ENANTIOSELECTIVE PREPARATION OF PHARMACEUTICALLY ACTIVE SULFOXIDES BY BIOOXIDATION

Abstract: A compound of formula (II) is prepared either as a single enantiomer or in an enantiomerically enriched form, wherein Het1 is (a) or (b) and Het2 is (c) or (d) and X is (e) or (f) wherein N in the benzimidazole moiety means that one of the carbon atoms substituted by R6-R9 optionally may be exchanged for an unsubstituted nitrogen atom; R1, R2 and R3 are the same or different and selected from hydrogen, alkyl, alkoxy optionally substituted by fluorine, alkylthio, alkoxyalkoxy, dialkylamino, piperidino, morpholino, halogen, phenylalkyl, phenylalkoxy; R4 and R4' are the same or different and selected from hydrogen, alkyl, aralkyl; R5 is hydrogen, halogen, trifluoromethyl, alkyl, alkoxy; R6-R9 are the same or different and selected from hydrogen, alkyl, alkoxy, halogen, haloalkoxy, alkylcarbonyl, alkoxycarbonyl, oxazolyl, trifluoroalkyl or adjacent groups R6-R9 may complete together with the carbon atoms to which they are attached optionally substitued ring structures; R10 is hydrogen or alkoxycarbonyloxymethyl; R11 is hydrogen or forms an alkylene chain together with R3; R12 and R13 are the same or different and selected from hydrogen, halogen or alkyl, by a method comprising stereoselective biooxidation of the pro-chiral sulfide counterpart compound.

Description: Enantioselective preparation of pharmaceutically active sulfoxides by biooxidation The present invention relates to a method of preparing compounds as defined below, either as a single enantiomer or in an enantiomerically enriched form, by biooxidation of their sulphide equivalents. Background to the Invention The racemic form of the compounds prepared by the method of the present invention are known compounds. Some of the compounds are also known in single enantiomeric form. The compounds are active HTCATPase inhibitors and they, including their pharmaceutically acceptable salts, are effective acid secretion inhibitors, and known for use as antiulcer agents. The compounds, which include the known compounds omeprazole (compound of formula (Ha) below), lansoprazole (compound of formula (He) below) and pantoprazole (compound of formula (lib) below), are known for example from European Patent Specifications EP 5129 and 124495, EP 174726 and EP 166287. These compounds, being sulfoxides, have an asymmetric centre in the sulfur atom, i.e. exist as two optical isomers (enantiomers). It is desirable to obtain compounds with improved pharmacokinetic and metabolic properties which will give an improved therapeutic profile such as a lower degree of interindividual variation. The separation of enantiomers of omeprazole in analytical scale is described in e.g. J. Chromatography, 532 (1990), 305-19.Also the separation of enantiomers of compounds, including omeprazole and pantoprazole, is described in German Patent Specification DE 4035455. Recently there has been a great deal of literature published relating to the synthesis of optically active compounds using biocatalysts. The majority of this work has been aimed at finding routes to single enantiomer forms of pharmaceuticals. The reactions receiving most attention have been those involved in the preparation of esters, acids and alcohols due to the general utility of these functionalities in synthesis and also because the biocatalysts are readily available. Studies on the synthesis of optically active sulfoxides are relatively rare partly due to the small number of pharmaceuticals containing sulfoxide groups and partly due to the fact that enzymes that react with the sulphur centre are not available commercially. The synthesis of optically active sulfoxides has been described in Holland, H.L. (1988) Chem. Rev. SS, 473-483 and Phillips, R.S. and Sheldon W.M., Enzyme Microb. Technol., 1981, Vol. 3, January, 9-18. Description of the Invention According to the present invention there is provided a method of preparing a compound of formula (II) either as a single enantiomer or in an enantiomerically enriched form: wherein and SUBSTITUTE SHEET and wherein: N in the benzimidazole moiety means that one of the carbon atoms substituted by R j -R, optionally may be exchanged for an unsubstituted nitrogen atom; R., R j and It, are the same or different and selected from hydrogen, alkyl, alkoxy optionally substituted by fluorine, alkylthio, alkoxyalkoxy, dialkylamino, piperidino, morpholino, halogen, phenylalkyl, phenylalkoxy; R and R 4 . are the same or different and selected from hydrogen, alkyl, aralkyl; R j is hydrogen, halogen, trifluoromethyl, alkyl, alkoxy; R j - R, are the same or different and selected from hydrogen, alkyl, alkoxy, halogen, haloalkoxy, alkylcarbonyl, alkoxycarbonyl, oxazolyl, trifluoroalkyl or adjacent groups R j - R, may complete together with the carbon atoms to which they are attached optionally substituted ring structures; R, 0 is hydrogen or alkoxycarbonyloxymethyl; SUBSTITUTE SHEET R.- is hydrogen or forms an alkylene chain together with R Ru and R^ are the same or different and selected from hydrogen, halogen or alkyl, which method comprises stereoselective biooxidation of the pro-chiral sulfide counterpart compound. The compounds of formula (II) are active HTCATPase inhibitors. By the method of the invention these compounds, which are sulfoxides, are obtained in single enantiomer form or such that one enantiomeric form is present in excess leading to an optically active product, by stereoselective biooxidation of the pro-chiral starting sulfide counterpart compound. In the above definitions alkyl groups or moieties may be branched or straight chained or comprise cyclic alkyl groups, for example cycloalkylalkyl. Preferably: and and SUBSTITUTE SHEET X is —CH— " R11 (wherein R . , R,, R, to R^ R. 0 and R,, are as defined above). Most preferably the compounds of formula (II) are compounds of the formula (Da) to (He): SUBSTITUTE SHEET n example of a compound of formula (II) wherein R. 0 is alkoxycarbonyloxymethyl The starring pro-chiral sulfides used in the method of the present invention are of the formula: wherein Hetj, X and Het j are as defined above. SUBSTITUTE SHEET In order to obtain each of the above compounds (ϋa)-(IIf), the following starting compounds of formula (la) to (If), respectively will be required: SUBSTITUTE SHEET The compounds prepared by the method of the invention possess a stereogenic (asymmetric) centre which is the sulfur atom which forms the sulfoxide group between the Het.-X-moiety and the Het j -moiety. The stereoselective biooxidation according to the present invention may be carried out using a microorganism or an enzyme system derivable therefrom. Suitable microorganisms may be selected from alkane oxidisers including Arthrnbartp r pefrolepphjigyp. Previbac erium paraffinolytimm. and AcinetobartPr species, alkene oxidisers such as Mycobacteriv-tπ. species, and a variety of fungal species particularly Penitiføwη species (Penicillium frequentans.. According to one embodiment of the invention the method comprises contacting the pro-chiral sulfide counterpart compound with a microorganism which is Penicillium frequentans Rhizopus stolonifer Cunninghamella elegans Ustiiagp maydis Arthrobacter petroleopha us Brevibacterium paraffinolyticum Adnetobacter sp. Mycobacterium sp. or Aspergillus niger Preferably the microorganism is: Penicillium frequentans BPFC 386, 585, 623, 733 Rhizopus stolonifer BPFC 1581 Ustilago maydis BPFC 1198, 6333 Arthipbacter petroleophagug ATCC 21494 Brevibacterium paraffinolyticum ATCC 21195 Actinetobacter sp. NCIMB 9871 Mycobacterium sp. BPCC 1174, 1178, 1179, 1186, 1187 or Aspergillus niger BPFC 32 The microorganisms may be grown on suitable medium containing an appropriate carbon source such as octane, ethene, cyclohexanone or glucose for example. The compounds of formula (II) are generally acid labile and thus the use of acid conditions is to be avoided. Generally the method according to the invention may be carried out at a pH of 7.6 to 8, suitably about 7.6, and at temperature of 25-35°C, suitably about 28°C. The present invention will now be illustrated with reference to the Examples. SUBSTITUTE SHEET EXAMPLE 1 The following microorganisms were screened for sulfoxidation activity against compounds of formula (la): Penicillium frequentans BPFC 386 Ppnirillium frequentans BPFC 585 PpninlliiiTr. frequentans BPFC 623 Penicillium frequentans BPFC 733 Rhizopus stolonifer BPFC 1581 Ustilago maydis BPFC 1198 Ustilago maydis BPFC 6333 Arthrobacter petroleophagus ATCC 21494 Brevibacterium paraffinolyticum ATCC 21195 Acinetobacter sp NCIMB 9871 Mycobacterium sp BPCC 1174 Mycobacterium sp BPCC 1178 Mycobacterium sp BPCC 1179 Mycobacterium sp BPCC 1186 Mycobacterium sp BPCC 1187 Growth Conditions The growth conditions for the above microorganisms were as follows. The following fungi: Ppnirilliiun frequentans BPFC 386 Penirillium frequentans BPFC 585 P niril um frequentans BPFC 623 Penicillium frequentans BPFC 733 SUBSTITUTE SHEET Rhizopus stolonifer BPFC 1581 Ustilago maydis BPFC 1198 Ustilago maydis BPFC 6333 were grown in 200 ml of sterile liquid medium (I) with the composition of (per litre) K j HPO, (1.9g), NaH j PO, 2HjO (2.02g), ammonium sulfate (1.8g), magnesium sulfate (0.2g), ferric chloride (0.97 mg), and trace elements solution (1 ml) pH 7.2. The composition of the trace elements solution used was as follows (in g/1): The above medium was supplemented with 0.2% w/v yeast extract and 2.2% w/v glucose. The medium contained in 1L baffled flasks was inoculated either by adding a suspension of spores in sterile distilled water or by the addition of a plug of agar containing the fungi from a Sabouraud Dextrose plate. Fungi were grown at 28°C on a rotary shaker at 150 rpm for 48 hours. With the exception of Ustilago maydis. the fungal biomass obtained from liquid culture was harvested by filtration on a Whatman Grade 113 filter paper and washed on the filter with 50 mM sodium phosphate buffer, pH7.6. Ustilago maydis was harvested by centrifuging for 20 minutes at 8,000 rpm and 4°C. The biomass was washed by resuspending in 50 mM sodium phosphate buffer, pH 7.6 and centrifuging as above. The bacteria were grown with the sources of carbon shown in Table 1: SUBSTITUTE SHEET TABLE ! The growth of Acinetobacter sp. NCIMB 9871 on cyclohexanone was performed in 100 ml of liquid medium (I) in a 500 ml baffled flask containing a centre well. Cyclohexanone was placed in the centre well. The microorganism was grown at 28°C on a rotary shaker at 150 rpm for 24-48 hours. Growth of Arthrobacter petroleophagus ATCC 21494 and Brevibacterium paraffinolyticum ATCC 21195 on octane was performed in 200 ml of liquid medium (I) containing 0.2% w/v yeast extract in a 1 L baffled flask. Octane (1ml) was added directly to the medium without sterilization. The above microorganisms were grown at 28°C on a rotary shaker at 150 rpm for 24-48 hours. Mycobacterium sp BPCC 1174, 1178, 1179, 1186 and 1187 were grown in 500 ml liquid medium (I) in a 2L non-baffled flask fitted with a rubber bung. The flask was partially evacuated and then charged with ethene. Growth was conducted at 28°C on a rotary shaker at 150 rpm for 7 days. Growth of Arthrobacter petroleophagus ATCC 21494 and Brevibacterium paraffinolyticum ATCC 21195 was also performed on glucose. Each microorganism was inoculated into 200 ml medium (I) containing 0.2% w/v yeast extract and 2.2% w/v glucose. Growth was performed at 28°C on a rotary shaker at 150 rpm for 24- 48 hours. SUBSTITUTE SHEET All bacteria were harvested from liquid medium by centrifuging at 8,000 rpm and 4°C for 20 minutes. Cells were washed by resuspending in 50 mM sodium phosphate buffer, pH 7.6 followed by centrifuging as above. Biooxidation Reactions Biotransformations were performed for each microorganism in 50mM sodium phosphate buffer, pH 7.6 with 5-10 g/1 dry cell weight and a substrate concentration of 1 g/1. The cells were incubated with the compound of formula (la) on a rotary shaker at 28°C for 18-20 hours. Samples were removed from the biotransformation and either centrifuged or filtered to remove biomass and analysed directly. Detection of Products The biooxidation of the compound of formula (la) was followed by reverse phase HPLC on a Spherisorb S5-ODS2 reverse phase column eluted with a 50:50 mixture of acetonitrile and 25mM sodium phosphate buffer, pH 7.6 at a flow rate of 0.8 ml/min. Under such conditions the compovmds of formulae (Ha) and (la) were well resolved with retention times of 5.2 and 9.8 minutes respectively. Both compounds were detected at a wavelength of 300 nm. The enantiomeric composition of the compound of formula (Ila) formed was investigated by the following method. After removal of biomass the aqueous media was extracted with two volumes of ammonia saturated dichloromethane. The pooled organic extracts were dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure to afford a pale brown solid. Then the enantiomeric composition of sulfoxide was determined by chiral HPLC on a Chiralpak AD Column under the following conditions: 1 SUBSTITUTE SHEET Column Chiralpack AD 250 mm x 4.6 mm interior diameter with 50 mm guard column Eluent Hexane:Ethanol:Methanol (40:55:5% V/V) Flow 1.0 ml/min Injection Volume 20μl Wavelength 300 ran Retention times Compound of formula (la) 5.1 min Compound of formula (Ha): (+) Enantiomer 8.5 min (-) Enantiomer 13.4 min The following results were obtained: IΔBLE2 SUBSTITUTE SHEET The enantiomeric excess value gives an indication of the relative amounts of each enantiomer obtained. The value is the difference between the relative percentages for the two enantiomers. Thus, for example, when the percentage of the (-) enantiomer of the formed sulfoxide is 97.5% and the percentage for the (+) enantiomer is 2.5%, the enantiomeric excess for the (-) enantiomer is 95%. With Arthrobacter petroleophagus ATCC 21494 and Brevibacterium paraffinolyticum ATCC 21195 the stereoselectivity of the biooxidation was unaffected by the choice of carbon source used for growth (octane and glucose). EXAMPLE 2 Compovmds of formula (Id) and (Ie) were screened against a range of microorganisms for the production of the corresponding sulfoxides. The growth of microorganisms and subsequent biotransformations were performed as in Example 1 except that the reaction times were as listed in Tables 5 and 6. Aspergillus niger BPFC 32 was grown in the same way as the fungi were grown in Example 1. Petectipn pf Products The biooxidation of the compovmds of formula (Id) and (Ie) was followed by reverse phase HPLC as in Example 1 except that the retention times were as follows: SUBSTITUTE SHEET T BLED The enantiomeric composition of the compounds of formula (Ed) and (He) was investigated by the method of Example 1 except in the chiral HPLC the solvent compositions, flow rates and retention times were as follows: TAPLE In Table 4 the first enantiomer eluted is referred to as enantiomer A and second as enantiomer B. The results are summarised in Tables 5 and 6. S UBSTITUTE SHEET TABLE 5 (E.e. means Enantiomeric excess) IΔB E£ SUBSTITUTE SHEET The oxidation of the compound of formula (Id) produced in all cases the "A" enantiomer of the compound of formula (lid) in excellent enantiomeric excess but in low yield. The four strains of Penicillium frequentans. previously shown to oxidise the compound of formula (la), failed to oxidise the compound of formula (Id). The oxidation of the compound of formula (Ie) produced fewer results. This compovmd proved to be particularly insoluble making the detection of product difficult. Whilst in a number of cases sulfoxide was produced, its concentration was too low to determine the enantiomeric excess. However two results were obtained with Mycobacterium sp. and Penicillium frequentans both affording sulfoxide of high enantiomeric excess but interestingly of opposite stereoselectivity. EXAMPLE 3 The microorganisms listed in Table 9 below were screened for sulfoxidation activity against compounds of formula (lb). They were grown under the same condition as in Examples 1 and 2. Biotransformations were performed following the protocol of Example 1 except that the dry cell weight was increased to approximately 20gL -1 and the reaction time was extended. Detection of Products The biooxidation of the compound of formula (lb) was followed by reverse phase HPLC as in Example 1 except that the retention times were as follows: SUBSTITUTE SHEET I LEJ. The enantiomeric composition of the compound of formula (Db) was investigated by the method of Example 1 except in the chiral HPLC the solvent composition, flow rate and retention time were as follows: TABLE S In Table 8 the first enantiomer eluted is referred to as enantiomer A and the second as enantiomer B. The results are summarised in the following table: IΔBLES SUBSTITUTE SHEET (E.e. means enantiomeric excess) The microorganisms listed in Table 9 were also screened under identical conditions for sulfoxidation of the compound of formula (Ic) but no product of formula (lie) could be detected. Deposits Of Microorganisms The following microorganisms were deposited at the National Collections of Industrial and Marine Bacteria Ltd (NCIMB), 23 St. Machar Drive, Aberdeen, Scodand AB2 1RY on 25 November 1994: 1. Mycobacterium sp BPCC 1174 Accession No. NCIMB 40695 2. Mycobacterium sp BPCC 1178 Accession No. NCIMB 40696 3. Mycobacterium sp BPCC 1179 Accession No. NCIMB 40697 4. Mycobacterium sp BPCC 1186 Accession No. NCIMB 40698 5. Mycobacterium sp BPCC 1187 Accession No. NCIMB 40699 The following microorganisms were deposited at the International Mycological Institute (IMP, Bakeham Lane, Englefield Green, Egham, Surrey TW209TY, England on 28 November 1994: 6. Penicillium frequentans BPFC 386 Accession No. IMICC 364802 7. Penicillium frequentans BPFC 585 Accession No. IMICC 364801 8. Penicillium frequentans BPFC 623 1 SUBSTITUTE SHEET 1 Accession No. IMICC 364800 9. Penicillium frequentans BPFC 733 Accession No. IMICC 364799 10. Rhizopus stolonifer BPFC 1581 Accession No. IMICC 364798 11. Ustilago maydis BPFC 1198 Accession No. IMICC 364797 12. Ustilago maydis BPFC 6333 Accession No. IMICC 364796 13. Asperigillus niger BPFC 32 Accession No. IMICC 364795 SUBSTITUTE SHEET | 22 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 136ά) A. Tbe indications made below relate to the microorganism referred to in tbe description on page 20 . line \S B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet ~~ Name of depositary institution The National Collections of Industrial and Marine Bacteria Limited Address of depositary institution Cinc -iing postal code anj country) 23 St Machar Drive ABERDEEN AB2 1RY Scotland, United Kingdom Date of deposit Accession Number Novembe r 25 , 1 994 N C I MB 4069 5 C ADDITIONAL INDICATIONS (leave blent if net applicable) Tbis informatioα b continued on an additional sheet ~~] In respect of all designated states in which such action is possible and to the extent that it is legal ly permissible under the law of the designated state, it is requested that a sample of the deposited mιcro-orgamsm( s) be made available only by the issue thereof to an independent expert , in accordance with the relevant patent legislation , e. g . EPC Rule 28( 4 ) , U. K . Rule 17( 3 ) , Australian Regulation 3.25( 3) and generally similar provisions mutatis mutandis for any other designated state . D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifύκh iu s art mot for eU έe pa i States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) Tbe indications listed below will be submitted lo the International Bureau later (speαfyti tfotnl nature βf the int.ca o*-m.g , 'A eαto Number ofDeposu~ For receiving Office use only For International Bureau use only IXJ Tbis sheet was received with tbe international application I \ Tbis sheet was received by the IntemsUonal Bureau on: Authorized ofDeer Authorized officer INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I'bis) A. The indications made below relate to the microorprusm referred to in the description on page 20 , line _J7 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet [ ] Name of depositary institution The National Collections of Industrial and Marine Bacteria Limited Address of depositary institution (includint postal code and country) 23 St Machar Drive ABERDEEN AB2 1RY Scotland, United Kingdom Date of deposit Accession Number November 25 , 1994 NCIMB 40696 C ADDITIONAL INDICATIONS (leave blank if not applicable) Tbis information β continued on an additional sheet Q In respect of all designated states in which such action is possible and to the extent that it is legally permissible under the law of the designated state, it is requested that a sample of the deposited mιcro-organιsm( s) be made available only by the issue thereof to an independent expert , in accordance with the relevant patent legislation, e. g. EPC Rule 28(4 ) , U. K . Rule 17( 3) , Australian Regulation 3.25( 3) and generally similar provisions mutatis mutandis for any other designated state. D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if It* iudicaόo* art not fc* ell ^ Steles) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) Tbe indications listed below will be submitted to tbe International Bureau later (spe fyihetαeerainatterecfiktinJicaύoiα eg. 'Accession Number mf[>tpostt~ For receiving Office use only For international Bureau use only I J Tbis sheet was received with the international application j I This sheet was received by the International Bureau < Authorized officer Authorized officer INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule l-bis) For receiving Office use only For International Bureau use only X){\ Tbis sheet was received with the international application | j Tbis sheet was received by tbe lntenu uoαal Bureau < Authorized officer Authorized officer cUαu ϋ 25 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I'bis) A. Tbe indications made below relate lo the microorganism referred lo in the description on page 20 . line 21 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet X| Name of depositary institution The National Collections of Industrial and Marine Bacteria Limited Address of depositary institution (including postal code and country) 23 St Machar Drive ABERDEEN AB2 1 RY Scotland , United Kingdom Date of de osit Accession Number November 25 , 1 994 NC IMB 40698 C ADDITIONAL INDICATIONS (leave blank if not applicable) Tbb information b continued on an additional sheet Q In respect of all designated states in which such action is possible and to the extent that it is legally permissible under the law of the designated state, it is requested that a sample of the deposited micro-organism(s) be made available only by the issue thereof to an independent expert, in accordance with the relevant patent legislation, e.g. EPC Rule 28(4), U.K. Rule 17(3), Australian Regulation 3.25(3) and generally similar provisions mutatis mutandis for any other designated state. D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (iflk mύcetionsereaotfor eU designated Stales) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if n t applicable) Tbe indications listed below will be submitted to the International Bureau later (ιpeάfyύιetcxtγ*lrmU.rcofύcιnd.c*lu ιt.g 'Accession Number of Deposit' For receiving Office use only For International Bureau use only This sheet was received with the internaiionsl application l l Tbi* *-eet was received by the International Bureau on: Authorized officer Authorized officer INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I 'bis) A. Tbe indications sde below relate lo the πncroorpnism referred lo in the description on pap 20 , line 23 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet ~~ Name of depositary institution The National Collections of Industrial and Marine Becteria Limited Address of depositary institution (includin postal code and country) 23 St Machar Drive ABERDEEN AB2 1RY Scotland, United Kingdom Dale of deposit Accession Number November 25 , 1994 \ IMB 40699 C. ADDITIONAL INDICATIONS (leave blank if not applicable) Tbb information b oontinued on an additional sheet In respect of all designated states in which such action is possible and to the extent that it is legally permissible under the law of the designated state , it is requested that a sample of the deposited micro-organism( s) be made available only by the issue thereof to an lnαependent expert , in accordance with the relevant patent legislation , e. g. EPC Rule 28(4 ) , U. K . Rule 17( 3 ) , Australian Regulation 3.25( 3 ) and generally similar provisions mutatis mutandis for any other designated state . D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (iflk i ations are net for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) Tbe indications listed below will be submitted lo tbe International Bureau later (specify the general nature of the indication eg. 'Accessio Number of Depot*") For International Bureau use only ~~j Tbis sheet was received by the International Bureau on: Authorized officer INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I ' bis) A. Tbe indications made below relate lo tbe microorprusm referred to in the description on pap 20 . line 28 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | X { Name of depositary institution International Mycological Institute Address of depositary institution (including postal code and country) Ba eha Lane Egham Surrey TW20 9TY, England, UK Date of deposit Accession Number November 28, 1994 IMICC 364802 C. ADDITIONAL INDICATIONS (leave blank if not applicable) Tbb information b oontinued on an additional sheet Q In respect of all designated states in which such action is possible and to the extent that it is legally permissible under the law of the designated state, it is requested that a sample of the deposited micro-organism(s) be made available only by the issue thereof to an independent expert, in accordance with the relevant patent legislation, e.g. EPC Rule 28(4), U.K. Rule 17(3), Australian Regulation 3.25(3) and generally similar provisions mutatis mutandis for any other designated state. D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indication are net for eU designated Stales) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) Tbe indications listed below will be submirted to the International Bureau later (ipecify the ge-σal nature of the indication e.g, 'Accession Number of Deposit") For receiving Office use only For International Bureau use only |y| Tbb sheet was received with the international application | I Tbb sheet waa received by the International Bureau on: Authorized officer Authorized officer Form FCr RO 134 Jul 199 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule \3bis) For receiving Office use only For International Bureau use only |yl Tbb sheet was received with the internationsl application | | Tbb sheet was received by the International Bureau on Authorized officer Authorized officer Form PCT RO 134 (July 1992) 29 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule \"bis) A. Tbe indications made below relate to the microorpnism referred to in tbe description on pap 21 . line 1 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet j X | Name of depositary institution International Mycological Institute Address of depositary institution (including postal code and country) Bakeham Lane Egham Surrey TW20 9TY, England, UK Date of deposit Accession Number November 28 , 1994 IMICC 364800 C ADDITIONAL INDICATIONS (leave blank if net βpplicabk) Tbb information b continued on an additional sheet Q In respect of all designated states in which such action is possible and to the extent that it is legally permissible under the law of the designated state , it is requested that a sample of the deposited micro-organism( s) be made available only by the issue thereof to an independent expert , in accordance with the relevant patent legislation , e . g. EPC Rule 28(4 ) , U . K . Rule 17 ( 3 ) , Australian Regulation 3.25( 3 ) and generally similar provisions mutatis mutandis for any other designated state . D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if ώe kdkaύo re net for ell designate Stales) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applkable) Tbe indications Ibled belo will be submitted lo the International Buresu Ister (specify Ike general nature of th indication e.g, 'Accession Number of Deposit") 30 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule \~bis) A. The indications made below relate to tbe microorpnism referred to in tbe description on pap 21 . line B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet P Name of depositary institution International Mycological Institute Address of depositary institution (including postal code and country) Bakeham Lane Egham Surrey TW20 9TY, England, UK Dale of deposit Accession Number November 28 , 1994 IMICC 364799 C. ADDITIONAL INDICATIONS (leave blank if not applkabk) Tbb information b continued on an additional aheet In respect of all designated states in which such action is possible and to the extent that it is legally permissible under the law of the designated state it is requested that a sample of the deposited mιcro-organιsm( s) be made available only by the issue thereof to an independent expert , in accordance with the relevant patent legislation , e. g. EPC Rule 28(4 ) , U. K . Rule 17( 3) , Australian Regulation 3.25( 3) and generally similar provisions mutatis mutandis for any other designated state. D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the mdkabon are not foe a designate Stales H. SEPARATE FURNISHING OF INDICATIONS (leave blank if n t applkable) Tbe indications listed belowwill be submitted lo the International Bureau later (specify Ike general nature of tke indication e.gm 'Accessio Number of Deposit") For receiving Office use only For International Bureau use only This sheet was received with the international application | | Tbb sheet was received by the International Bureau o Authorized officer Authorized officer CUOΛ Λ Form PCT/RO/134 Jul 1992 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule \3bis) The indications made below relate to tbe microorpnbm referred to in tbe description on page 21 , li„e _J> B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | ** x) Name of depositary institution International Mycological Institute Address of depositary institution (including postal code and country) Bakeham Lane Egham Surrey TW20 9TY, England, UK Date of deposit Accession Number November 28, 1994 IMICC 364798 C ADDITIONAL INDICATIONS (leave bknk if not applkable) Tbb information b continued on an additional sheet [~ In respect of all designated states in which such action is possible and to the extent that it is legally permissible under the law of the designated state, it is requested that a sample of the deposited micro-organism( s) be made available only by the issue thereof to an independent expert , in accordance with the relevant patent legislation, e. g. EPC Rule 28(4 ) , U. K. Rule 17( 3 ) , Australian Regulation 3.25( 3) and generally similar provisions mutatis mutandis for any other designated state. D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if ike inalαimen are net ftirtO designated Stales) E. SEPARATE FURNISHING OF INDICATIONS (leave bknk if net applkable) Tbe indications listed belowwill be submilted to tbe International Bureau later (specify the general nature of the indication e.g , 'Accession Number of Deposit") For receiving Office use only For International Bureau use only jVJ Tbb sheet was received with the international application | | Tbb sheet was received by the International Bureau on: Authorized officer Authorized officer INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13Wι) A. Tbe indications made below relate to the microorpnism referred to in the description on pap 21 . line B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | X| Name of depositary institution International Mycological Institute Address of depositary institution (including postal code end country) Bakeham Lane Egha Surrey TW20 9TY, England, UK Date of deposit Accession Number November 28 , 1994 IMICC 364797 C ADDITIONAL INDICATIONS (leave bknk if not applkable) Tbb information b continued on an additional sheet In respect of all designated states in which such action is possible and to the extent that it is legally permissible under the law of the designated state it is requested that a sample of the deposited micro-organism(s) be made available only by the issue thereof to an independent expert, in accordance with the relevant patent legislation, e.g. EPC Rule 28(4), U.K. Rule 17(3), Australian Regulation 3.25(3) and generally similar provisions mutatis mutandis for any other designated state. D. DESIGNATEDSTATESFORWHICHINDICATIONSAREMADE (if the wdicaύon arc aαlfc+aU designated Stales) E. SEPARATEFURNISHINGOFINDICATIONS (lea-, blank if not applkabk) Tbe mdicslions Ibled below will be submitted lo tbe lnteπuiional Bureau later (specify the general ature of ike indication e.g, "Accession Number of Deposit") For International Bureau use only I ) Tb sheet was received by the International Bureau on; Authorized officer INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 12Φis) For receiving Office use only For International Bureau use only | 1 Tbis sheet was received with the international application [ I Tbb sheet was received by tbe International Bureau on: Authorized officer Authorized officer Form PCT/RCVl-M J INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I3bis) For International Bureau use only j j Thb sheet waa received by the International Bureau o Authorized officer

Claims: 1. A method of preparing a compound of formula (II) either as a singk enantiomer or in an enantiomerically enriched form: wherein: and and wherein: N in the benzimidazole moiety means that one of the carbon atoms substituted by R.-R, optionally may be exchanged for an unsubstituted nitrogen atom; SUBSTITUTE SHEET R,, R, and R, are the same or different and selected from hydrogen, alkyl, alkoxy optionally substituted by fluorine, alkylthio, alkoxyalkoxy, dialkylamino, piperidino, morpholino, halogen, phenylalkyl, phenylalkoxy; R 4 and R 4 . are the same or different and selected from hydrogen, alkyl, aralkyl; s is hydrogen, halogen, trifluoromethyl, alkyl, alkoxy; R, - R, are the same or different and selected from hydrogen, alkyl, alkoxy, halogen, haloalkoxy, alkylcarbonyl, alkoxycarbonyl, oxazolyl, trϋluoroalkyl or adjacent groups R 6 - R, may complete together with the carbon atoms to which they are attached optionally substituted ring structures; R-o is hydrogen or alkoxycarbonyloxymethyl; R„ is hydrogen or forms an alkylene chain together with R,; R. 2 and R_ 3 are the same or different and selected from hydrogen, halogen or alkyl; which method comprises stereoselective biooxidation of the pro-chiral sulfide counterpart compound. 2. A method according to claim 1 wherein: and SUBSTITUTE SHEET and wherein R,, R., R,, R 6 -R,, R IO and R,. are as defined in claim 1. 3. A method to claim 1 or 2 wherein the compound of formula (II) is a compound of formula: SUBSTITUTE SHEET 4. A method according to any one of the previous claims wherein a single enantiomer of the compound of formula (II) is prepared. 5. A method according to claim 3 wherein there is prepared a compound of formula (Ha) and the biooxidation is carried out with a microorganism which is Penicillium frequentans Brevibacterium paraffinolyticum or Mycobacterium sp. 6. A method according to claim 3 wherein there is prepared a compound of formula (lib) and the biooxidation is carried out with a microorganism which is: Aspergillus niger or Ustilago maydis. SUBSTITUTE SHEET 7. A method according to claim 3 wherein there is prepared a compound of formula (lid) and the biooxidation is carried out with a microorganism which is Mycobacterium sp. Arthrobacter petroleophagus Brevibacterium paraffinolyticum or Ustilago maydis. 8. A method according to claim 3 wherein there is prepared a compound of formula (He) and the bioxidation is carried out with a microorganism which is: Mycobacterium sp. Penicillium frequentans 9. A method according to claim 1 substantially as described in any one of the Examples. 10. A compound of formula II, as a single enantiomer or in enantiomerically enriched form, as defined in claim 1 prepared by the method claimed in any one of claims 1 to 9. SUBSTITUTE SHEET

Inventors: HOLT ROBERT, LINDBERG PER, REEVE CHRISTOPHER, TAYLOR STEPHEN, HOLT, ROBERT, LINDBERG, PER, REEVE, CHRISTOPHER, TAYLOR, STEPHEN
Applicants: ASTRA AB, HOLT ROBERT, LINDBERG PER, REEVE CHRISTOPHER, TAYLOR STEPHEN, ASTRA AKTIEBOLAG, HOLT, ROBERT, LINDBERG, PER, REEVE, CHRISTOPHER, TAYLOR, STEPHEN
Application year: 1995
Main classification: C12P11
IPC codes: A61K31, A61P1, C07D401, C12P17, C12P41, C12R1