Abstract:
Enantiomeric or enantiomerically enriched H +  K +  ATPase inhibiting pyridinylsulfinyl--benzimidazoles are prepared using microorganisms or microbial enzyme systems to enantioselectively biooxidize corresponding prochiral sulfide compounds and isolating the pharmaceutically active enantiomer or enantiomerically enriched sulfoxide form.

Description:
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 H +  K +  ATPase 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 (IIa) below), lansoprazole (compound of formula (IIc) below) and pantoprazole (compound of formula (IIb) 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. 88, 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: ##STR1## wherein Het 1  is ##STR2## and Het 2  is ##STR3## and X is ##STR4## wherein: N in the benzimidazole moiety means that one of the carbon atoms substituted by R 6  -R 9  optionally may be exchanged for an unsubstituted nitrogen atom; 
     R 1 , R 2  and R 3  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&#39;   are the same or different and selected from hydrogen, alkyl, aralkyl; 
     R 5  is hydrogen, halogen, trifluoromethyl, alkyl, alkoxy; 
     R 6  -R 9  are the same or different and selected from hydrogen, alkyl, alkoxy, halogen, haloalkoxy, alkylcarbonyl, alkoxycarbonyl, oxazolyl, trifluoroalkyl or adjacent groups R 6  -R 9  may complete together with the carbon atoms to which they are attached optionally substituted ring structures; 
     R 10  is hydrogen or alkoxycarbonyloxymethyl; 
     R 11  is hydrogen or forms an alkylene chain together with R 3  ; 
     R 12  and R 13  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 H +  K +  ATPase 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: 
     Het 1  is ##STR5## and Het 2  is ##STR6## and ##STR7## (wherein R 1 , R 2 , R 3 , R 6  to R 9 , R 10  and R 11  are as defined above). 
     Most preferably the compounds of formula (II) are compounds of the formula (IIa) to (IIe): ##STR8## 
     An example of a compound of formula (II) wherein R 10  is alkoxycarbonyloxymethyl is ##STR9## 
     The starting prochiral sulfides used in the method of the present invention are of the formula: 
     
         Het.sub.1 --X--S--Het.sub.2                                (I) 
    
     wherein Het 1 , X and Het 2  are as defined above. 
     In order to obtain each of the above compounds (IIa)-(IIf), the following starting compounds of formula (Ia) to (If), respectively will be required: ##STR10## 
     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 1  --X-moiety and the Het 2  -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 Arthrobacter petroleophagus, Brevibacterium paraffinolyticum, and Acinetobacter species, alkene oxidisers such as Mycobacterium species, and a variety of fungal species particularly Penicillium 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 
     Ustilago maydis 
     Arthrobacter petroleophagus 
     Brevibacterium paraffinolyticum 
     Acinetobacter 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 
     Arthrobacter petroleophagus 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. 
    
    
     EXAMPLE 1 
     The following microorganisms were screened for sulfoxidation activity against compounds of formula (Ia): 
     Penicillium frequentans BPFC 386 
     Penicillium frequentans BPFC 585 
     Penicillium 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: 
     Penicillium frequentans BPFC 386 
     Penicillium frequentans BPFC 585 
     Penicillium frequentans BPFC 623 
     Penicillium frequentans BPFC 733 
     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 liter) K 2  HPO 4  (1.9 g), NaH 2  PO 4  2H 2  O (2.02 g), ammonium sulfate (1.8 g), magnesium sulfate (0.2 g), 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/l): 
     
         ______________________________________  CuSO.sub.4.5H.sub.2 O           0.02  MnSO.sub.4.4H.sub.2 O           0.1  ZnSO.sub.4.7H.sub.2 O           0.1  CaCO.sub.3           1.8______________________________________ 
    
     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: 
     
                       TABLE 1______________________________________Microorganism             Carbon Source______________________________________Arthrobacter petroleophagus ATCC 21494                     OctaneBrevibacterium paraffinolyticum ATCC 21195                     OctaneAcinetobacter sp NCIMB 9871                     CyclohexanoneMycobacterium sp BPCC 1174, 1178, 1179, 1186, 1187                     Ethene______________________________________ 
    
     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. Cydohexanone 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 1L baffled flask. Octane (1 ml) 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. 
     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 50 mM sodium phosphate buffer, pH 7.6 with 5-10 g/l dry cell weight and a substrate concentration of 1 g/l. The cells were incubated with the compound of formula (Ia) 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 (Ia) was followed by reverse phase HPLC on a Spherisorb S5-ODS2 reverse phase column eluted with a 50:50 mixture of acetonitrile and 25 mM sodium phosphate buffer, pH 7.6 at a flow rate of 0.8 ml/min. Under such conditions the compounds of formulae (IIa) and (Ia) 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 (IIa) 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: 
     Column Chiralpack AD 250 mm×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 20 μl 
     Wavelength 300 nm 
     Retention times 
     Compound of formula (Ia) 5.1 min 
     Compound of formula (IIa): 
     (+) Enantiomer 8.5 min 
     (-) Enantiomer 13.4 min 
     The following results were obtained: 
     
                       TABLE 2______________________________________           Compound  Enantio-           of Formula                     meric    EnantiomerMicroorganism   (IIa) (ppm)                     excess (%)                              ((+) or (-))______________________________________Penicillium frequentans           23        &gt;99      (-)BPFC 386Penicillium frequentans           2.1       &gt;99      (-)BPFC 585Penicillium frequentans           3.0       95       (-)BPFC 623Penicillium frequentans           2.6       87       (-)BPFC 733Rhizopus stolonifer BPFC 1581           3.0       56       (-)Ustilago maydis BPFC 1198           8.0       88       (-)Ustilago maydis BPFC 8333           34.0      99       (-)Arthrobacter petroleophagus           24.0      96       (-)ATCC 21494Brevibacterium paraffinolyticum           13.0      &gt;99      (-)ATCC 21195Acinetobacter sp NCIMB 9871           0.4       17       (-)Mycobacterium sp BPCC 1174           10.0      97       (-)Mycobacterium sp BPCC 1178           3.3       93       (-)Mycobacterium sp BPCC 1179           9.0       96       (-)Mycobacterium sp BPCC 1186           11.0      97       (-)Mycobacterium sp BPCC 1187           6.0       96       (-)______________________________________ 
    
     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 
     Compounds 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. 
     Detection of Products 
     The biooxidation of the compounds of formula (Id) and (Ie) was followed by reverse phase HPLC as in Example 1 except that the retention times were as follows: 
     
                       TABLE 3______________________________________Compound of formula           Retention time (min)______________________________________Id              13.7IId             5.0Ie              9.4IIe             4.3______________________________________ 
    
     The enantiomeric composition of the compounds of formula (IId) and (IIe) was investigated by the method of Example 1 except in the chiral HPLC the solvent compositions, flow rates and retention times were as follows: 
     
                       TABLE 4______________________________________Com-pound                  Flow rateof formula  Solvent Compositon                  (ml/min) Retention Time______________________________________IId    Hexane/Ethanol  1.0      12.9 (Enantiomer A)  (70:30% v/v)             21.7 (Enantiomer B)  Hexane/Ethanol/Methanol                  1.0       7.4 (Enantiomer A)  (40.55:5% v/v)           10.6 (Enantiomer B)IIe    Hexane/Ethanol  1.0      26.0 (Enantiomer A)  (70:30% v/v)             30.5 (Enantiomer B)______________________________________ 
    
     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. 
     
                                           TABLE 5__________________________________________________________________________                    Aqueous                    concentration (PPM)                    Compound                          Compound               Reaction                    of formula                          of formula                                E.e.Microorganism       time (h)                    (Id)  (IId) %  Enantiomer__________________________________________________________________________Mycobacterium sp. BPCC 1174               42   5     16.7  &gt;99                                   AMycobacterium sp. BPCC 1178               42   5.9   14.4  &gt;99                                   AMycobacterium sp. BPCC 1179               42   6.6   17.4  &gt;99                                   AMycobacterium sp. BPCC 1186               42   4.8   42    &gt;99                                   AMycobacterium sp. BPCC 1187               42   7.4   18.3  &gt;99                                   AArthrobacter petroleophagus ATCC 21494               42   3.5   6.6   &gt;99                                   ABrevibacterium paraffinolyticum ATCC 21195               42   2.6   21.7  &gt;99                                   AUstilago maydis BPFC 1198               18   6.7   45    &gt;99                                   AUstilago maydis BPFC 6333               18   4.6   43    &gt;99                                   AAspergillus niger BPFC 32               42   5.6   2.7   -- --Penicillium frequentans BPFC 386               18   5     0     -- --Penicillium frequentans BPFC 585               48   5.2   0     -- --Penicillium frequentans BPFC 623               48   4.5   0     -- --Penicillium frequentans BPFC 733               18   3.5   0     -- --__________________________________________________________________________ (E.e. means Enantiomeric excess) 
    
     
                                           TABLE 6__________________________________________________________________________                    Aqueous                    concentration (PPM)                    Compound                          Compound               Reaction                    of formula                          of formula                                E.e.Microorganism       time (h)                    (Ie)  (IIe) (%)                                   Enantiomer__________________________________________________________________________Mycobacterium sp. BPCC 1179               42   1.6   3.3   &gt;99                                   AArthrobacter petroleophagus ATCC 21494               42   3.2   0     -- --Brevibacterium paraffinolyticum ATCC 21195               72   4.0   1.6   -- --Ustilago maydis BPFC 1198               18   2.3   0     -- --Ustilago maydis BPFC 6333               72   3.2   0     -- --Asergillus niger BPFC 32               72   3.7   9.2   -- --Penicillium frequentans BPFC 386               72   3.1   0.5   -- --Penicillium frequentans BPFC 585               48   3.2   3.2   -- --Penicillium frequentans BPFC 623               48   2.9   1.5   83.4-                                   BPenicillium frequentans BPFC 733               18   3.2   0        --__________________________________________________________________________ 
    
     The oxidation of the compound of formula (Id) produced in all cases the &#34;A&#34; enantiomer of the compound of formula (IId) in excellent enantiomeric excess but in low yield. The four strains of Penicillium frequentans previously shown to oxidise the compound of formula (Ia), failed to oxidise the compound of formula (Id). 
     The oxidation of the compound of formula (Ie) produced fewer results. This compound 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 (Ib). 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 20 gL -1  and the reaction time was extended. 
     Detection of Products 
     The biooxidation of the compound of formula (Ib) was followed by reverse phase HPLC as in Example 1 except that the retention times were as follows: 
     
                       TABLE 7______________________________________Compound of formula           Retention time (min)______________________________________Ib              8.1IIb             4.2______________________________________ 
    
     The enantiomeric composition of the compound of formula (IIb) 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 8______________________________________Solvent composition        Flow Rate (ml/min)                     Retention times (min)______________________________________Hexane/ethanol (70:30%)        1.0          32.3 (Enantiomer A)                     36.6 (Enantiomer B)______________________________________ 
    
     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: 
     
                                           TABLE 9__________________________________________________________________________                    Aqueous                    concentration (PPM)                    Compound                          Compound               Reaction                    of formula                          of formula                                E.e.Microorganism       time (h)                    (Ib)  (IIb) %  Enantiomer__________________________________________________________________________Mycobacterium sp. BPCC 1178               72   8.6   3.4   8.2                                   BBrevibacterium paraffinolyticum ATCC 21195               72   8.4   4.0   26.6                                   BUstilago maydis BPFC 6333               72   8.2   4.3   &gt;99                                   AAspergillus niger BPFC 32               72   5.6   28.0  &gt;99                                   APenicillium frequentans BPFC 386               72   8.4   4.5   -- --Penicillium frequentans BPFC 585               48   6.5   11.4  -- --Penicillium frequentans BPFC 623               48   7.7   6.5   -- --__________________________________________________________________________ (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 (IIc) 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, Scotland AB2 1RY on 25 Nov. 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 (IMI), Bakeham Lane, Englefield Green, Egham, Surrey TW20 9TY, England on 28 Nov. 1994: 
     6. Penicillium frequentans BPFC 386 Accession No. IMICC 364802 
     7. Penicillium frequentans BPFC 585 Accession No. IMICC 364801 
     8. Penicillium frequentans BPFC 623 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