GB2358859A - Synthesis of isosorbide compounds - Google Patents

Abstract

A method of synthesising a compound of the following formula (1): <EMI ID=1.1 HE=26 WI=57 LX=1216 LY=532 TI=CF> <PC>comprises: <SL> <LI>(i) treating a compound of the following formula (2): <EMI ID=1.2 HE=24 WI=49 LX=1267 LY=958 TI=CF> in the presence of an enzyme effective to selectively acylate the compound of formula (2) at the 2-position to produce a compound of the following formula (4): <EMI ID=1.3 HE=24 WI=48 LX=1202 LY=1473 TI=CF> <LI>(ii) treating the resulting compound of formula (4) with a nitrating agent to form a compound according to the following formula (5): <EMI ID=1.4 HE=24 WI=47 LX=1226 LY=1927 TI=CF> and <LI>(iii) treating the compound of the formula (5) with a hydrolysing agent to remove the protecting -OCOR<SP>5</SP> ester group from the 2-position to form the above compound according to formula (1). </SL> R<SB>1</SB>-R<SB>5</SB> are substituents as defined in the specification.

Description

2358859 1 COMPOUND SYNTHESIS This invention relates to the synthesis of

chemical compounds, in particular the synthesis of isosorbide-5mononitrate and substituted analogues thereof. More specifically, the invention relates to the preparation of isosorbide-5-mononitrate via enzymatic esterification of isosorbide. More generally, the invention also relates to intermediate enzymatic protection selectively at the 2position of isosorbide and its substituted analogues in the preparation of 5-substituted derivatives thereof.

Isosorbide (1,4:3,6-dianhydro-D-glucitol) is one of the hexitol class of bicylic heterocyles derived from simple sugars, which in recent years has attracted increasing interest, particularly as regards its biological activity. This growing importance particularly of certain analogues of isosorbide is attributable to the reactivity of the hydroxyl groups, which as shown in the structural formula of isosorbide below are, relative to the ring juncture, exo at the 2-position and endo at the 5- position:

HO 0 2 0. 1 1 1 OH Isosorbide The other two main compounds of the hexitol class, which differ from isosorbide solely in the stereochemistry of the hydroxyl groups, are isoidide (1,4:3,6-dianhydro-Liditol) in which the hydroxyl groups are both exo relative to the ring juncture, and isomannide (1,4:3,6-dianhydro- Dmannitol) in which the hydroxyl groups both adopt an endo 2 conformation.

The nature of the endo 2-hydroxyl group of isosorbide leads to internal hydrogen bonding between this hydroxyl group and the opposite ether ring oxygen. This increases the reactivity of the 5-endo position, thus leading to an interesting synthetic problem to the protection of the 2position.

Isosorbide-5-mononitrate is currently the most commercially important analogue of isosorbide, which is an important vasodilator useful in cardiac treatment, for example for treating angina. Known synthetic routes to isosorbide-5-mononitrate are, however, inefficient, in particular because of the difficulty of protecting the 2position. This leads to an expensive product.

Various other synthetic routes to isosorbide-5mononitrate are also known, but to date all these other preparative methods suffer from disadvantages, whether it be expense, excessive time of reaction, low selectivity or low yield.

For example, Seemayer et al in Tetrahedron Asymmetry, 1992, 3, 1123 and United States Patent US-A-5538891 describe the enzymatic hydrolysis of the diester analogue of isosorbide through the use of lipase from Pseudomonas fluorescens. Isosorbide is initially diacetylated using acetic anhydride and pyridine and the resulting di-acetate is then treated with the enzyme Pseudomonas fluorescens to give the 2-monoacetate. After nitration at the 5-position with nitric acid and acetic anhydride and deprotection of the 2-position with potassium carbonate and methanol, the 5-mononitrate is obtained. This however is a four-stage process involving an inefficient route to protection of the 3 2-position.

The preparation of i so sorbide- 5-mononi t rate from isosorbide dinitrate is also a commercially important route. For example, in Ind. J. Chem., 1997, 36B, 793 Chandrasekaran et al disclose a process involving chemoselective reduction of isosorbide dinitrate to its 5mononitrate in a 70% yield using benzyltriethylammonium tetrathiomolybdate. In a previous paper from the same first author, in Synthesis, 1994, 1032, a corresponding process was achieved using a titanium borohydride complex which gave isosorbide-5-mononitrate in a 57% yield. The cost of such reduction complexes, however, makes such processes unfeasible on anything other than a laboratory scale.

In Gazzetta Chimica Italiana, 1987, 117, 173, Lucchi et al describe the preparation of isosorbide-5-mononitrate using a reductive system comprising zinc and acetic acid in an inert medium. Yields of 44% are reported. However, a drawback of this method is the need for addition of acetic acid at regular time intervals over a 24 hour period.

Catalytic hydrogenation of isosorbide-2,5-dinitrate has also been proposed in the literature for the preparation of isosorbide-5- mononitrate, for example in published European patent application EP-A- 0201067. In this disclosure palladium on carbon (10%) in the presence of nickel chloride as a selector is used, and is reported to give a 61% yield of the desired 5-mononitrate. Also, as disclosed in US-A-4381400, hydrazine derivatives have also shown some potential, but a drawback of this route is that yields favour the formation of isosorbide-2- mononitrate over isosorbide-5-mononitrate.

The bioconversion of isosorbide dinitrate has been studied by Ropena et al, as reported in Appl. Microbial.

4 Biotechnol, 1988, 27, 358. The study identified two Cunninghamella strains for effecting chemo-selective reduction of the dinitrate and good selectivity towards the preparation of either i sosorbide-2 -mononi t rate or isosorbide-5mononitrate is reported.

Another route to isosorbide-5-mononitrate from the 2, 5-dinitrate is reported by Anteunis et al in Org. Mag. Res., 1971, 3, 693, and involves heating the dinitrate in a closed vessel with hydrochloric acid. However, only limited reduction yields are obtained and furthermore the process is not selective as between the 2- and 5-mononitrates.

The literature also contains disclosures of selective alkylations of isosorbide. The most productive of these methods with respect to yield is reported by Stoss et al, in Synthesis Com., 1987, 174 and European Patent application EP-A-0057847. The preparation involves acetylation with acetic anhydride followed by distillation over alkali metal carbonate, hydroxide or methylate, or lead oxide, yielding up to 70% of the desired product. However, from a practical point of view and in particular an industrial viewpoint, the use of large scale distillation apparatus can incur excessive capital expenditure. A more practical solution to this problem is reported by Cetovic et al, in Synthesis, 1989, 610. This method selectively esterifies isosorbide with carboxylic acid derivatives in the presence of 4 -dime thyl aminopyridine (DMAP). The yields for the functionalised isosorbide were of the order of 68 to 85% of the 2protected diol. This approach is an example of the utilisation of the sterically unhindered 2- exo hydroxyl group which is esterified in preference to the more sterically crowded 5-endo moiety. The process however suffers from problems relating to the expense and toxicity of the reagents used.

Another approach to the selective alkylation of isosorbide is reported by Abenhaim et al in Carbohydrate Research 1994, 261, 255. Under direct alkylation using allyl, propyl and benzyl protecting groups, 40% yields of the 2-exo-benzyl derivative was reported. This reference also disclosed results of selective mono-acylation of isosorbide and it also suggests the use of a counter-ion for guiding the selectivity of the reaction towards the 2exo moiety.

In Bull. Soc. Chim. Fr., 1988, 3, 567, Lem et al report the protection of the 2-hydroxyl function of isosorbide, preliminary dialkylation of the carbohydrate, followed by treatment with pivalyl -chloride, to yield the required 2exo adduct. The yields for this route were reasonable, but it was not so promising as regards selectivity for the 2exo product.

As mentioned above, none of the above discussed known routes to isosorbide-5-mononitrate is free from one or more problems or disadvantages as regards yield, selectivity, time of reaction or expense. There is therefore a need for a new synthetic route to isosorbide-5mononitrate which represents an improvement on the prior art as discussed above.

With this object in mind, the present invention has been devised and involves the unexpected usefulness of particular enzymes as a means of directing nitration of the isosorbide moiety selectively towards the 5position, by selective protection of the 2-position.

Accordingly, in a first aspect the present invention provides a method of synthesising a compound of the following formula (1):

6 0 2 NO 0 R4 -- i R3 0 1 n,R 1 (1) in which: R' is H or optionally substituted straight or branched chain Cl- C30 carboxyalkyl, CI-C30 sulphoxyalkyl, C3C30 carboxycycloalkyl,C3-C30 sulphoxycycloalkyl, carboxyheterocyclic, sulphoxyheterocyclic, C3-C30 carboxycycloalkenyl or sulphoxycycloalkenyl, CS-C30 carboxycycloalkynyl or sulphoxycycloalkynyl, C2-C30 carboxyalkynyl or sulphoxyalkynyl group, C4-C30 carboxyaromatic or sulphoxyaromatic, C4-C30 carboxyheteroaromatic or sulphoxyheteroaromatic group, wherein in any of the said hereto atom- containing groups the hetero atom is selected from the group consisting of 0, S and N and in the case of any of the aforementioned groups being substituted there are present one or more substituents independently selected from the group consisting of halogen, cyano, Cl-C30 alkyll C2- C30 alkenyl, C4-C30 aromatic, CI-C30 ether, Cl-C3. ester, CI-C30 sulphonate ester, nitro, CI-C30 ketonef Cl-C30 thioether and/or one or more pharmaceutically active groups such as 2acetoxybenzoate, 2N-(31- trifluoromethylphenyl) aminobenzoate, (S)6-methoxy-c(-methyl-2- naphthaleneacetate and (S)-1-[N-[1-(ethoxycarbonyl)-3-phenylpropyll-L- alanyl]L-proline carboxylate; and each of R' and R' is independently selected from H or optionally substituted straight or branched chain CI- C30 alkyl, Cl-C30 carboxyalkyl, Cl-C3. sulphoxyalkyl, Cl-C30 alkoxy, C3- C30 cycloalkyl, C3-C30 carboxycycloalkyl, C3-C30 sulphoxycycloalkyl, C3- C30 cycloalkoxy, heterocyclic, carboxyheterocyclic, 7 sulphoxyheterocyclic, oxyheterocyclict C3-C30 cycloalkenyl, carboxycycloalkenyl, sulphoxycvcloalkenyl or cycloalkenoxy, C8-C30 cycloalkynyl, carboxycycloalkynyl, sulphoxycycloalkynyl or cycloalkynoxy, C2-C30 alkynyl, carboxyalkynyl, sulphoxyalkynyl or alkynoxy group, C4-C3. aromatic, carboxyaromatic, sulphoxyaromatic or aryloxy, C,C30 heteroaromatic, carboxyheteroaromatic, sulphoxyheteroaromatic or heteroaryloxy group, wherein in any of the said hereto atom-containing groups the hetero atom is selected from the group consisting of 0, S, and N and in the case of any of the aforementioned groups being substituted there are present one or more substituents independently selected from the group consisting of halogen, cyano, CI-C30 alkyl, C2-C,., alkenyl, C4- C30 aromatic, CI-C30 ether, ClC30 ester, CI-C30 sulphonate ester, nitro, Cl-C30 ketone, Cl-C3. thioether and/or one or more pharmaceutical ly active groups such as 2acetoxybenzoate, 2-N(3'trifluoromethylphenyl)aminobenzoate, (S)-6-methoxy-cxmethyl - 2 naphthal eneace tat e and (S)-1-[N-El(ethoxycarbonyl) - 3-phenylpropyl 1 L-alanyl] -L-pro line carboxylate; the method comprising:

(i) treating a compound of the following formula (2):

R.20 0 R4- R' 0 (2); OR' in which R 2 is H or optionally substituted straight or 8 branched chain C--C30 carboxyalkyl, CI-C30 sulphoxyalkyl, C3C30 carboxycvcloalkyl, C3-C30 sulphoxycycloalkyl, carboxyheterocyclic, sulphoxyheterocyclic, C3-C30 carboxycycloalkenyl or sulphoxycycloalkenyl, C8-C30 carboxycycloalkynyl or sulphoxycycloalkynyl, C2-C30 carboxyalkynyl or sulphoxyalkynyl group, C4-C-10 carboxyaromatic or sulphoxyaromatic, C4C30 carboxyheteroaromatic or sulphoxyheteroaromatic group, wherein in any of the said hetero atom-containing groups the hetero atom is selected from the group consisting of 0, S and N and in the case of any of the aforementioned groups being substituted there are present one or more substituents independently selected from the group consisting of halogen, cyano. Cl-C3. alkyl, C2-C30 alkenyl, C4-C30 aromatic, CI-C30 ether, CI-CM ester, CI-C30 sulphonate ester, nitro, CI-C30 ketone., CI-C30 thioether and/or one or more pharmaceutically active groups such as 2 acetoxybenzoate, 2-N- (3 1 trifluoromethylphenyl)aminobenzoate, (S)-6methoxy-c(methyl -2 -naphthaleneace tat e and (S)-1-EN-[l(ethoxycarbonyl)3-phenylpropyl]-Lalanyll-L-proline carboxylate, and R', R 3 and R 4 are as defined above; with an acylating agent of the following formula (3) 0 Y R5) X 1001 in which X is 0 or S, and Y is a group selected from (3) 9 R 7 R 7 R 7 c c 1,11 1 L-R6 R "\\R 6 \ 6 R R 6 N \1, R 7 or N=R 6 in which each of R', R6, R 7 and R8 is independently selected from H, substituted or unsubstituted straight or branched chain C,-CM alkyl, C,-C, o carboxyalkyl, CI-C30 sulphoxyalkyl, C,-C,o alkoxy, C3-C30 cycloalkyl, C3-C3. carboxycycloalkyl, C,C3C sulphoxycycloalkyl, C3-C30 cycloalkoxy, Cl-C30 haloalkyl (where the halo functionality may be mono-, di- or trisubstituted chloro, fluoro, bromo, or iodo groups) heterocylic, carboxyheterocyclic, sulphoxyheterocyclic, oxyheterocyclic, C3-C30 cycloalkenyl, carboxycycloalkenyl, sulphoxycycloalkenyl or cycloalkenoxy. C8-C30 cycloalkynyl, carboxycycloalkynl, sulphoxyalkynyl or cycloalkynoxy, C2-C30 alkynyl, carboxyalkynyl, sulphoxyalkynyl or alkynoxy group, C4- C30 aromatic, carboxyaromatic, sulphoxyaromatic or aryloxy, C4-C30 heteroaromatic, carboxyheteroaromatic, sulphoxyheteroaromatic or heteroaryloxy group, wherein in any of the said hetero atom-containing groups the het-eroatom is selected from the group consisting of 0, S, or N and in the case of the aforementioned groups being substituted there are present one or more substituents selected from the group consisting of halogen, cyano, Cl-C30 alkyl, C-I-C30 alkenyl, C4-C30 aromatic, Cl-C30 ether, Cl-C30 ester, CI-C30 sulphonate ester, nitro, CI-C30 ketone, CI-C30 thioether, and/or one or more pharmaceutically active groups such as 2acetoxybenzoate, 2-N(31trifluoromethylphenyl)aminobenzoate, (S)6-methoxyamethyl-2-naphthaleneacetate and (S)-1-[N-[l(ethoxycarbonyl) -3phenylpropyl 1 -L-alanyl] -L-proline carboxylate; or in the formula of Y, C=R 7 may represent a carbonyl (i.e. C=O) group; or in the formula of Y represented as containing both R' and R7 the group R 7 may be absent and R6, or the C to which it is bonded, is bonded directly to R5 to form a corresponding cyclic structure; and where in the case of R' or R 7 being double bonded to C or N (as the case may be) the said R' and R 7 group is the Trene" group corresponding thereto; in the presence of an enzyme effective to selectively acylate the compound of formula (2) at the 2-position to produce a compound of the following formula (4):

R2C) 0 J R 0 1 oco-.,.5 (4); (ii) treating the resulting compound of formula (4) with a nitrating agent to form a compound according to the following formula (5):

07ING 0 - _p 3 R 4 0 0 C 0,R's (5); and 11 (iii) treating the compound of formula (5) with a hydrolysing agent to remove the protecting -OCOR5 ester group from the 2position to form the above compound according to formula (1).

Preferably according to the invention the enzyme that is used to effect the 2-selective acylation is one or more enzymes selected from the group consisting of: pig (or hog) liver esterase, horse liver esterase, Subtilisin carlsberg and Rhizopus oryzae.

Unexpectedly, it has been found that by using the above enzymes in particular, an especially good balance good yield and high selectivity for the 2-protected intermediate may be achieved.

In a second aspect the invention provides a compound according to formula (1) as defined above as or when produced by the method of the first aspect of the invention.

The invention further provides, in a third aspect, the use of an enzyme selected from the group consisting of pig (or hog) liver esterase, horse liver esterase, Subtilisin carlsberg and Rhizopus oryzae for effecting acylation selectively at the 2-position of a compound according to formula (2) in the presence of an acylating agent, particularly that according to formula (3).

Additionally, and more generally, the present invention provides, in a broader fourth aspect, a method of introducing a protecting group into a compound according to formula (2) as defined above selectively at the 2position, comprising treating the compound of formula (2) with a protecting group- introducing agent in the presence of an enzyme effective to introduce the protecting group 12 selectively at the 2-position, which enzyme is preferably selected from the group consisting of: pig (or hog) liver esterase, horse liver esterase, Subtilisin carlsberg and Rhizopus oryzae.

Preferred features and embodiments of the present invention in its various aspects will now be described in detail.

The compound according to formula (1) as defined above, which compound is preferably that which is prepared by the method of the invention, is preferably isosorbide-5mononitrate, i.e. that compound in which R' is hydrogen, as are both R 3 and R 4, and also as is R 2 in formula (2) above.

Various acylating agents may be used for introducing the acyl (ester) protecting group. Vinyl acetate and vinyl butyrate (ie. where R' is, respectively, methyl and propyl and R' is H in formula (3) above) are particularly preferred. They also have the advantage that they can be converted during the course of the reaction to acetaldehyde and thus be lost from the system owing to its volatility. Generally, vinyl esters are preferred for use as the acylating agent in the invention because the vinyl alcohol, once produced as a by-product in the process, spontaneously tautomerises to the equivalent aldehyde, thus ensuring that the reverse reaction is not favoured. Various other acylating agents may also be suitable, for example vinyl butyrate, 2,2,2- trichloroethylbutyrate, 2,2,2trifluoroethylbutyrate, S ethyl thiooctanoate, biacetyl monooxime acetate, isopropenyl acetate, 1ethoxyvinyl acetate, diketene, acetic anhydride or succinic acid anhydride. All these acylating agents are employable either singly or in combination with one or more others. Whilst not wishing to be bound by theory, it is possible that, as between different acylating agents, a modest 13 improvement in selectivity may be obtained as a function of the increase in size of the acyl unit.

According to the invention, the enzyme which is used to effect the acylation of the isosorbide moiety selectively (or, more accurately, more favourably) in the 2-position is preferably selected from the group consisting of: pig (or hog) liver esterase, horse liver esterase, Subtilisin carlsberg and Rhizopus oryzae. As will be described further in the Examples hereinbelow, these particular enzymes were found unexpectedly to be especially effective at promoting good selectivity of acylation at the 2-position in combination with good yields of the desired product.

This unexpectedness is especially because the existing literature for related esterases and lipases (in particular Tetrahedron Asymmetry (1997), 8, 425 and US-A-5538891) teaches the regiospecific resolution of esters through the hydrolysis mechanism and thus teaches away from the use of these particular enzymes in accordance with this invention.

These preferred enzymes may be derived from their respective animal sources by techniques readily available to persons skilled in the art and described in the literature. Such enzymes are also commercially available, for example from Altus Biologics, Inc. As used herein, the reference to "pig" and "hog" liver esterases refers to the same enzyme species, but from different sources ("pig" being derived from European sources and "hog." from US sources).

When pig liver esterase is used as the enzyme, it may be preferable to use it in a modified form, for example through the covalent attachment of methoxypolyethylene glycol (MPEG), in order to overcome characteristic losses 14 of activation in organic solvents. This is taught for example by Ruppert et al in Tetrahedron Asymmetry, 1997, -8, 3657, or Heiss et al in Tetrahedron Letters, 1995, 36, 3833.

In the practicing of the present invention the relative amounts of enzyme which may be used, as a weight proportion of the substrate (ie. the compound of formula (2)) may vary from typically from about 1 to about 500%. Particularly preferred, from an economic viewpoint, is the approximate range 110%. Similarly, the amount of acylating agent used (relative to the substrate ie. the compound of formula (2)) may typically vary from about 1 to about 10 molar equivalents. Particularly preferred, from an economic viewpoint, is the approximate range 1 to 5 molar equivalents.

The method of the invention may typically be carried out under conditions of room temperature and pressure.

The starting materials for the reaction method of the invention are all readily available from commercial sources, as will be readily apparent to persons skilled in the art. The sources of the various preferred enzymes for use in the invention have been mentioned above, and in addition isosorbide in particular may be obtained in >98% purity from commercial sources. By way of an example of one known synthetic route to isosorbide from D-glucitol as the appropriate starting sugar, reference is made to the disclosure by Fleche, et al, starch/starke 1986, 38, 26.

The method of the invention is preferably carried out in an aqueousorganic solvent. A predominately organic solvent medium is preferred in order to dissolve the organic reagents in the starting mixture, but an amount of water in the solvent phase is generally required to enable the enzyme to effectively perform its catalyst function. Chloroform is a preferred organic solvent for solubility reasons, although other suitable organic solvent components may include THF, dichloromethane, iso-octane, t-butanol, acetone, t-butyl-methyl ether, toluene, acetonitrile and dimethyl formamide. Any combination of one or more organic solvent components and water may be used as the preferred solvent medium. Preferably the solvent phase of the reaction mixture comprises total organic component(s) in an amount of from about 1 to about 40% by weight of the total solvent phase, and water in a corresponding amount of from about 0.1 to about 20% by weight.

The amount of water present in the solvent phase will generally be selected in accordance with the particular enzyme employed, given that typically optimum reactivity of different enzymes will require different levels of water in the solvent phase. This is taught in more detail in the Examples hereinbelow.

Preferred features and embodiments of the invention in its various aspects will now be described in further detail, by way of non-limiting example only, in the following Examples.

Examples

For the purpose of detection and characterisation of the various products of interest in the Examples below, techniques employed included any suitable combination of 'HNMR and 13 CNMR spectroscopy, gas chromatography, elemental analysis and melting point determination. Notes about each of these techniques are set out below:

(1) Nuclear Mapnetic Resonance Spectra (NMR) The 'HNMR and 13 CNMR spectra for isosorbide have been fully 16 characterised, for example by Hopton et al, in Can. J. Chem., 1969, 47, 2395. It is noteworthy to mention that the spectrum of isosorbide is complicated with respect to the other parent compounds isomannide and isoidide, owing to the loss of symmetry arising from the endo-exo nature of the hydroxyl groups. This characteristic induces inequivalence of the hydrogens within the two rings.

The observed 'HNMR spectrum for isosorbide-5mononitrate is as follows: 'HNMR (300 MHz, WC13) 5, ppm: 2.1(1H,s,OH), 3.98(4H,m) 4.37(1H,s), 4. 42(1H,d,J=4.95), 5.0(1H,t,J=5.08 and 4.95), 5.35(1H,M).

The 13WMR spectrum for isosorbide-5-mononitrate, according to SYNTHESIS, (1994), 1032, is as follows:

"WMR (300MHZ, CDC13)51 PPM 69.12, 75.50, 75.65, 81.07, 81.32, 88.69.

(2) Gas ChromatoaraPhy A SE-30 column was used, with the following temperature settings: Col; 16WC, inj:180'c, det:24WC.

(3) Elemental analysis A Carlo Erba strumentazione mod. 1106 CHN analyzer was used according to the standard procedure.

(4) MeltinQ point determination A GallenKamp (UK) electrothermal melting point unit was used accordingly to the standard procedure.

Exam.ple 1 ScreeninQ of Enzymes A range of different enzymes were screened in order to 17 determine those particularly preferred for use in the invention in terms of their optimum combination of high selectivity and good yield of the desired 2-protected intermediate.

A 3.3 mg/ml stock solution of isosorbide was prepared by dissolving isosorbide (100 mg, 0.68 mmol) in chloroform (30 ml). From this stock solution 25 1 ml aliquots were removed and placed into vials containing 10mg samples of 25 different enzymes, as shown in Table 1 below. Vinyl acetate (500 il, 5.42 mmol) was added to each vial and the mixtures were stirred at room temperature. The presence of reaction components was monitored by GC. The results are set out in Table 1 below.

Table 1

Lipase No 2-Acetyl -F% 5-Acetyl Diacetyl Isosorbide Pig liver esterase 32.95 - - 67.04 (47.91) 12) (52.10) (21 Pseudomonas cepacia 10.8 - 89.2 Candida rugosa 11.50 4.94 - 83.57 (13.01) (2) (5.83) (2) (81. 16) (2) Penicillin Acylase 2.136 - - 97.13 Aspe.rgillus niger 1.96 4.91 93.13 Mucor rnelhel - 16.17 - 83.34 Candida antartica B 4.73 31.11 - 64.16 Humicola lanuginosa 2.48 8.24 - 89.28 Bacillus protease 7.79 - - 92.21 Candida rugosa (3) 16.829 4.997 76.628 0.703 Subtilisin 71.05 14.96 1.59 5.69 carlsberg (4) (81.06) (2) (11.66) (2) (13. 99) (21 Pseudomonas 16.09 81.69 - Capacia(5) Rhizopus delmar 1.92 4.93 0.79 87.76 Rhizopus oryzae 8.01 - - 91.99 Chrom.bobacterium 1.11 5.29 93.61 vicosum Alcallgenes species 10.91 72.48 12.76 3.85 Geotricum candidum 1.6 - - 98.4 Mucor javanicus - - 0.602 99.398 Aspergillus oryzae 0.51 2.01 - 97.48 protease Candida rugosa 0.17 - 0.64 96.3 esterase Notes:

(1) Reaction Time: approx 5 days. (2) Figures in brackets represent reassay after a further 36 hrs.

(3) ChiroCle CTI- CR (dry), ex Altus Biologics, Inc. (4) ChiroCle CT1 -BL (dry), ex Altus Biologics, Inc (5) ChiroCleCTI-pC (dry), ex Altus Biologics, Inc.

A further enzyme screening experiment was conducted as follows:

A 3 mg/ml stock solution of isosorbide was prepared by dissolving isosorbide (15 mg) in dichloromethane (5 ml) Four further different enzymes (10 mg) were weighed into separate vials and 1 ml of the isosorbide solution was added to each vial using a Gilson pipette. Vinyl acetate (500 pl, 5.42 mmol) was added and all the mixtures were stirred at room temperature. The presence of reaction components was monitored by GC. The results are set out in Table 2 below.

Table 2

Esterase % Isosorbide %2-Acetyl %5-Acetyl Mucor Miehei 78.2 - 21.8 Horse liver 93.75 6.25 - 19 Bacillus Sp. 85 15 Hog liver 78 22 - From Tables 1 and 2 above it can be seen that four of the enzymes screened showed a good combination of yield and selectivity, making them preferred enzymes for use in the invention. These were: pig (hog) liver esterase, Subtilisin carlsberg, Rhizopus oryzae and horse liver esterase.

Example 2

This example illustrates application of the invention to the preparation of isosorbide-5-mononitrate, using pig liver esterase as the enzyme and vinyl acetate as the 2acylating protecting agent.

(a) Preparation of isosorbide-2-acetate Isosorbide (50 mg, 0.34 mmol) was dissolved in dichloromethane (4 ml). Pig liver esterase (PLE) (250 mg) and vinyl acetate (0.5 ml, 5.4 =ol) were added and the reaction mixture was stirred at room temperature for 9 days. The enzyme was gravity filtered and washed with dichloromethane. The products were separated by column chromatography (silica, ethyl acetate: petroleum ether 1:1) to yield isosorbide-2 -acetate (38.8 mg, 56.3%) as a white crystalline solid. The product was characterised by NMR as follows:

HNMR (300 MHz, CDC13) 5, ppm:2.06(3H,s), 2.60 (1H,d,OH,J=7.3), 3.58(1H,dd, J=2 x 6.0), 3.89 (1H,dd,J=2 x 6.0), 4.02 (2H,m), 4.32 (1H,m), 4.49 (1H,d, J=4.4), 4.63 (1H,t, J=5.1 and 4.8), 5.22 (1H,s). 13 CNMR (300 MHz, WC13) 5, ppm: 20.91, 72.41, 73.72, 78.54, 82.08, 85.76, 170.05.

(b) Preparation of Is osorb ide - 5-Mononitrate- 2 -Acetate I sosorbide-2 -acetate (35.0 mg, 0.18 mmol) from step (a) in dichloromethane (1 ml) was added dropwise into a solution of nitric acid (69% w/w aqueous solution, 21p11) and acetic anhydride (100%, 85 pil) at OOC. The solution was thenstirred for a further 20 minutes at room temperature. Dichloromethane (50il) and water (80 1l) were added and the phases separated. The organic layer was extracted with ammonium hydroxide solution (10%) until neutral. It was then dried over M9S04 and the solvent removed under vacuum to yield i sosorbide-5-mononi trate-2 - acetate (32.5 mg, 74.7%) as a white crystalline solid. The product was characterised by NMR as follows:

HNMR (300 MHz, CDC13) 5, ppm: 2.05(3H,s), 3.90 (1HddJ=5.5 and 5.6), 4.05 (3H,m), 4.50 (1H,d,J=4.9), 5.0 (1H,t,J=5.5 and 5.4), 5.2 (1Hs), 5.4 (1H,m).

(c) Preparation of Isosorbide-5-Mononitrate I sosorbide- 5-mononitrate-2 -acetate (30 mg, 0.13 mmol) from step (b) was dissolved in methanol (4 ml). Potassium carbonate (8 mg) was added and the reaction mixture was stirred at room temperature for 12 hours. Water (10 ml) was added and the organic layer extracted with ethyl acetate (7 x 5 ml). The combined layers were then dried over M9S04 and the solvent removed under vacuum to yield isosorbide-5mononitrate (25.3 mg, 94.9%) as a white crystalline solid. (m.p. 89.60 C, Lit. 89.90OC). The final product was characterised by NMR as follows:

HNMR (300 MHz, CDC13) 51 PPM: 1.85(1H,OH), 3.85 (4H,m,) 4.38 (1H,s), 4.42 (1H,d,J=5.0), 5.0 (1H,tJ=5.2 and 5.1), 5.40 (1H,m) 13 CNMR (300 MHz, CDC13) 6, ppm: 69.218, 75.718, 75.827, 81.177, 81.461, 88.900.

Example 3

21 This example illustrates application of the invention to the preparation of isosorbide-5-mononitrate, using Subtilisin carlsberg as the enzyme and vinyl butyrate as the 2-acylating protecting agent.

(a) Preparation of Isosorbide-2-Butyrate Isosorbide (lg, 6.8 mmol) was dissolved in tetrahydrofuran (50 ml). Chiroclec-BL (50 mg) and vinyl butyrate (2.1 ml, 16.54 mmol) were added and the components were stirred at room temperature for 27 hours. The enzyme was gravity filtered and washed with tetrahydrofuran. The reaction components were separated by column chromatography (silica, petroleum ether:ethyl acetate, 3.1) to yield isosorbide-2butyrate (900 mg, 62%), as a white crystalline solid (m. p. 49.5OC). The product was characterised by NMR as follows:

HNMR (300 MHz, WC13) 5, ppm: 0. 95 (3H, t, J=7. 3 and 7. 4), 1. 67 (2H, m), 2.3 (2H, t, J=7. 5 and 7. 3), 2. 63 (1H, d, OH, J=6.5), 3.57 (1H, dd, J=6. 0 and 6.0), 3.89 (1H,dd,J=6.0 and 6.0), 4.02 (2H,d), 4.28 (1Hm), 4. 48 (1H,d,J=4.4), 4.63 (1H,t,J=5.0 and 4.7), 5.25 (1H,s). 1ICNMR (300 MHz, CDC13) 5, ppm: 13.49, 18.28, 35.96, 72.32, 73.55, 73.70, 78.17, 81.97, 85. 72, 172.628.

(b) Preparation of Isosorbide-5-Mononitrate Isosorbide-2butyrate (500 mg, 2.31 mmol) from step (a) in dichloromethane (2 ml) was added dropwise to a solution of nitric acid (324 il) and acetic anhydride (1.272 pl) at O'C. The solution was then stirred for 20 minutes at room temperature. Dichloromethanie and water were added and the phases separated. The organic phase was extracted with ammonium hydroxide solution (10%) until neutral. The combined layers were dried over M9S04 and then the solvent was removed under vacuum, to give isosorbide-2-butyrate-5 mononitrate as the intermediate product.

22 The crude isosorbide-2-butyrate-5-mononitrate was dissolved in methanol (15 ml). Potassium carbonate (75 mg) was added and the mixture was stirred at room temperature for 14 hours. The reaction mixture was extracted with ethyl acetate (7 x 10 ml) and the combined layers were dried over M9S04. The solvent was removed under vacuum, the product was purified by column chromatography (silica, ethyl acetate) to yield pure isosorbide-5-mononitrate (0.3771 g, 85.4%(2 steps)) as a white crystalline solid (m.p. 89.9'C, Lit. 89.89IC). The final product was characterised by NMR and elemental analysis as follows:

HNMR (200 MHz, CDC13) 6, ppm: 2.02(1H,sOH), 3.97 (4H,m) 4.37 (1H,s), 4.42 (1H,dJ=.8), 5.01 (1Ht.,J=5.1 and 4.94), 5.40 (1H,m) 13 CNMR (300 MHz, CDC13) 5, ppm: 69.218, 75.732, 75.834, 81.177, 81.454, 88.907. Elemental analysis found: % (calc. for C1.H1405 (37.70), H 4.74 (4.75), N 7.33(7.33).

): C 37.82 Exam-ple 4 This example illustrates the use of a different solvent, tbutanol, for the preparation of the isosorbide-2-butyrate intermediate in an alternative to step (a) in Example 3 above.

(a) Preoaration of Isosorbide-2-Butyrate Isosorbide (lg, 6.84 mmol) was dissolved in t-butanol (50m1) at 35'c. Vinyl butyrate (2.6m1, 20.52 mmol) and Carlsberg subtilisin (50mg) were added and the reaction mixture was stirred at this temperature until complete. After 92 hours the reaction was assessed by gas chromatography and gave the following resulting weight yields:

23 Isosorbide Isosorbide-2-butyrate I so so rbide- 5-butyrate 8.6% 77.0% 14. 4 %- The isosorbide-2-butyrate intermediate could be separated and applied to equivalent subsequent process steps in the production of isosorbide-5- mononitrate as in Example 3 above.

Example 5

This example illustrates application of the invention to the selective protection of isosorbide at the 2-position using Subtilisin carlsberg enzyme and 2,2,2-trichloroethylbutyrate.

Isosorbide (100 mg, 0.68 mmol) was dissolved in tetrahydrofuran (5 ml). To this solution Chiroclec-BLT1 (95 mg) and 2,2,2,trichloroethylbutyrate (350 1l, 2.04 mmol) were added and the mixture was stirred at room temperature for 92 hours. The enzyme was gravity filtered and washed with tetrahydrofuran. Purification by column chromatography (silica, dichloromethane: methanol, 96:4) gave isosorbide-2butyrate (83 mg, 55.9%). The product was characterised by NMR as follows:

HNMR (300 MHz, WC13) 51 PPM: 0.93(3H,t,J=7.5 and 7.5), 1.60 (2H,m), 2.30 (2H,t,J=7.5 and 7.2), 2.57 (1H,d,OH,J=7.5), 3.55, (1H,dd,J=5.7 and 6.0), 3.88 (1H,dd,J=6.0 and 6.3), 4.02 (2H,d), 4.28 (1H,m), 4.47 (1Hd,J=4.2), 4. 62 (1H,t,J=4.8 and 4.8), 5.25 (1H,s). 13 WMR (300 MHz, CDC13) 51 PPM: 13. 540, 18.330, 36.003, 72.355, 73.612, 73.745, 78.207, 82.007, 85.763, 172. 667.

Exam-ple 6: Effect of water concentration in the reaction mixture on the reactivitV of enzymes 24 If enzymes are to perform effectively as biocatalysts, an amount of water is required. Without intending to be limited by theory, water molecules are thought to surround the enzyme, forming a barrier between it and the organic solvent, preventing deactivation of the enzyme. This is taught for example by Theil et al, in Tetrahedron, 1991, 47, 7569. The following experiments were carried out on three of the preferred enzymes screened for in Example 1 above to determine optimum concentrations of water in the reaction mixtures for preferred methods according to the invention involving these three particular enzymes.

(a) Effect of water on -pig liver esterase (PLE) Three reaction vials were set up containing a mixture of tetrahydrofuran (10 ml), varying amounts of isosorbide and PLE as shown in Table 3 below, and to each vial an amount of water was added as shown in Table 3 below (as % by weight of the total reaction components). Vinyl acetate (400 pl, 4.3 mmol) was added to each vial and the reaction mixture was stirred at room temperature. The presence of reaction products was monitored by GC. The results are set out in Table 3 below.

Table 3

Vial No % wt Isosorbide PLE (mg) % 2- % 5- % water (mg) hcetyl Rcetyl Isosorbide 1 1 150.1 75.2 7.00 4.00 B9.00 2 2 150.4 75.1 8.70 - 91.30 3 3 150.0 75.1 4.25 95.80 Notes:

1 Average enzyme sub = 0. 50.

2. Conversion time = 196 hours.

The results in Table 3 above for PLE show that in THF transformation occurs at approximately 2% by weight by water. It followed from this observation that a 5-fold excess of PLE substrate was required for acceptable yields to be obtained.

(b) Effect of water on Subtilisin carlsberg (Chiroclec - BLTmI) The procedure in (a) above for PLE was repeated, but using vials containing 1.5 ml of THF, Chiroclec-BL T11 as the enzyme and 138 il (0.75 mmol) vinyl acetate. The results are shown in Table 4 below.

Table 4

Vial Isosorbide % wt Enzyme % 2- % 5- % % Di No (mg) water (mg) Acetyl Acetyl Isosorbide acetyl 1 28.0 1.0 9.8 70.72 15.88 - 13.40 2 26.0 1.5 10.8 70.33 17.30 2.30 10.05 3 27.8 2.0 11.0 62.25 18.28 15.50 4.00 4 28.0 2.5 9.9 52.50 10.97 36.50 - 27.3 3.0 9.8 38.51 10.80 50.70 Table notes: (1) Average enzyme sub = 0.37. (2) Conversion time = 91 hours (vial no. 1), 118 hours (vials 2 to 5).

As can be seen from the results in Table 4 above, the reaction yield was high after 5 days at a water concentration of 1% wt but the larger the water concentration, the lower the yield, as shown for vials numbers 1 to 5. Without intending to be limited by theory, the reason for this may be due to the competing hydrolysis reaction that occurs with excess water, thus regenerating the enzyme starting material.

26 (c) Effect of water on Rhizopus orvzae The procedure in (a) above for PLE was repeated, but using 1.7 ml THF, Rhizopus oryzae as the enzyme and 138 1l (0.75 mmol) vinyl acetate. The results are set out in Table 5 below.

Table 5

Vial Isosorbid %wt Enzyme %2-.:5- % % Di No e water (mg) Acetyl Acetyl Isosorbid acetyl (mg) e 1 25.0 1.0 10.6 2,50 - 97.50 - 2 27.1 1.5 11.0 2.50 - 97.50 - 3 26.6 2.0 10.9 8.83 - 91.17 - 4 24.8 2.5 12.0 22.69 77.31 - 25.0 3.0 12.3 28.01 - 71.99 - Notes:

(1) Average enzyme sub = 0.44.

(2) Conversion time = 100.4 hours.

The results in Table higher level of water is above indicate that generally a required for effective results using Rhizopous oryzae as compared with the other preferred enzymes discussed above. A total conversion of approximately 28% over four days was achieved at a 3% wt water level.

27

Claims (20)

Claims

1. A method of synthesising a compound of the following formula (l):

02NO _,0 R4-- R3 0n "I" ORI (1) in which: R' is H or optionally substituted straight or branched chain CI-C30 carboxyalkyl, Cl-C30 sulphoxyalkyl, C3 C30 carboxycycloalkyl,C3-C30 sulphoxycycloalkyl, carboxyheterocyclic, sulphoxyheterocyclic, C3-C30 carboxycycloalkenyl or sulphoxycycloalkenyl, C8-C30 carboxycycloalkynyl or sulphoxycycloalkynyl, C2-C30 carboxyalkynyl or sulphoxyalkynyl group, C4-C30 carboxyaromatic or sulphoxyaromatic, C4-C30 carboxyheteroaromatic or sulphoxyheteroaromatic group, wherein in any of the said hereto atom-containing groups the hetero atom is selected from the group consisting of 0, S and N and in the case of any of the aforementioned groups being substituted there are present one or more substituents independently selected from the group consisting of halogen, cyano, CI-C30 alkyl, C2C30 alkenyl, C4-C30 aromatic, CI-C30 ether, Cl-C3. ester, CI-C30 sulphonate ester, nitro, Cl-C30 ketone, Cl-CM thioether and/or one or more pharmaceutically active groups; and each of R' and R' is independently selected from H or optionally substituted straight or branched chain CI-C30 alkyl, Cl-C30 carboxyalkyl, Cl-C3. sulphoxyalkyl, CI-C30 alkoxy, C3-C30 cycloalkyl, C3-C30 carboxycycloalkyl, C3-C30 sulphoxycycloalkyl, C3-C30 cycloalkoxy, heterocyclic, carboxyheterocyclic, sulphoxyheterocyclic, oxyheterocyclic/ C3-C30 cycloalkenyl, carboxycycloalkenyl, sulphoxycycloalkenyl or cycloalkenoxy, 28 C8-C30 cycloalkynyl, carboxycycloalkynyl, sulphoxycycloalkynyl or cycloalkynoxy, C2-C.30 alkynyl, carboxyalkynyl, sulphoxyalkynyl or alkynoxy group, C4-C30 aromatic, carboxyaromatic, sulphoxyaromatic or aryloxy, C4 C30 heteroaromatic. carboxyheteroaromatic, sulphoxyheteroaromatic or heteroaryloxy group, wherein in any of the said hereto atom-containing groups the hetero atom is selected from the group consisting of 0, S, and N and in the case of any of the aforementioned groups being substituted there are present one or more substituents independently selected from the group consisting of halogen, cyano. Cl-C30 alkyl, C2-C30 alkenyl, C4-C30 aromatic, Cl-C3. ether, Cl-C30 ester, Cl-C3. sulphonate ester, nitro, CI-C30 ketone, CI-C30 thioether and/or one or more pharmaceutical ly active groups; the method comprising:

(i) treating a compound of the following formula (2):

R20 -- 0 R4-<n R3 (2); 0 -I..

OR1 in which R 2 is H or optionally substituted straight or branched chain CI- C30 carboxyalkyl, Cl-C30 sulphoxyalkyl, C3C30 carboxycycloalkyl, C3-C30 sulphoxycycloalkyl, carboxyheterocyclic, sulphoxyheterocyclic, C3-C30 carboxycycloalkenyl or sulphoxycycloalkenyl, C8-C30 carboxycycloalkynyl or sulphoxycycloalkynyl, C2-C30 carboxyalkynyl or sulphoxyalkynyl group, C4-C30 carboxyaromatic or sulphoxyaromatic, C4-C30 carboxyheteroaromatic or sulphoxyheteroaromatic group, wherein in any of the said hetero atom-containing groups the hetero atom is selected from the group consisting of 0, 29 S and N and in the case of any of the aforementioned groups being substituted there are present one or more substituents independently selected from the group consisting of halogen, cyano, CI-C30 alkyl, C2- C30 alkenyl, C4-C30 aromatic, CI-C30 ether, CI-C30 ester, CI-C30 sulphonate ester, nitro, CI-C30 ketone, Cl-C30 thioether and/or one or more pharmaceutically active groups; and R', R 3 and R 4 are as defined above; with an acylating agent of the following formula (3) 0 Y (3); R5 X in which X is 0 or S, and Y is a group selected from R7 R7 1 11, R7 1 c c C -R8 \ R6 \6 \R6 R6 \ R7 or N=R6 in which each of R', R', R' and R' is independently selected from H, substituted or unsubstituted straight or branched chain C1-C30 alkyl, Cl-C30 carboxyalkyl, Cl-C30 sulphoxyalkyl, CI-C30 alkoxy, C3-C30 cycloalkyl, C3-C30 carboxycycloalkyl, C3 C30 sulphoxycycloalkyl, C3-C30 cycloalkoxy, CI-C30 haloalkyl (where the halo functionality may be mono-, di- or trisubstituted chloro, fluoro, bromo, or iodo groups), heterocylic, carboxyheterocyclic, sulphoxyheterocyclic, oxyheterocyclic, C3-C30 cycloalkenyl, carboxycycloalkenyl, sulphoxycycloalkenyl or cycloalkenoxy, C8-C30 cycloalkynyl, carboxycycloalkynl, sulphoxyalkynyl or cycloalkynoxy, alkynyl, carboxyalkynyl, sulphoxyalkynyl or alkynoxy group. C4-C30 aromatic, carboxyaromatic, sulphoxyaromatic or aryloxy, C4-C30 heteroaromatic, carboxyheteroaromatic, sulphoxyheteroaromatic or heteroaryloxy group, wherein in any of the said hetero atom-containing groups the heteroatom is selected from the group consisting of 0, S, or N and in the case of the aforementioned groups being substituted there are present one or more substituents selected from the group consisting of halogen, cyano, Cl-C30 alkyl, C2- C30 alkenyl, C4-C30 aromatic, Cl-CH ether, CI-C30 ester, CI-C30 sulphonate ester, nitro, Cl-C30 ketone, CI-C30 thioether, and/or one or more pharmaceutical ly active groups; or in the formula of Y, C=R 7 may represent a carbonyl (i.e. C=O) group; or in the formula of Y represented as containing both R' and R 7 the group R7 may be absent and R6, or the C to which it is bonded, is bonded directly to R 5 to form a corresponding cyclic structure; and where in the case of R6 or R 7 being double bonded to C or N (as the case may be) the said R 6 and R 7 group is the frenell group corresponding thereto; in the presence of an enzyme effective to selectively acylate the compound of formula (2) at the 2-position to produce a compound of the following formula (4):

R20 0 R4--n R3 (4); 0 0COR5 (ii) treating the resulting compound of formula ( nitrating agent to form a compound according to the following formula (5):

4) with a 31 02NO R41 0 R3 (5); and 0 0COR5 (iii) treating the compound of formula (5) with a hydrolysing agent to remove the protecting -OCOR5 ester group from the 2-position to form the above compound according to formula (1).

2. A method according to claim 1 wherein the pharmaceutically active group is selected from the group consisting of 2-acetoxybenzoate, 2-N- (3'tri f luoromethylphenyl) aminobenz o ate, (S)6methoxy-amethyl - 2 - naphthal eneacetate and (S)-1-[N-[l(ethoxycarbonyl) -3-phenylpropyl 1 -L- alanyl 1 L-proline carboxylate.

3. A method according to claim 1 or claim 2 wherein R', R 2, R 3 and R 4 are hydrogen.

4. A method according to any preceding claim wherein the enzyme is one or more enzymes selected from the group consisting of pig liver esterase, hog liver esterase, horse liver esterase, Subtilisin carlsberg and Rhizopus oryzae.

5. A method according to any preceding claim wherein the 30 acylating agent is one or more vinyl esters.

6. A method according to claim 5 wherein the acylating agent is vinyl acetate, vinyl butyrate or a mixture of both.

A method according to any one of claims 1 to 4 wherein 32 the acylating agent comprises one or more compounds selected from 2,2,2trichloroethylbutyrate, 2,2,2trifluoro-ethylbutyrate, Sethylthiooctanoate, biacetyl monooxime acetate, isopropenyl acetate, 1ethoxyvinyl acetate, diketene, acetic anhydride and succinic acid anhydride.

8. A method according to any preceding claim wherein the amount of the enzyme used in the method is 1 to 500% by 10 weight of the compound of formula (2).

9. A method according to claim 8 wherein the amount of the enzyme used in the method is 1 to 10% by weight of the compound of formula (2).

10. A method according to any preceding claim wherein the amount of acylating agent used in the method is 1 to 10 molar equivalents with respect to the compound of formula (2).

11. A method according to claim 10 wherein the amount of acylating agent used in the method is 1 to 5 molar equivalents.

12. A method according to any preceding claim wherein the reactions are carried out at room temperature and pressure.

13. The use of an enzyme selected from, the group consisting of pig liver esterase, hog liver esterase, horse liver esterase, Subtilisin carlsberg and Rhizopus oryzae for effecting acylation selectively at the 2position of a compound according to formula (2) defined in any one of claims 1 to 3 in the presence of an acylating agent.

14. The use according to claim 13 wherein the acylating agent is a compound according to formula (3) defined in 33 claim 1.

15. The use according to claim 14 wherein the acylating agent is one or more vinyl esters.

16. The use according to claim 15 wherein the acylating agent is vinyl acetate, vinyl butyrate or a mixture of both.

17. The use according to claim 14 wherein the acylating agent comprises one or more compounds selected from 2,2,2trichloroethylbutyrate, 2,2,2trifluoro-ethylbutyrate, Sethyl thi ooctano ate, biacetyl monooxime acetate, isopropenyl acetate, 1 - ethoxyvinyl acetate, diketene, acetic anhydride and succinic acid anhydride.

18. A method of introducing a protecting group into a compound according to formula (2) defined in any one of claims 1 to 3 selectively at the 2position, comprising treating the compound according to formula (2) with a protecting group- introducing agent in the presence of an enzyme effective to introduce the protecting group selectively at the 2-position.

19. A method according to claim 18 wherein the enzyme is one or more enzymes selected from the group consisting of pig liver esterase, hog liver esterase, horse liver esterase, Subtilisin carlsberg and Rhizopus oryzae.

20. A method synthesising a compound according to formula (1) defined in claim 1 substantially as herein described.