (5%) into second stage seed fermentations employing TNC medium (500 mL/ 2 L flask) incubated at 30° C with agitation (200 φm). Following 72 hours of growth, second stage seed cultures were used to inoculate (5 % v/v) fermentors containing 5-10 liters of BTX-1-X02 medium (1% Tastone 154, 2% NZ amine, 12% cerelose, 0.5 mL SAG-471 antifoam/L medium; pH 5.5). The culture was propagated in the fermentor at 30° C, with agitation (impellor speed; 600-1000 φm) and aeration (airflow; 3-10 Lpm) under pressure (5 psi). Dissolved oxygen was maintained at or above 30% by adjusting agitation speed and aeration rates. Culture growth was monitored by analyzing off gas for carbon dioxide and medium pH was adjusted prior to inoculation but was not controlled during culture propagation and ketone reduction. Ketone reduction was initiated by adding compound π (10 g/L) dissolved in dimethyl sulfoxide (400 mg/mL) directly to cultures when the off gas attained an initial concentration of 3.9%. Cerelose (3% w/v) was added to the ongoing fermentation when the off gas attained both an initial concentration of 2.5 %, and a maximum carbon dioxide evolution. Additional 6% cerelose was added following maximum carbon dioxide evolution when the off gas contained 2% carbon dioxide. Samples of fermentation broth were taken following 24-72 hours of incubation with compound π and extracted with TBME (1 :4). Extracts were diluted with ethanol (1 :1) prior to analysis by reverse-phase HPLC. Approximately 80-90% of compound π was reduced to compound I following 48-72 hours of incubation. Table 3. Reduction of Compound 11 (4-10 g/L) by Z. bailii ATCC 38924 in flask fermentations. Example 6. Reduction of FBBA-phenyloxazolidinone conjugate by selected microbes including Schizosaccharomyces octosporus ATCC #2479. The general method for identifying the stereoselective microbial reduction of the condensation product compound IV, formed from 4-(4- Fluorobenzoyl)butyric acid (FBBA) and phenyloxazolidinone, to compound ru for use as a synthetic precursor for the production of 1 -(4- fluorophenyl)-3(R) -[3(S)-hydroxy-3-(4-fluoro-phenyl)propyl)]-4(S)-(4- hydroxyphenyl)-2-azetidinone is described below. m Seed cultures of yeast, filamentous fungi, and bacteria were grown in 125 mL flasks containing 25 mL of YPD (1% yeast extract, 2% peptone, 2% dextrose; pH 5.5), SIM6 (3.5% soy flour, 5% white potato dextrose, 0.5% cerelose, 2mg/ cobalt chloride , 0.5% calcium carbonate; pH 6.0) and NYC (0.8% nutrient broth, 2% yeast extract, 2% cerelose; pH 7.0) media respectively, for 72 hours at 30°C with agitation (175-250 φm) prior to inoculation (4 % v/v) into flask fermentations. In all fermentations, medium pH was adjusted prior to inoculation but was not controlled during culture propagation and ketone reduction. Ketone reduction was initiated by adding compound IV (1 g/L) dissolved in methanol (100 mg mL) directly to cultures following 24 hours of growth in (25mL YPD/125 mL flask for yeast and filamentous fungi or 25mL NYC /125 mL flask for bacteria) incubated at 30° C with agitation (250 rpm). Synthesis and enantiomeric excess of compound HI were determined by chiral HPLC using samples of fermentation broth extracted with TBME (1 :2) following 24-48 hours incubation with compound IV. Cultures capable of reducing compound IV yielding compound HI are summarized in Table 4. Schizosaccharomyces octosporus ATCC 2479 afforded the highest yield of compound HI with the desired enantiomeric purity from 1 g/L of compound IV. Table 4. Microbes capable of reducing compound IV (FBBA- phenyloxazolidinone conjugate) to compound III (S,S diastereomer) with > 95% de. Genus species ATCC strain # Rhodococcus erythropolis 4277 Rhodococcus globerulus 21505 Rhodococcus rhodochrous 9356 Mucor mucedo 20094 Mucor racemosus 7924 Mucor circinelloides 1207a 7941 56641 Rhizopus oryzae 11145 24563 Aspergillus carneus 16798 20231 Aspergillus niveus 12276 Schizosaccharomyces octosporus 2479 4206 Multiple flask bioconversions were conducted employing S. octosporus ATCC 2479 to generate material for chemical characterization. Seed cultures were grown in YPD medium (25 mLJ125 mL flask) for 72 hours at 30° C with agitation prior to inoculation (4 % v/v) into flask fermentations (20 X 100 ml_ 300mL). Medium pH was adjusted prior to inoculation but was not controlled during culture propagation and ketone reduction. Compound IV (1 g/L dissolved in methanol at 100 mg/mL) was added to flask fermentations after 24 hours of growth at 30°C with agitation (250 φm). Compound IV was reduced to compound m in 20-40% yield. Degradative hydrolysis of compound IV was also observed. The fermentation broth was pooled, centrifuged to remove cells and the supematant extracted with ethyl acetate (1:0.5). The extract was washed twice with an equal volume of salt solution (6M sodium chloride) followed by deionized distilled water. Anhydrous magnesium sulfate was added to the ethyl acetate extract to remove residual water, the extract was filtered and the filtrate concentrated by evaporation. Extract concentrate was subjected to purification by silica gel chromatography (1X 12 inch column bed). Material was eluted from the column with a solution of methylene chloride:ethyl acetate: acetic acid (90:10:0.5). Peak fractions containing products were pooled and concentrated by evaporation, followed by separation on preparative thin layer chromatography plates employing a solution of ethyl acetate: hexane: acetic acid (60:40:0.5). Two products were isolated from the fermentation broth using this procedure and shown to be the desired reduced alcohol conjugate compound HI and the phenyloxazolidinone reagent used to synthesize compound IV, as determined by HPLC and NMR analyses.

Claims: WHAT IS CLAIMED IS: 1. A stereoselective reduction of a compound of formula π to compound of formula I reductase which comprises adding a compound of formula π to a culture broth of Zygosaccharomyces bailii ATCC 38924, incubating the resulting mixture, and isolating a hydroxy compound of formula I. 2. A stereoselective reduction of a compound of formula IV to compound of formula m microbial reductase rv m which comprises adding a compound of formula IV to a culture broth of Schizosaccharomyces octosporus ATCC 2479, incubating the resulting mixture, and isolating a hydroxy compound of formula III.

Inventors: HOMANN MICHAEL J, PREVITE EDWARD, HOMANN, MICHAEL, J., PREVITE, EDWARD
Applicants: SCHERING CORP, SCHERING CORPORATION
Application year: 1996
Main classification: C12P7
IPC codes: C12P7, C12P17, C12R1