EP0466793A1

EP0466793A1 - Cyclohexadienediols and their use

Info

Publication number: EP0466793A1
Application number: EP19900906283
Authority: EP
Inventors: Christopher Thomas Stable Cottage Evans; Douglas William Ellerslie Sutton Lane Ribbons; Steven David Thomas; Stanley Michael Combe Cottage Roberts
Original assignee: ENZYMATIX Ltd
Current assignee: ENZYMATIX Ltd
Priority date: 1989-04-14
Filing date: 1990-04-17
Publication date: 1992-01-22
Also published as: WO1990012798A2; WO1990012798A3; AU5438090A

Abstract

Les cyclohexadiènes-cis-diols, dont de nombreux composés à carboxyle substitué sont nouveaux, et qui peuvent être préparés par la biotransformation, sont utilisés pour la synthèse (i) de lactames bicycliques (destinés aux nucléosides isocydiques anti-viraux), (ii) d'analogues de phénylglycine, et (iii) de complexes métalliques d'organotransition.Cyclohexadienes-cis-diols, of which many compounds with substituted carboxyl are new, and which can be prepared by biotransformation, are used for the synthesis (i) of bicyclic lactams (intended for the anti-viral isocydic nucleosides), (ii) phenylglycine analogs, and (iii) metal organotransition complexes.

Description

CYCLOHEXADIENEDIOLS AND THEIR USE

Field of the Invention

This invention relates to cyclohexadienediols which are of utility as chiral synthons.

Background of the Invention

Cyclohexadiene-cis-diols are known. They can be prepared by microbiological transformation of benzene and substituted analogues, including benzoic acids. The diols may be represented by formula I (when R₂=R₃=H), although the term cyclohexadiene-cis-diol compound will be used to describe all compounds of formula I.

Summary of the Invention

The cyclohexadiene-cis-diols of formula I may be converted to lactams of formula II which are themselves precursors of anti-viral compounds, to amino-acids of formula Ilia which are analogues of phenylglycine, to transition metal complexes, and also to compounds as shown in the accompanying Chart. All these conversions are facilitated by the ready availability of the starting material.

Many compounds of formula la (representing formulae IA, IB, IC and ID) are novel, as given in the compound per se claims.

Description of the Invention

The diols at the centre of the accompanying Chart can be produced by microbiological transformation from the corresponding (R) -substituted-benzoic acid A, B, C or D, or a more reduced precursor such as the

corresponding toluene, benzyl alcohol or benzaldehyde.

From benzoic acids A, there is 2,3-reglio

substitution; from benzoic acids B, 1,2-regio; and from phthalic acids C/D, 4,5-regio substitution. Microorganisms which can be used are given in the table of "strains" (below).

Other compounds of the invention include the

derivatives indicated in the reaction scheme, i.e. esters of the primary microbiological product a. The

modifications which are shown represent processes which are generally known. represents R₁ as alkyl; and

R₃ respectively represent the alkyl groups of each of R₂ and R₃ as alkylcarbonyl.

Ethers of formula I (R₂ and/or R₃ as alkyl) can be prepared by alkylation. R₂ and R₃, in this case, are preferably each CH-, or together are -C(CH₃)₂-.

Route i represents chemical acylation; routes i and ii represent biological acylation (using a lipase/ether, R₁COOH).

An example of biological hydrolysis or

stereospecific deacylation, suitable for route iii, is reaction of the substituted benzoate diester with water in the presence of a suitable lipase and co-solvent (or emulsifier). Suitable lipases include those isolated from strains of Candida, Pseudomonas, Rhizopus and

Aspergillus, together with hydrolases in other microbes found in tissue extracts such as those derived from pig and beef pancreas. Suitable co-solvents include ethers (e.g. diethyl ether), ketones (e.g. acetone), CFC's and hydrocarbons alkanes (such as toluene, hexane or

isooctane).

An example of chemical esterification suitable for route iv is reaction with a C_1-8 alkanol (

₁OH) in the presence of acid or base.

An example of biological esterification, suitable for route iv, is reaction with a suitable alkanol (e.g. C_1-8 ethanol) in the presence of a suitable lipase such as those listed above. Suitable co-solvents include those listed above. Water is preferably excluded from the bulk phase.

The esters can be subjected to trans-esterification, and interconverted. The esterified products can be acylated, and vice versa. An example of biological transesterification is reaction of the diester derivative with another

acid moiety CO₂-) in the presence of a suitable lipase

such as one of those listed above. Suitable co-solvents again include those listed above.

For esterification and transesterification, careful choice of lipase and conditions is necessary if the benzoic acid moiety is not to act both as donor and acceptor. An acylated compound can be esterified, and vice versa, by the given procedures.

Compounds of the invention are dienes. They will therefore undergo Diels-Alder-type cyclo-addition

reactions with an alkene or other dienophile, in known manner, to give stereospecific products which are also compounds of the invention.

Compounds of the invention can be transformed chemically or, in some cases, biologically to further compounds of interest, e.g. because of useful

functionality, by a varity of procedures including:

(i) oxidation, e.g. sterospecifically, to the corresponding mono or di-epoxide;

(ii) reduction, to the corresponding cyclohexene or cyclohexane;

(iii) ring-contraction, to a 5-membered ring, e.g. of the type found in prostaglandins;

(iv) ring-opening, initially to highly functional conjugated dialdehydes.

Compounds of the invention may lose the chirality due to ring-substitution, on further reaction. The molecule as a whole may remain chiral, if there if a chiral substituent.

The compounds of this invention can be used as chiral or prochiral intermediates in the synthesis of pharmaceuticals and agrochemicals and as the raw material for polyarylene-type polymers. Compounds of formula IA are particularly suitable for modification as substrates for the Strecker reaction, i.e. using NH₃ and HCN, or any of the various appropriate modifications described in the Merck Index, to provide analogues of phenylglycine. Modification from the acid/ester to aldehyde group can be conducted by

conventional means. The products are semi-synthetic penicillin-type compounds having additional chiral centres.

Compounds of formula la are also useful as

intermediates in the preparation of inositol phosphates which are key secondary messengers in mammalian

metabolism, mediating the effects of extracellular hormones on cellular pathways. Research based on

inositol phosphate is in areas as diverse as control of diabetes and clinical depression.

Inositol triphosphate/tetraphosphates have until recently been available only by extraction from natural sources. Thus, only the parent molecules are produced, while the generation of analogues with differing

substitution patterns and therefore different properties is taxing.

Recently, Ley et al generated inositol phosphate (1,4,5) P₃ and two derivatives of formula VII using benzene cis-glycol as a starting material. This

synthetic route makes new potential therapeutics

available for the first time. There is no other chemical or biological route to these molecules.

The availability of compounds of formula la extends the range of possible substituted inositol derivatives.

Various 9-substituted purines are known as antiviral and anti-neoplastic agents. One such compound, known as AZT, has been used for the treatment of AIDS. More recently, a compound known as Carbovir (see formula V) has been disclosed by Vince et al, Biochem. and Biophys. Res. Com. 156 No. 2 (1988) 1046-1053, as a potent and selective anti-HIV agent.

Carbovir may be synthesised from the known γ-lactam, 2-azabicyclo [2.2.1] hept-5-en-3-one. The synthesis, from the corresponding ring-opened amino-acid, is described in GB-A-2217320. The γ-lactam can be prepared by reacting cyclopentadiene with tosyl cyanide.

Active compounds analogous to Carbovir are described in EP-A-0325460, US-A-4268672 and US-A-4742064. Their synthesis is from the same γ-lactam.

A compound of formula I can be converted to a corresponding lactam of formula II which itself can be converted by known means to a novel amino-acid of formula III and thence to a novel carbocyclic nucleoside of formula IV (Z is a purine, e.g. adenine or guanine). In the lactams and amino-acids, and in the further synthetic sequence, and if necessary or desired, the OH groups may be protected during preparation and/or use, e.g. as a 2,2-propylenedioxy group. Any susceptible group R may also be protected.

The compounds of formula IV may be prepared by known means, e.g. as described above for Carbovir, from lactams of formula II. If desired, the OH groups may be retained or functionalised. Lactams of formula II may be prepared from cyclohexadienediols of formula I, e.g. by reaction with tosyl cyanide or chlorosulphonyl isocyanate.

The reaction of the lactam with a material having lactamase activity gives a compound of formula III. If desired, this compound can be reacted with an acylating agent such as acetic acid or acetic anhydride to give the corresponding N-acyl compound.

If the lactamase reaction is conducted in the presence of water, the illustrated aldehyde is prepared. Alternatively, a nucleophile may be used to introduce an alkyl or other group directly. For example, the

nucleophile is methanol. Compounds of formula I may also be used to prepare organotransition metal (M) complexes of formula V, wherein L is a ligand and p an integer, specific examples are given as formulae Vi, Vii and Viii. The attachment of a transition metal to the diene moiety adds an additional dimension to the synthetic potential of the dienediols. Transition metal complexes are undergoing rapid development as reagents for organic synthesis, and offer unique reactivities, frequently under exceptionally mild conditions and with high stereocontrol. A major problem in conventional approaches to these complexes is the limited access to homochiral material. The

microbially-derived dienediols resolve this problem for diene ligands and, at the same time, attachment of the metal can stabilise the inherently labile ligand. Thus, the combination of the two chemistries offers

considerable scope for synthetic development.

The utility of compounds of formula I depends largely on their preparation by means of

biotransformation. The general attributes which make their use so attractive are the access they provide to optically pure compounds, chemically labile products, novel transformations, regio-controlled reactions, chemospecific transformations, substrate specificity (high specificity for a part of a substrate molecule and low specificity for the remainder), and new mutants with slightly differing specificities and thereby increasing the spectrum of substrates.

Biotransformations can provide a ready source of many chiral starting materials, as above, but an

additional feature of the broad substrate specificity is the potential for application at a much later stage in a synthetic sequence such that generation of the sensitive dienediol functionality can be delayed to a more

appropriate point. The following Preparations and Examples illustrate the invention.

Nutrient Broth

The nutrient broth used, purchased from Oxoid Ltd., Basingstoke, Hants, England, was reconstituted in

distilled water to give the following composition;

Lab-Lemco powder (1.0 g.1^-1), yeast extract (2.0 g.1^-1), peptone (5.0 g.1^-1) and sodi ride (5.0 g.1^-1). The solution was sterilised by autoclaving at 121°C for 1 hour prior to use.

Trace Element Solution

The trace element solution used has the following composition; citric acid (100 g.1^-1), CaCl₂.2H₂O (4.38 g.1^-1), FeSO₄.7H₂O (8.0 g.1^-1), ZnSO₄.5H₂O (0.2 g.1^-1), CuSO₄.5H₂O (0.4 g.1^-1), CoCl₂.6H₂O (0.04 g.1^-1),

NaMO₄.2H₂O (0.04 g.1^-1), H₃BO₄ (0.004 g.1^-1). The solution was sterilised by autoclaving at 121°C for 30 minutes prior to use.

Minimal Salts Medium

The composition of minimal salt

Na₂HPO₄ 0.860 g.1^-1

KH₂PO₄ 0.531 g.1^-1

NH ₄C1 0.535 g.1^-1

K₂SO₄ 0.174 g.1^-1

MgSO₄.7H₂O 0.037 g.1^-1

CaCl₂.2H₂O 0.00735 g.1^-1

Trace Element

Solution 1 ml.1^-1

FeSO₄.7H₂O (0.1M) 0.2 ml

pH to 6.8

Preparation of Mutant Strain 1

A strain, HG5000, derived from a Pseudomonas testosteroni wild-type was isolated by enrichment culture in mineral salts medium (ASM) containing o-phthalic acid as sole source of carbon and energy. Mutagenesis of strain HG5000 with ethanemethanesulphonate, and recovery of surviving organisms on minimal salts medium containing 0.5% sodium succinate, gave strains which were further selected for an ability to grow in the presence of

4 , 5-dihydroxyphthalate but not on o-phthalic acid. One strain with this phenotype was characterised as deficient in an active dihydrodiol reductase. This strain,

designated Halo 1, was found to accumulate cis-1,2- dihydroxy-4,5-dicarboxycyclohexa-3,5-diene when grown in the presence of o-phthalic acid when glucose was present as a source of carbon and energy.

Example 1 cis-1,2-dihydroxy-4,5-dicarboxy-3-chlorocyclohexa-3,5-diene (IC)

Mutant Halo 1 was grown overnight at 30°C with shaking in 2x250 ml of nutrient broth. The 500 ml of culture was then used to inoculate 4.5 litres of a medium containing succinic acid (4.72 g.1^-1), MgSO₄.7H₂O (0.25 g.1^-1), KH₂PO₄ (3.0 g.1^-1), yeast extract (0.5 g.1^-1) trace element solution (10 ml 1^-1) and P2000 antifoam (1 ml 1^-1), adjusted to pH 6.8 with a 50% (v/v) aqueous ammonia solution (S.G. 880). This was stirred at 400 rpm, maintained at 30.5°C, and air was added at 2.5

1.min^-1. A pH of 6.8 was maintained by automatic

titration with 30% (v/v) aqueous phosphoric acid and 50% (v/v) aqueous ammonia (S.G. 880). All solutions with the exception of the aqueous ammonia were sterilised by autoclaving at 121 °C for 30 minutes prior to use. After 6 hours, the optical density of the broth measured at 600 nm in a 1 cm path length cell was 5-6.

To 5 litres of culture an aqueous solution of succinic acid (69 g.1^-1), disodium 3-chlorophthalate (26.25 g.1^-1), aqueous ammonia (S.G. 880, 6 ml.1^-1) and MgSO₄.7H₂O(1.0 g.1^-1) was fed at a flow rate equivalent to 17 mM.h^-1 for disodium chlorophthalate. A pH of 6.8 was maintained by automatic titration with 50% (v/v) aqueous ammonia (S.G. 880), and dissolved oxygen tension was maintained at above 50% saturation by automatic adjustment of fermentor impeller speed. The volume of broth was kept constant by removing broth at the same rate as the feed solution was added. Product formation was monitored by HPLC and reached 70 mM after 25 hours, at which point the feed was stopped and the culture was left for a further 2 hours to enable residual substrate to be converted to product.

The cells were harvested by centrifugation (10,000 g, 30 mins) and the supernatant was concentrated from 5 litres to 0.5 litres under vacuum by rotary evaporation at 45°C. The pH of the concentrate was then dropped to 2.0 by adding phosphoric acid, and it was repeatedly extracted with 5 volumes of ethyl acetate in the presence of anhydrous magnesium sulphate. The pH of the aqueous concentrate was readjusted to 2.0 between extractions, with further phosphoric acid. The ethyl acetate

fractions containing product were then evaporated to dryness to yield a crystalline powder which, following washing in cold ether, was found to be pure title

compound.

Preparation of Mutant Strain 2

A strain, HG1001, derived from a Pseudomonas putida wild-type was isolated by enrichment culture in a minimal salts medium containing limonene as sole source of carbon and energy. It grows in the presence of p-cymene and metabolises this substrate via the p-cymene pathway

(DeFrank and Ribbons 1977a,b, J. Bacteriol. 129 1356 and 1365). A mutant strain HG1006 was derived from HG1001 following sequential selection for growth in the presence of two p-cymene analogues, p-toluic acid and

p-tert-butylbenzoic acid. This strain, which was shown to be constitutive for the p-cymene pathway, was then exposed to the mutagen NTG. Mutants were isolated on a minimal salts medium containing 0.5% sodium succinate and were selected by an inability to grow in the presence of p-cymene, cumate, p-toluate and p-tert-butylbenzoate.

One of the strains selected in this manner,

designated Halo 2, was characterised as deficient in an active dihydrodiol dehydrogenase. This enzyme normally catalyses the oxidation, with NAD, of the p-cymene pathway intermediate 2R,3S-cis-dihydroxy-4-isopropyl- cyclohexa-4,6-diene-1-carboxylic acid.

Example 2 4-bromo-cis-2,3-dihydroxycyclohexa-4,6-diene- 1-carboxylic acid (IA)

Mutant Halo 2 was grown overnight at 30°C with shaking in 2x250 ml of nutrient broth. The 500 ml of culture was then used to inoculate 4.5 litres of a medium containing glucose (10.0 g.1^-1), NH₄SO₄ (1.0 g.1^-1), MgSO₄.7H₂O (0.25 g.1^-1), KH₂PO₄ (3.0 g.1^-1), trace element solution (10 ml 1^-1) and P2000 antifoam (1 ml 1^-1), adjusted to pH 6.8 with a 50% (v/v) aqueous ammonia solution (S.G. 880) in distilled water. This was stirred at 400 rpm, maintained at 30.5°C, and air was added at 2.5 1.min . A pH of 6.8 was maintained by automatic titration with 30% (v/v) aqueous phosphoric acid and 50% (v/v) aqueous ammonia (S.G. 880). All solutions with the exception of the aqueous ammonia were sterilised by autoclaving at 121°C for 30 minutes prior to use. After 6 hours, the optical density of the broth measured at 600 nm in a 1 cm path length cell was 5-6.

To 5 litres of culture an aqueous solution of sodium 4-bromobenzoate (29.3 g.1^-1), glucose (62.8 g.1^-1), citric acid (1.0 g.1^-1) and MgSO₄.7H₂O(1.0 g.1^-1) was fed at a flow rate equivalent to 4 mM.h^-1 for sodium

4-bromobenzoate and 10 mM.h for glucose. The feed solution without glucose was sterilised by autoclaving at 121°C for 30 minutes prior to use. The glucose. sterilised in a similar manner, was then added and the solutions were mixed to give the composition above. A pH of 6.8 was maintained by automatic titration with 50% (v/v) aqueous ammonia (S.G. 880), and dissolved oxygen tension was maintained at above 50% saturation by

automatic adjustment of fermentor impeller speed. The volume of broth was kept constant by removing broth at the same rate as the feed solution was added. Product formation was monitored by HPLC and reached 24 mM after 18 hours, at which point the feed was stopped and the culture was left for a further 2 hours to enable residual substrate to be converted to product.

The cells were harvested by centrifugation (10,000 g, 30 mins) and the supernatant was concentrated from 5 litres to 0.5 litres under vacuum by rotary evaporation at 45°C. The pH of the concentrate was then dropped to 4.0 by adding phosphoric acid, and it was repeatedly extracted with 5 volumes of cold ethyl acetate. The pH of the aqueous concentrate was readjusted to 4.0 between extractions, with further phosphoric acid. The ethyl acetate fractions containing product were then evaporated to dryness to yield a crystalline powder which, following washing in cold ether, was found to be pure title

compound.

Preparation of Mutant Strain 3

A strain of Pseudomonas putida U (ATCC 17514) was isolated by its ability to grow in the presence of benzoic acid as the sole source of carbon and energy. Another characteristic of the organism was an inability to grow in the presence of 2-fluorobenzoic acid even when benzoic acid was also present. Mutagenesis of this strain in the presence of NTG and recovery of surviving organisms on a minimal salts medium containing 0.5% sodium succinate gave strains which were further selected for their ability to grow in the presence of 2-fluorobenzoic acid as sole source of carbon and energy. One strain with this phenotype was shown to be incapable of growth on benzoic acid as sole source of carbon and energy and was characterised as deficient in an active dihydrodiol reductase, such that cis-1,2-dihydroxycyclohexa-3,5-dienecarboxylic acid accumulated when the strain was incubated with benzoic acid in the presence of glucose as a source of carbon and energy. This mutant strain was designated Halo 3.

Example 3 cis-1,2-dihydroxy-2-carboxy-4-trifluoromethylcyclohexa-3,5-diene (IB)

Mutant Halo 3 was grown overnight at 30°C with shaking in 2x250 ml of nutrient broth. The 500 ml of culture was then used to inoculate 4.5 litres of a medium containing glucose (10.0 g.1^-1), NH₄SO₄ (1.0 g.1^-1), MgSO₄.7H₂O (0.25 g.1^-1), KH₂PO₄ (3.0 g.1^-1), trace element solution (10 ml 1^-1) and P2000 antifoam (1 ml 1^-1) adjusted to pH 6.8 with a 50% (v/v) aqueous ammonia solution (S.G. 880). This was stirred at 400 rpm, maintained at 30.5°C, and air was added at 2.5 1.min^-1. Sodium benzoate (5 mM) was then injected into the

fermenter to induce the culture to produce the aromatic dioxygenase enzyme. A pH of 6.8 was maintained by automatic titration with 30% (v/v) aqueous phosphoric acid and 50% (v/v) aqueous ammonia (S.G. 880). All solutions with the exception of the aqueous ammonia were sterilised by autoclaving at 121°C for 30 minutes prior to use. After 6 hours, the optical density of the broth measured at 600 nm in a 1 cm path length cell was 5-6.

To 5 litres of culture an aqueous solution of sodium 4-trifluoromethylbenzoate (25 g.1^-1), glucose (81.3 g.1^-1), citric acid (1.0 g.1^-1) and MgSO₄.7H₂O (1.0 g.1^-1) was fed at a flow rate equivalent to 5 mM.h^-1 for sodium 4-trifluoromethylbenzoate and 13 mM.h for glucose. The feed solution without glucose was sterilised by autoclaving at 121°C for 30 minutes prior to use. The glucose, sterilised in a similar manner, was then added and the solutions were mixed to give the composition above. A pH of 6.8 was maintained by automatic titration with 50% (v/v) aqueous ammonia (S.G. 880), and dissolved oxygen tension was maintained at above 50% saturation by automatic adjustment of fermentor impeller speed. The volume of broth was kept constant by removing broth at the same rate as the feed solution was added. Product formation was monitored by HPLC and reached 28 mM after 24 hours, at which point the feed was stopped and the culture was left for a further 2 hours to enable residual substrate to be converted to product.

The cells were harvested by centrifugation (10,000 g, 30 mins) and the supernatant was concentrated from 5 litres to 0.5 litres under vacuum by rotary evaporation at 45°C. The pH of the concentrate was then dropped to 2.2 by adding phosphoric acid, and it was repeatedly extracted with 5 volumes of cold ethyl acetate. The pH of the aqueous concentrate was readjusted to 2.2 between extractions, with further phosphoric acid. The ethyl acetate fractions containing product were then evaporated to dryness to yield a crystalline powder which, following washing in cold ether, was found to be pure title

compound.

Example 4

The iron carbonyl complex of formula Vi is prepared by reacting the corresponding uncomplexed compound with Fe(CO)₉. The complex is reacted with (Ph) ₃C-BF₄ and NH₄PF₆, and then with NaBH₄, to remove one methoxy group; the product is reacted with CF₃COOH/NH₄PF₆, to remove the other methoxy group (20% yield). The product is then reacted with NaCH (COOMe)₂ to give the compound of formula Vli stereospecifically, in 72% yield. This Example illustrates the utility of the

catalysts in preparing intermediates for chiral alkaloid synthesis.

Example 5

To a solution of the 2,3-dihydrotoluenediolacetonide (500 mg, 3.0 mmol) in dichloromethane (2 ml) was added p-toluenesulphonyl cyanide (580 mg, 3.2 mmol), and the mixture was stirred at ambient temperature.

After 72 h, glacial acetic acid (0.5 ml) was added, followed after 1 min by ice-water (2 ml), and the mixture was shaken. The aqueous phase was neutralised with saturated aqueous sodium bicarbonate (2 x 1.5 ml) and extracted continuously with dichloromethane. The adduct (II: n=1, R=Me, R₂+R₃ = -C(CH₃)₂-) was obtained upon concentration of the combined organic phases.

Claims

1. Use of a cyclohexadiene-cis-diol compound of the formula

wherein m is 0, 1, 2, 3 or 4;

each R is independently selected from C_1-24 alkyl, halogen, alkylthio, alkylsilyl, alkylselenyl, formyl, C_1-6 haloalkyl (especially monohalo-, dihalo- or

trihalomethyl), CN, C_2-6 alkenyl, C_2-6 alkynyl, C_1-6 hydroxyalkyl, (C_1-6 alkyl) carbonyl, C_1-6 alkoxy, COOH, (C_1-8 alkoxy) carbonyl, trihalomethoxy and C_1-10 aryl, provided also that (when n is 2, 3 or 4) two adjacent groups R may be joined to form part of a fused saturated or unsaturated carbocyclic or heterocyclic 5 or

6-membered ring which may itself be substituted by any group as defined above for R; and

either R₂ and R₃ are independently selected from H, C_1-8 alkyl and (C_1-8 alkyl) carbonylor R ₂ and R₃, together form a C_3-8 acetonide;

for the preparation of a corresponding bicyclic lactam of the formula

e.g. by reaction with tosyl cyanide or chlorosulphonyl isocyanate or a reagent having the same function.

2. Use of a cyclohexadiene-cis-diol compound of the formula

representing compounds of the formulae

wherein R₂ and R₃ are as defined in claim 1; n is 1, 2, 3 or (for IB) 4; R₁ is H or C_1-8 alkyl; and R' and R" are independently as defined for R in claim 1, but are not COOR₁;

for the preparation of a corresponding amino-acid of the formula

e.g. by conversion of the COOR₁ group to the

corresponding aldehyde and subsequent Strecker reaction.

3. Use of a cyclohexadiene-cis-diol compound of formula I as defined in claim 1, for the preparation of a

corresponding transition metal complex.

4. Use according to claim 1 or claim 3, wherein the cyclohexadiene-cis-diol is of formula la as defined in claim 2.

5. Use according to any preceding claim, wherein R₁, R₂ and R₃ are each H.

6. A cyclohexadiene-cis-diol of formula IA as defined in claim 2, provided that, when R₁, R₂ and R₃ are H and n=1, R' is not alkyl, CF₃, halogen, phenyl or

hydroxyphenyl at the 4-position.

7. A compound as claimed in claim 6, wherein n is 1 and R¹ is F, I, CH₂Cl or CH₂Br.

8. A compound as claimed in claim 7, wherein R' is at the 4-position.

9. A compound as claimed in claim 6, wherein n is 2 and the R's are at the 4 and 5-positions.

10. A compound as claimed in claim 9, wherein the R's are CH₃,CH₃, Cl,Cl, 4-CH₃,5-Br, 4-CH₃,5-F or 4-CH₃,5-Cl.

11. A cyclohexadiene-cis-diol compound of formula IB as defined in claim 2, provided that, when R₁, R₂ and R₃ are H, (i) n is not 1 if R' is F, 3-CH₃, 4-CH₃, 3-C₂H or 5-Cl, and (ii) n is not 2 if the two R' groups are each F or are 3,4-di-CH₃.

12. A compound as claimed in claim 11, wherein n is 1 and R' is Cl, Br or CF₃.

13. A cyclohexadiene-cis-diol compound of formula IC or ID as defined in claim 2, provided that, when R₁, R₂ and R, are H, R' and R" are not both H, and R' is not F when R" is H and the two OH groups are above the plane of the ring.

14. A compound as claimed in claim 13, wherein one of R' and R" is H and the other is H, F, Cl, Br, I, CH₃ or CF₃.

15. A compound as claimed in any of claims 6 to 10, wherein R₁, R₂ and R₃ are each H.

16. Use according to any of claims 1 to 5, wherein the cyclohexadiene-cis-diol compound is as defined in any of claims 5 to 15.

17. Use according to any of claims 1 to 4 and 16, wherein the cyclohexadiene-cis-diol has been prepared by transforming the corresponding (R')_n-substituted-benzoic or phthalic acid in the presence of a micro-organism characterised by the ability to effect the

transformation.