PHARMACEUTICALS

Abstract: A process for the preparation of an intermediate (II) which is useful in the preparation of compounds having antiviral activity.

Description: PHARMACEUTICALS The present invention relates to a process for the preparation of an intermediate which is useful in the preparation of compounds having antiviral activity. EP-A-141927 (Beecham Group p.I.e.) describes the compound, penciclovir and its use as an antiviral agent and EP-A-182024 (Beecham Group p.I.e.) describes its pro-drug, famciclovir. Penciclovir and famciclovir are of formulae (A) and (B) respectively: HO - CH„ - CH - CH 0 - OH (A) (B) The method of preparation of the above compounds involves the reaction of 2-amino-6-chloropurine of formula (C) : with a side-chain intermediate of formula (D) Q (D) wherein Q is a leaving group, such as hydroxy or halo, for example iodo or bromo. The side-chain intermediate of formula (D) is not readily available and is prepared by a multi-stage synthesis. Although the compound of formula (D) but wherein Q is replaced by acetate i.e. of formula (I) : can be prepared from commercially available triethyl 1,1,2-ethanetricarboxylate by a modification of the method of .J. Bailey et.. .al., J. Org. Chem., 1962, it is not possible to selectively convert one of the acetoxy groups to a leaving group, since they are all in a similar chemical environment and chemical hydrolysis is unlikely to differentiate between the functionalities. A novel process has now been discovered which converts a compound of formula (I) , the triacetate, to the diacetate of formula (II) : (ID by using a microbial hydrolase. Accordingly, the present invention provides a process for the preparation of a compound of formula (II) as hereinbefore defined, which process comprises the treatment of a compound of formula (I) , as hereinbefore defined, with a microbial hydrolase. Suitable microbial hydrolases are to be found in microorganisms which include Penicillia and Bacteria, in particular Penicillium frequentans IMI 92265. The organisms are grown in a nutrient broth or other suitable medium at a temperature of 10-50°C, preferably around 20-40°C, according to the nature of the organism employed. The reactions can be carried out in this medium, or in an alternative medium or salt solution after separation and transfer of the cells. Alternatively, purified or partially purified enzymes can be used after cell disruption and fractionation of the cells. The reaction is carried out at a pH in the range 3-10, preferably 5.0-8.0, usually at a temperature of 10-40°C, or 20-40°C. The enzyme transformation may be improved in yield in the presence of ammonium sulphate, preferably 1 molar. Typically, enzyme in cell bound or cell-free form can be immobilised and re-used. In the case of Penicillium frequentans IMl 92265 this can be done several times without significant loss of activity. The substrate concentration may be increased to 1% or greater w/v. Hence a continuous production method can be envisaged, passing the substrate over the immobilised enzyme in a column, loop reactor or similar reactor. The product is purified by extraction into a suitable solvent following which it may optionally be chromatographed. The process has the advantage that minimal amounts of by-product of formula (III) are produced. (III) The following Examples illustrate the invention. It will be appreciated that yields may be improved by carrying out strain improvement on the organism, by known techniques. Example 1 Culture of Penicillium frequentans IMl 92265 Two loopsful of mycelium from a slope of Penicillium frequentans IMl 92265 were mixed in 4ml of 0.02% Tween 80 in water and 2ml of the suspension was used to inoculate 40ml of seed medium in a 250ml Erlenmeyer flask. The seed medium comprised (% w/v) cornsteep liquor (4%); treacle (2.6%); CaC0 3 (0.5%) and distillers' solubles (Scotafeed) (2%) in deionised water adjusted to pH 5.2. After shaking at 240rpm at 28<C for 72h, 1.5ml of the seed broth was used to inoculate 40ml of production medium in 250ml Erlenmeyer flasks. This medium comprised (% w/v) glucose (3%), nutrient broth (0.8%); yeast extract (0.2%) and malt extract (0.3%) in deionised water adjusted to pH 5.6. These flasks were incubated, with shaking at 240rpm, at 28°C, for 72h. Example 2 Reqioselective ester hydrolysis with whole cells of Penicillium frequentans IMl 92265 The triacetate of formula (I) was added to cultured shake flasks of Penicillium frequentans IMl 92265, produced as in example 1, to give a final substrate concentration of 4mg ml- . The flasks were then shaken at 240rpm and 28<C and finally assayed for products after centrifugation and extraction of the supernatant with chloroform. The organic phase was assayed by hplc. After incubation of the substrate with the microorganism for 6hr, a 15% conversion of su,;strate to diacetate product (II) was indicated and the ratio of product (II) to its regioisomer (III) was 91:9. After 9h incubation a 6% conversion to product was indicated and the ratio of product (II) to its regioisomer (III) was 97:3. Example 3 Extraction and partial purification of a reqioselective esterase from Penicillium frequentans IMl 92265 The mycelium from 1.61 of Penicillium frequentans IMl 92265 culture broth, produced as in example 1, was separated by centrifugation (16,000xg, 10 min., 4°C) after cooling to 4°C. The biomass was resuspended to 650ml in 0.1M Tris buffer at pH 7.5 and the cells disrupted using a French press (1250psi) in 60ml batches of cell slurry. After centrifugation (23,000xg, 20 min., 4°C) the supernatant was adjusted to pH 7.1 by the addition of IM sodium hydroxide solution and a solution of 3.65g of streptomycin sulphate in 5ml water was added with stirring: after 0.5h at 0°C the cell extract was centrifuged (39,000xg, 20 min., 4°C) and the separated supernatant brought to a 60% saturation level of ammonium sulphate by the addition of solid ammonium sulphate while stirring. After 15 min. at 0°C then centrifugation (23,000xg, 10 min., °C) , the separated supernatant was brought to 80% saturation of ammonium sulphate as before. After 15 min. at 0°C re-centrifugation as before and separation of the su p ernatant left a precipitate which was dissolved in ".5ml 25mM Tris buffer, pH 7.5. This solution was desalted on Sephadex PD10 columns according to the manufacturers instructions eluting with 25mM Tris buffer, pH 7.5. The desalted protein preparation was made up to 20ml volume with deionised water and applied to an anion exchange resin. column (DEAE-Trisacryl, 7.5 x " .8cm) which had been pre-equilibrated with 25mM Tris buffer, pH 7.5. Elution was with this buffer and, over 8h, a linear gradient of 0 - 300mM ammonium sulphate solution in the same buffer. Column fractions were assayed for esterase activity by incubating 250μl of column protein fraction with 250μl of a 4mg ml- 1 solution of triacetate (I) in 0.2M Tris buffer pH 7.5 with 0.4M ammonium sulphate. After 18h at ambient temperature the solution was extracted with 125μl chloroform which was assayed by hplc. Two protein fractions were isolated which showed 80% conversion of (I) to the product (II) and neither showed more than 11% of the regioisomer (III) in the mixture of diacetate (II) and (III) . Column protein fractions were optionally concentrated by ultrafiltration and the protein concentrations determined (Method of M.M. Bradford, Anal. Biochem., 1974, 72 . , 248-254) . Example 4 Reqioselective ester hydrolysis with enzyme preparation from Penicillium frequentans IMl 92665 To 125μl of a 4mg ml- 1 solution of triacetate (I) in 1.2M Tris buffer pH 7.5 was added 250μl of an enzyme preparation described in example 3 with a protein concentration of 2.67 mg ml- 1 , and 125 μl of 4M ammonium sulphate solution in 12mM Tris buffer pH 7.5. After gentle mixing the reactions were incubated at 20°C for 4h then the products extracted into 125μl chloroform and assayed by hplc. A conversion of 97% substrate (I) to diacetate (II) was found and the ratio of product (II) to its regioisomer (III) was 95:5. Example 5 Effect of ammonium sulphate on enzyme catalysed ester hydrolysis An esterase preparation was obtained by the procedure described in example 3 with a protein concentration of 3.56mg ml . A 250μl aliquot of this was added to each of two mixtures of 5mg of triacetate (I) in 125μl of 4M ammonium sulphate solution in 12mM Tris buffer pH 7.5 and to the other reaction was added 125μl 12mM Tris buffer pH 7.5. After gentle mixing for 19h at 20° the reactions were extracted into 250μl chloroform and the organic phase assayed by hplc. Reaction in the presence of ammonium sulphate showed at 71% conversion of triacetate (I) to diacetate (II) whereas in the absence of ammonium sulphate the conversion was 28%. In the presence of ammonium sulphate the ratio of diacetate (II) to its regioisomer (III) was 94.6 whereas in the absence of ammonium sulphate the ratio was 82:18. Example 6 Immobilisation of the esterase with cyanogen bromide activated Sepharose An enzyme preparation with a protein concentration of 1.74mg 1 ml-- * - was prepared as m example 3 and 1.25ml of this was mixed with 1.25ml of 'coupling buffer' (solution of 0.1M sodium bicarbonate and 0.5M sodium chloride at pH 8.3) . Chromatography on a Sephadex PD10 column in this solution gave 3.5ml of a solution of enzyme from which the Tris buffer had been removed. This solution was added to 0.6ml of cyanogen bromide activated Sepharose 4B which had been prepared according to the manufacturer's instructions. After mixing for 2h the gel was allowed to settle and the supernatant discarded. To 5 the gel was added 4ml of O.IM glycine in 'coupling buffer' and the suspension mixed for a further 1.5h at ambient temperature. The gel was then washed sequentially with 'coupling buffer', 0.4M acetate buffer at pH 4.0 in 0.5M sodium chloride solution, and 'coupling buffer'. The gel 10 was stored in suspension at 4°C. Example 7 Repeated use of immobilised regioselective esterase 15 Gel immobilised enzyme was prepared as described in example 6 immobilising 5 mg of protein per ml of gel, and 144 ml of this was washed with an equal volume of O.IM Tris buffer, pH 7.5, containing IM ammonium sulphate. To the gel was added 20 72 ml of 4M ammonium sulphate solution and a solution of 600 mg of triacetate (I) in 72 ml of 0.4M Tris buffer, pH 7.5. The reaction mixture was shaken at 14°C for 5h before centrifugation and removal of the supernatant. The 25 immobilised enzyme preparation was further washed with 0.IM Tris buffer, pH 7.5, containing IM ammonium sulphate and the combined supernatants extracted into chloroform to yield, after drying and evaporation, 403 mg of a clear oil. This oil was shown to comprise (II) and (III) in a ratio of 94:6 0 respectively. The immobilised enzyme was resuspended in 0.1M Tris buffer, pH 7.5, containing IM ammonium sulphate and stored at 4°C. The same batch was reused as described above on a further nine occasions giving similar results in each case. A aliquot of gel immobilised enzyme was used under the above described conditions on sixteen occasions with no detectable reduction in activity or regioselectivity as assayed by hplc. Example 8 Immobilisation of the esterase with Phenyl Sepharose To 600μl of solid Phenyl Sepharose CL-4B gel was added 625μl of 4M ammonium sulphate solution and 625μl of 0.4M Tris buffer pH 7.5. To this was added 1.25ml of an enzyme preparation with a protein concentration of 1.74mg l- which was prepared as in example 3. The suspension was mixed gently for 30 min. at ambient temperature, centrifuged and the gel washed twice with 4ml of IM ammonium sulphate in O.IM Tris buffer pH 7.5.

Claims: Claims 1. A process for the preparation of a compound of formula (ID : (ID which process comprises the treatment of a compound of formula (I) : (I) with a microbial hydrolase. 2. A process according to claim 1 wherein the microbial hydrolase is from a Penicillia or Bacteria. 3. A process according to claim 2 wherein the microbial hydrolase is from a Penicillium frequentans IMl 92265. 4. A process according to any one of claims 1 to 3 wherein the reaction takes place in the presence of ammonium sulphate. 5. A process according to any one of claim 1 to 4 wherein the enzyme is immobilised and reused in a continuous production method.

Inventors: GRINTER TREVOR JOHN, SIME JOHN THOMAS, WORONIECKI STEFAN ROLAND, GRINTER, TREVOR, JOHN, SIME, JOHN, THOMAS, WORONIECKI, STEFAN, ROLAND
Applicants: BEECHAM GROUP PLC, GRINTER TREVOR JOHN, GRINTER, TREVOR, JOHN
Application year: 1991
Main classification: C12P7
IPC codes: C12N9, C12P7, C12R1