WO2000058500A1

WO2000058500A1 - THE PREPARATION OF trans-4-AMINO-2-CYCLOPENTENE-1-CARBOXYLIC ACID DERIVATIVES

Info

Publication number: WO2000058500A1
Application number: PCT/GB2000/001141
Authority: WO
Inventors: Stephen John Clifford Taylor; Michael Lloyd
Original assignee: Chirotech Technology Limited
Priority date: 1999-03-26
Filing date: 2000-03-24
Publication date: 2000-10-05
Also published as: EP1165829A1; AU3445400A; GB9907082D0

Abstract

A process for the preparation of an enantiomerically enriched trans-1,4-disubstituted 2-cyclopentene of formula (4), substantially free of the corresponding cis isomer, comprises selective hydrolysis of a mixture of diastereomeric esters (5) and (8) or their opposite enantiomers, in the presence of an enzyme, wherein R1 is H or alkyl, R2 is alkoxy, alkyl, aryl or H, R3 is H or any non-interfering organic group, and R4 is alkyl.

Description

THE PREPARATION OF tra -4-AMINO-2-CYCLOPENTENE-l-CA-RBOXYLIC

ACID DERTVATINES Field of the Invention

This invention relates to the synthesis of single enantiomers of trans-4- aminocyclopent-2-ene-l-carboxylic acid derivatives by way of an enzymic separation of cis- and trαns-isomers. Background of the Invention γ-Aminobutyric acid (GABA) is an important naturally occurring biologically active molecule which increases the permeability of postsynaptic membranes to chloride ions. An increase in chloride conductance leads to membrane hyperpolarization which increases the threshold for the triggering of an action potential. The result of this is that GABA serves as an inhibitory transmitter in the central nervous system, and GABA receptors, of which there are several, are targets for new molecules which are agonists or antagonists of these. Hence there is much commercial interest in analogues of GABA which are pharmacologically active and modify the behaviour of the central nervous system. An example of such a drug is baclofen, a GAB Ag receptor agonist, which was introduced 25 years ago for the treatment of spasticity. In particular, with the discovery of different GABA receptors, more sophisticated and selective compounds are sought by pharmaceutical companies to act as selective cognitive enhancers, antidepressents, anxiolytic or epilepsy drugs.

Many GABA analogues have been described in the literature; see, for example, Dickenson et al., Νeuroscience Letters, 1988, 86, 351-5; Allan et al, Aust. J. Chem., 1979, 32, 2517-21; and Allan et al, Aust. J. Chem, 1986, 39, 855-64. The last of these papers describes compounds such as trα«s-4-aminocyclopent-2-ene- 1 -carboxylic acid (1), a constrained GABA analogue, and its synthesis in stereochemically pure form. This synthesis is outlined below: HO-_j

(1) (+H1Λ. Λ)

8.7% yield

This route was used for the unprotected (lR,4R)-isomer of 1, but could not be used to obtain the (lS,4S)-isomer due to poor recovery from the crystallisation and the lack of availability of the opposite isopropylidene ribonolactone enantiomer for use as a chiral auxiliary. Instead, the D-pantolactone diastereomeric ester was synthesised, from which the (lS,4S)-N-phthaloyl ester could be crystallised. However, access to the free amino acid was not possible since mild acid hydrolysis merely opened the lactone ring, and harsher conditions resulted in cis-trans isomerisation. This chemistry has several disadvantages besides the inability to synthesise one of a pair of the enantiomeric amino acids. It requires high temperature (200°C) molten epimerisation, involves a low yielding crystallisation step and uses toxic reagents in the deprotection.

Epimerisation of esters derivatives of c/s-4-aminocyclopent-2-ene-l-carboxylic acid can be effected under milder conditions than those reported by Allan et al. for the N- phthaloyl acid. For example, Kam and Oppenheimer (Journal of Organic Chemistry, 1981, 46, 3268-72) report partial epimerisation of the racemic methyl ester using ΝaOH/methanol, followed by separation of the resulting 1 : 1 mixture of cis- and trans- isomers by reverse-phase preparative HPLC. The latter technique is not easily applied on a large scale. The carbocyclic nucleoside abacavir, a potent reverse transcriptase inhibitor, is synthesised using enantiomerically pure (-)-2-azabicyclo[2.2.1 ]hept-5-en-3-one as a chiral building block. This drug has been produced at multi-tonne scale, and an economical bioresolution of 2-azabicyclo[2.2. l]hept-5-en-3-one, to provide the chiral building block as a single enantiomer, is disclosed in WO-A-98/10075. This uses a cloned lactamase at high volume efficiency:

500g/l >98% ee (+) >98% ee (-)

The residual (-)-lactam and the (+)-amino acid product can be converted into single enantiomer N-Boc ct-s-amino esters 2 and 3 using standard chemical methods.

ΝHBOC

2) (3)

There are isolated reports in the literature that biocatalysis can be used as a means to effect separation of diastereomeric mixtures. For examples, see Wang et al, J. Org. Chem., 1998, 63, 4850-3; Hiroya et al, Synthesis, 1995, 379-81; Mulzer et al, Liebigs Ann. Chem., 1992, 1131-5. Summary of the Invention This invention is based on the discovery that enantiomerically enriched derivatives of trαns-4-aminocyclopent-2-ene- 1 -carboxylic acid can be prepared in synthetically useful quantities from the corresponding cis isomers. This is achieved by a two-step process, giving a compound of formula (4) starting from a compound of formula (5)

(5)

The process of the invention can be conducted by base-induced epimerisation at

C-l, followed by enzymic separation of the resultant diastereomeric mixture. In formulae (4) and (5), R¹ is H or alkyl, R² is alkoxy, alkyl, aryl or H, R³ is H or any non-interfering organic group, and R⁴ is alkyl. The invention also includes the use of the opposite enantiomers of compounds (4) and (5). Description of the Invention

The exact nature of the groups R is not critical to the invention. Any suitable alkoxy, alkyl or non-interfering group can be used and can be readily determined by one skilled in the art. It may have up to 6, 10 or 20 C atoms. R² is preferably alkoxy. R³ is preferably H. In a particularly preferred embodiment of the present invention, R² is tβrt- butoxy, R³ is H, and R⁴ is methyl or ethyl.

For the epimerisation step, the readily available N-Boc c/^'s-amino ester 2 (or 3, see below) may be treated with methanol and catalytic sodium methoxide, as depicted for 2:

HBoc

(2) MeO_jC^_/ ^HBoc ca.0.1 part

1 part

Whilst this gave a 1 :1 mixture of cis and trans diastereoisomers, there was also formation of the 1,2-coηjugated ester, comprising 5-10% of the mixture, which was unavoidable even at 0°C. Kam and Oppenheimer (reference as above) report a similar observation. Efficient separation of this complex product mixture to isolate pure the trans isomer is not possible by crystallisation, and difficult on any appreciable scale by chromatographic methods. Therefore, it was necessary to identify a method for selective reaction of one component of the mixture to facilitate separation.

MeO_jC^ /s^ HBOC H0₂C,_// / _χ .NHBOC

\ / (3) Candida rugosa lipase \ / MeOzCv /^^NHBOC Me0₂C /\ _v.NHBOC

W

Su risingly, when mixtures of diastereoisomers were screened for selective hydrolysis using isolated enzymes that in one enantiomeric series, derived from starting material (3), the cis isomer was hydrolysed, whereas in the other series from (2) the trans isomer was hydrolysed. The most selective enzyme was a lipase derived from Candida rugosa, which was simultaneously able to select between the cis and trans isomers and the conjugated and non-conjugated esters, with very little hydrolysis of conjugated ester detected. The enzyme did not recognise the stereochemistry at the -NHBOC position, which is surprising, since it is a relatively bulky group, but instead provided a separation of each enantiomeric series of isomers on the basis of the absolute stereochemistry at the carboxyl ester alone. It preferred in each case to hydroiyse the (R)-carboxyl ester. This is illustrated schematically above. Other suitable selective enzymes can readily be identified by routine experimentation, based on the information provided herein.

Thus, this invention provides a convenient and scaleable route to protected forms of both enantiomers of tr «s-4-aminocyclopent-2-ene- 1 -carboxylic acid. The route is also efficient in that the remaining cis isomer can be recovered and epimerised further for another separation. In preferred embodiments of the invention, as depicted above, N-Boc amino acid (6) and N-Boc amino ester (7) are obtained. These compounds can be readily deprotected to yield the free amino acids. Alternatively, N-Boc protection is convenient for use of (6) and (7) as chiral scaffolds, to generate single isomer libraries for initial lead identification as part of a drug discovery programme. The following Examples illustrate the invention. Example 1 Synthesis of N-Boc-(-Vamino acid methyl ester (2)

A solution of (-)-2-azabicyclo[2.2.1]hept-5-en-3-one (200g, 1.83mol) in 800ml methanol was placed into a two litre jacketed vessel. The solution was cooled to below

5°C by means of a circulator, and thionyl chloride (147ml, 2.01mol) was added dropwise to the stirred solution. After the addition was complete, stirring was continued overnight at 5°C. The reaction mixture was then concentrated under reduced pressure to yield 324g (99%) of an off white solid.

A suspension of (-)-4-aminocyclopent-2-ene-l-carboxylic acid methyl ester hydrochloride (324g, 1.83mol) in 800ml of dichloromethane was stirred by means of an overhead stirrer in a 5 litre jacketed vessel. The vessel was cooled to 2°C by means of a circulator, and triethylamine (307ml, 2.2mol) was added dropwise to the reaction. Upon completion of the triethylamine addition, a solution of di-t-butyl dicarbonate (400g, 1.83mol) in 600ml of dichloromethane was added dropwise to the reaction over a period of 1.5 hours. The reaction was left stirring for a further 1 hour, after this time 500ml of water were added and the organic phase was extracted with 3 x 1 litre of IM potassium hydrogen sulfate and 1 litre of saturated brine solution. The organic phase was dried over magnesium sulfate and solvent was removed under reduced pressure to yield 43 Og (98%) of an off-white solid. Example 2 Epimerisation of N-Boc-C-Vamino acid methyl ester (2

A solution of 1.82g (7.55mmol) of N-Boc-(-)-amino acid methyl ester (2) in 30ml of methanol was cooled to 0°C using an ice bath. To the cooled mixture was added 2ml of 25% wt. solution of sodium methoxide in methanol, which was also cooled to 0°C. The resulting mixture was stirred for 120 minutes, maintaining the temperature at 0°C throughout. The reaction mixture was quenched using 4ml of glacial acetic acid and solvent was removed by rotary evaporator to yield 1.6g of a yellow oil which solidified on standing. GC-MS analysis indicated that the solid consisted of a 1 :1 mixture of cis and trans epimers in addition to a small amount (<10%) of the 1,2-conjugated ester. GC-MS gave: retention time 16.59 minutes (cis epimer); 17.16 minutes (trans epimer); 17.91 (conjugated ester) m/z (cis epimer): 185, 168, 141, 126, 82 m/z (trans epimer): 185, 168, 141, 126, 82

Epimerisation of N-Boc (+)-amino acid methyl ester (3) was carried out using the same method and resulted in an equivalent set of products. Example 3

Enzymic Hydrolysis Screen of cis and trans Epimers

A total of five enzymes was screened to evaluate their potential for selectively hydrolysing either the cis or trans epimer. The enzymes used were: pig pancreatic lipase (PPL), Candida rugosa lipase (CCL), lipase PS, Chirazyme L9 and Chirazyme L2. In each case, 50mg of substrate was placed into a scintillation vial with 5ml of 50mM potassium phosphate buffer, pH 7 and 20mg of enzyme. All five vials were continuously agitated at 26°C in a water bath/shaker. After 18 hours all reactions were extracted with 5ml ethyl acetate. The organic extracts were dried over magnesium sulfate and analysed by GC-MS. Analysis revealed that lipase CCL gave the greatest selectivity and scale up reactions were performed using this enzyme. Of the other enzymes screened, lipase PS exhibited some degree of selectivity, Chirazyme L2 hydrolysed both epimers, and Chirazyme L9 and lipase PPL both failed to hydroiyse either epimer. Example 4 Enzymic Resolution of a mixture of N-Boc (+Vaminoester (3 and its C-l epimer

0.6g (2.5mmol) of the epimeric mixture, 60ml of 50mM phosphate buffer, pH7 and 250mg lipase CCL Type Nil (Sigma) were placed into a 250ml conical flask. The reagents were left to shake in a water bath/shaker at 26°C for 24 hours, after which time the reaction was extracted with 3 x 50ml ethyl acetate. The combined organic extracts were washed with 50ml of saturated sodium bicarbonate solution and dried over magnesium sulfate. Removal of solvents by rotary evaporator yielded 260mg of an off-white solid. GC-MS analysis indicated that the recovered material was almost exclusively the trans- aminoester (7).

The carboxylic acid resulting from the hydrolysis of the cis epimer was recovered by acidification of the aqueous phase with 1.2M HCl and subsequent extraction with 3 x 50ml ethyl acetate. The combined extracts were dried over magnesium sulfate and removal of solvent yielded a colourless oil that solidified on standing. ΝMR analysis confirmed the identity of the carboxylic acid and indicated a 4:1 cis.trans ratio. Example 5 Enzymic Resolution of a mixture of N-Boc C-Vaminoester (2 and its C-l epimer lg (4.15mmol) of the epimeric mixture, 100ml of 50mM potassium phosphate buffer, pH7, and 400mg lipase CCL Type V were placed into a 250ml conical flask. The reagents were left to shake in a water bath/shaker at 26°C for 24 hours, after which time the reaction was extracted with 3 x 50ml ethyl acetate. The combined organic extracts were washed with 50ml of saturated sodium bicarbonate solution and dried over magnesium sulfate. Removal of solvents by rotary evaporator yielded 220mg of a yellow oil. GC-MS analysis indicated that the recovered material was almost exclusively the cis- aminoester (2).

The trø/w-aminoacid (6) resulting from the hydrolysis of the C-l epimer was recovered by acidification of the aqueous phase with 1.2M HCl and subsequent extraction with 3 x 50ml ethyl acetate. The combined extracts were dried over magnesium sulfate and removal of solvent yielded 270mg of a white solid. NMR analysis confirmed the identity of the carboxylic acid and indicated a 3: 1 trans: cis ratio.

Claims

1. A process for the preparation of an enantiomerically enriched trans- 1,4- disubstituted cyclopentene of formula (4), substantially free of the corresponding cis isomer, which comprises selective hydrolysis of a mixture of diastereomeric esters (5) and (8)

(4) (5) (8)

or their opposite enantiomers, in the presence of an enzyme, wherein R¹ is H or alkyl, R² is alkoxy, alkyl, aryl or H, R³ is H or any non-interfering organic group, and R⁴ is alkyl.

2. A process according to claim 1 , wherein R¹ = R⁴ and compound (5) or its opposite enantiomer is hydrolysed.

3. A process according to claim 1 , wherein R¹ is H and compound (8) or its opposite is hydrolysed.

4. A process according to any preceding claim, wherein R² is alkoxy.

5. A process according to any preceding claim, wherein R³ is H.

6. A process according to claim 5, wherein R² is tert-butoxy, and R⁴ is methyl or ethyl.

7. A process according to any preceding claim, wherein the enzyme displays specificity for the (lR)-carboxylic ester.

8. A process according to claim 7, wherein the enzyme is Candida rugosa lipase.

9. A process according to any preceding claim, wherein the mixture of compounds

(5) and (8) is prepared by partial epimerisation of (5) by reaction with a base.

10. A process according to any preceding claim, wherein compound (4) of at least 80%) enantiomeric excess is prepared.

11. A process according to claim 10, wherein compound (4) of at least 95% enantiomeric excess is prepared.