WO1995003421A1

WO1995003421A1 - Process for the enzymatic preparation of optically active n-substituted-3-pyrrolidinol

Info

Publication number: WO1995003421A1
Application number: PCT/NL1994/000162
Authority: WO
Inventors: Adrianus Franciscus Peter Van Tilburg; Theodorus Johannes Godfried Maria Van Dooren; Catharina Hubertina Maria Schepers; Bernardus Hendrik Nicolaas Dassen; Quirinus Bernardus Broxterman
Original assignee: Dsm N.V.
Priority date: 1993-07-19
Filing date: 1994-07-13
Publication date: 1995-02-02
Also published as: AU7468894A; BE1007297A3

Abstract

The invention relates to a process for the preparation of optically active N-substituted-3-pyrrolidinol of general formula (1), wherein R represents a linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl group, optionally substituted with one or more halogen atoms, or wherein R = OR2, wherein R2 may have the same meaning as stated above for R, via enantioselective enzymatic hydrolysis of a corresponding N-substituted-3-acyloxypyrrolidine of general formula (2), wherein R1 represents an acid residue, for instance a linear, cyclic or branched alkyl, alkenyl, aryl-aralkyl or aralkyl group, optionally substituted with one or more halogen atoms and for the preparation of an optically active N-substituted-3-acyloxypyrrolidine compound, wherein a mixture of the corresponding N-substituted-3-pyrrolidinol is subjected to an enzymatic enantioselective esterification by means of an enzyme with ester hydrolase activity.

Description

PROCESS FOR THE ENZYMATIC PREPARATION OF OPTICALLY ACTIVE N-SUBSTITUTED-3- PYRROLIDINOL

The invention relates to a process for the preparation of an optically active N-substituted-3- pyrrolidinol, wherein a mixture of the enantiomers of a corresponding N-substituted-3-acyloxypyrrolidine compound is subjected to an enzymatic enantioselective hydrolysis by means of an enzyme with ester hydrolase activity. From JP-A-1141600 the enantioselective enzymatic hydrolysis of N-benzyl-3-acyloxypyrrolidine is known.

The process described in JP-A-1141600, however, has the drawback that the enzymatic hydrolysis is carried out at low substrate concentrations. Moreover, the resulting optically active N-benzyl-3-pyrrolidinol can only be converted into optically active 3-pyrrolidinol via hydrogenolysis, which in practice requires special provisions.

Optically active 3-pyrrolidinol, in particular (R)-3-pyrrolidinol, is a known intermediate product for several pharmaceutics. It is therefore of importance to obtain the optically active 3-pyrrolidinol with a high enantiomeric excess. In practice it is often advantageous, if not required, to make use of 3-pyrrolidinol with the N atom protected for the preparation of optically active 3- pyrrolidinol.

The enantiomeric excess, which is a measure of the enantiomeric purity and is abbreviated to 'e.e. ', is a commonly used variable. Briefly, the enantiomeric excess is equal to the difference between the quantities of enantiomers divided by the sum of the quantities of enantiomers, which quotient can be expressed as a percentage figure by multiplying it with 100. The object of the invention is to provide a process enabling optically active Ν-substituted-3- pyrrolidinol with a high e.e. to be prepared via enzymatic hydrolysis, also at a high substrate concentration, which N-substituted-3-pyrrolidinol can be easily converted into optically active 3-pyrrolidinol.

This is achieved according to the invention if the N-substituted-3-pyrrolidinol is a compound of the general formula (1):

,C = 0

where R represents H, a linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl group or where R = OR², where R² can have the same meaning as stated in the foregoing for R, and if as N-substituted-3- acyloxypyrrolidine compound an ester is used of the general formula (2):

C = 0

where R is as defined above and where R¹ represents an acid residue, for instance H, a linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl group.

In particular, the linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl groups in R or R¹ of formula(s) (1) and/or (2) contain, independently of each other, 1-10 C atoms; the linear or branched alkyl, aryl, alkaryl or aralkyl groups in R or R¹ may also be substituted, independently of each other, with one or more halogen atoms.

For, surprisingly, it has been found that if as N substituent an acyl group or an acyloxy group, preferably an acyl group, is used, a very selective enzymatic hydrolysis can be realized in a commercially attractive process and, in addition, a product is obtained which can be easily hydrolized to 3-pyrrolidinol alcohol.

The reaction conditions for the enantioselective hydrolysis are not very critical. The hydrolysis is mostly carried out in an aqueous medium, but mixtures of water with organic solvents can also be used. It has also appeared to be well possible, however, to effect the hydrolysis with the substrate not being dissolved in a solvent. Water miscible and water immiscible solvents such as DMF (dimethylformamide) , DMSO (dimethyl sulphoxide), ethanol, methanol, toluene, hexane, isooctane, MTBE (methyl t-butyl ether), dichloromethane, etc. may be present in the reaction mixture. The concentration of the substrate in the reaction mixture is preferably as high as possible. In practice it appears that substrate concentrations in the reaction mixture of more than 25 wt. , in particular more than 50 wt.%, are possible. It has also been found that the corresponding enantioselective (trans)esterification proceeds extremely well, too. The invention therefore also relates to a process for the preparation of an optically active N- substituted-3-acyloxypyrrolidine compound, wherein a mixture of the enantiomers of the corresponding N- substituted-3-pyrrolidinol is subjected to an enzymatic enantioselective (trans)esterification in the presence of a suitable enzymatic esterification agent by means of an enzyme with ester hydrolase activity, with application of an Ν-substituted-3-pyrrolidinol of formula (1), in which R is defined as in claim 1, and wherein the N-substituted-3- acyloxypyrrolidine compound is an ester according to formula (2), in which R and R¹ are defined as in claim 1. The enantioselective enzymatic (trans)esterification of N-benzyl-3-pyrrolidinol is described in JP-A-4131093. Besides the drawbacks of a low substrate concentration during the enzymatic reaction and not quite easy conversion to 3-pyrrolidinol, referred to above in relation to JP-A-1141600, the process of JP-A- 4131093 has the additional drawback that product with a lower e.e. is obtained.

Further, from Tetrahedron: Asymmetry Vol. 3, No. 8, pp. 1049-1054, 1992, is known an enzymatic hydrolysis or esterification of another substrate, viz. 3- (hydroxymethyl)piperidine, or a corresponding ester, by means of lipase. In this case, too, the enzymatic (transJesterification or hydrolysis is carried out at low substrate concentrations, which results in a commercially less attractive process. Moreover, from said article it appears that separation of the reaction products by means of extraction - which is considered to be a much easier form of processing than the proposed selective crystallization - is hardly possible.

The enzymatic (trans)esterification may be effected both in a pure substrate and in a suitable organic solvent with a very low water content. Suitable solvents are for instance ketones, alkanes, cyclic ethers, dialkyl ethers, chlorine-containing alkanes and alkyl aromatics, in particular methylethyl ketone, methylisobutyl ketone, 2-pentanone, isooctane, dioxane, di-n-butyl ether, toluene, hexane, cyclohexane, dichloromethane, preference being given to dichloromethane, 2-pentanone and methylethyl ketone. The concentration of the substrate in the reaction mixture preferably is as high as possible. In practice it appears that substrate concentrations in the reaction mixture of more than 25 wt.%, relative to the total quantity of solvent and substrate, in particular more than 50 wt.%, are possible. Preferably, a substrate concentration of more than 90% is applied. In addition, the reaction mixture also contains a suitable enzymatic esterification agent in which the substrate may in principle also be dissolved. Surprisingly, it has been found that high substrate concentrations can be used in the processes according to the invention.

The applicant has further found that the separation of the reaction products can be simply effected by means of extraction. For it has appeared that it is possible to first isolate the ester if a suitable extraction agent is selected. Optionally, the alcohol can be isolated next, by choosing another extraction agent. For surprisingly it has been found that even if both the ester and the alcohol are separately soluble in the extraction agent, the ester only was extracted and the alcohol remained behind in the rest of the reaction mixture when an appropriately selected extraction agent was applied to the water-containing reaction mixture. Suitable extraction agents with which to extract the ester appeared to be, among others, apolar solvents such as for instance alkanes with 5 or more C atoms and dialkyl ethers with 4 or more C atoms; examples of particularly suitable extraction agents for isolation of the ester are hexane and MTBE (methyl-tert-butyl ether). Suitable extraction agents for isolation of the alcohol are for instance chlorine-containing alkanes with 3 or fewer C atoms and alkyl aromatics with 7-10 C atoms; examples of particularly suitable extraction agents for isolation of the alcohol are dichloromethane and toluene.

The enzymes that can be used in the hydrolysis or (trans)esterification are enzymes with ester hydrolase activity. For the enzymatic (trans)esterification the same enzymes (microorganisms) can in principle be used as for the hydrolysis. Known groups are lipases and esterases. Examples of suitable enzymes are from the genera Candida, Mucor, Rhizopus, Pseudomonas, Penicillum, Chromobacterium, Asperσillus and Humicola.

Such enzymes can be obtained through generally known cultivation methods. Many of them are produced on a technical scale and are commercially available. The enzyme preparation as used in the present invention is not restricted by purity and the like, and may be either a coarse enzyme solution or a purified enzyme, but it may also consist of (permeabilized and/or immobilized) cells possessing the desired activity or of a homogenate of cells possessing such activity. The enzyme may also be used in immobilized form or in chemically modified form. If there is also any undesirable opposite enzyme activity present in the enzyme preparation used, it is recommendable to remove or suppress this undesirable activity in order to obtain maximum enantioselectivity. The invention is not restricted in any way by the form in which the enzyme is used for the present invention. In the framework of the invention it is of course also possible to use an enzyme originating from a mutant or a genetically modified microorganism.

Chromobacterium viscosu , Cutinase from Fusarium solani pisi and lipase originating from Candida antarctica have been found to be particularly suitable. The latter lipase may be produced for instance through recombinant

DΝA technology. The gen coding for the lipase concerned is then heterologously expressed in a host microorganism, for instance Asperσillus orvzae. The lipase of Candida antarctica is commercially available, for instance under the brandnames SP435 and SP525 of ΝOVO. Cutinase from Fusarium solani pisi is e.g. described by Martinez, de Geus, Lauwereys, Mathyssens and Cambillau in Nature, Vol. 356, 16 april 1992, p. 615-618.

It has been found that the lipase of Candida antarctica is virtually fully selective, which makes it possible to obtain both enantiomers of the ester as well as the alcohol with a high e.e. The cutinase from Fusarium solani pisi showed an extremely high activity.

The process according to the invention is not restricted to a specific type of reactor and can for instance be carried out in a continuous, a batch, a fed- batch or a membrane reactor.

In the process according to the invention a (virtually) racemic mixture of N-substituted-3- pyrrolidinol will mostly be started from if an enantioselective enzymatic (trans)esterification is carried out, or from N-substituted-3-acyloxypyrrolidine if an enantioselective enzymatic hydrolysis is carried out. By a 'virtually racemic mixture' is understood in the framework of the invention a mixture of enantiomers with an e.e. of less than 10%.

The starting compounds can be prepared in a known way via N-acylation of racemic 3-pyrrolidinol, followed by esterification of the product thus obtained. The enzymatic hydrolysis or (trans)esterification, respectively, will mostly be carried out, although this is no critical parameter, at a temperature of 0-50°C, preferably at a temperature of 25- 45°C. The pressure at which the reaction is carried out can vary within wide limits. In practice the reaction will mostly be carried out at atmospheric pressure. The pH at which the reaction is carried out is not critical either and will mostly be between 3 and 10, preferably between 6 and 9.

Suitable solvents that can be used in the enzymatic hydrolysis or (trans)esterification are for instance chloroform, dichloromethane, benzene, xylene, trichloromethane, isopropyl ether, 3-pentanone, methyl- tert-butyl ether, methylisobutyl ketone, cyclohexanone, isooctane, ethyl acetate, and others.

In the enantioselective enzymatic (trans)esterification all known esterification agents suitable for such processes may in principle be used. Examples of such esterification agents are acids in activated form such as for instance anhydrides and esters. It has been found that selection of the starting compounds makes it possible to design a commercially attractive route for the preparation of optically active 3-pyrrolidinol. The invention therefore also relates to the N-acyl-3-pyrrolidinol compounds and the N-acyl-3- acyloxy- pyrrolidine compounds applied in the process according to the invention, as well as to the (R)- and

(S)-enantiomers thereof. More in particular, the invention relates to the compounds (R,S)-, (R)- and (S)-3- pyrrolidinol of the general formula (1):

l c - 0 R^

where R represents a linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl group, on the understanding that R ≠ CH₃ or non-substituted phenyl; and to the compounds (R,S)-, (R)- and. (S)-3-acyloxypyrrolidine of the general formula (2):

C = 0

where R represents H, a linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl group and where R¹ represents an acid residue, for instance H, a linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl group.

In particular, the linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl groups in R or R¹ of formula(s) (1) and/or (2) contain, independently of each other, 1-10 C atoms; the linear, cyclic or branched alkyl, aryl, alkaryl or aralkyl groups in R or R¹ may also be substituted, independently of each other, with one or more halogen atoms.

Preference is given to compounds in which R and R¹ are identical, since these compounds are easily prepared using one acylation agent. Further, it can be said that, roughly, on the one hand an improved process operation is obtained if R and/or R¹ contain more C atoms, while on the other, operating with compounds whose R and R¹ contain fewer C atoms is commercially more attractive. Preferably, the compounds applied and/or obtained in the process according to the inventions are such that R and/or R¹ contain 2-5 carbon atoms. The best results are achieved if R and R¹ both represent ethyl or propyl.

The invention will now be further elucidated by means of the following examples, without however being restricted thereto. Example I

Enzymatic hydrolysis

4.95 g of racemic Ν-butyryl-3-pyrrolidinyl butyrate was added to 20 ml of Kpi (potassium phosphate) buffer of pH 8. After addition of 1000 mg of Candida antarctica lipase (SP 435; NOVO) the pH was kept constant at 8 by means of automatic titration with 1 N potassium hydroxide solution. The temperature was kept at 30°C. The reaction was stopped by filtration of the reaction mixture, the biocatalyst remaining behind as residue. The biocatalyst was washed with 2 * 10 ml water. After setting the pH to 3 the (S)-N-butyryl-3-pyrrolidinyl butyrate was isolated from the recovered water layers by means of continuous extraction with hexane. Next, by means of continuous extraction with toluene, the (R)-N-butyryl-3- pyrrolidinol was isolated from the remaining water layer. The isolated (R)-N-butyryl-3-pyrrolidinol in toluene was hydrolized to (R)-3-pyrrolidinol as hydrochloric acid salt at 100°C for 17 hours by means of 6 N hydrochloric acid, after which the water that was present was removed azeotropically with toluene. The (R)-3-pyrrolidinol.HCl then crystallized out and was obtained, after filtration, as hygroscopic crystals having an e.e. > 98%.

Example II

Enzymatic hydrolysis

23.3 g of racemic N-butyryl-3-pyrrolidinyl butyrate was added to 2.5 ml of Kpi buffer of pH 8. After addition of 1980 mg of Candida antarctica lipase (SP 435; NOVO) the pH was kept constant at 8 by means of automatic titration. The temperature was kept at 40°C. The reaction was stopped by filtration of the reaction mixture, the biocatalyst remaining behind as residue. The biocatalyst was washed with 2 * 10 ml water. The e.e. of the (R)-N- butyryl-3-pyrrolidinol formed was 91%. Example III Enzymatic hydrolysis

24.1 g of racemic N-butyryl-3-pyrrolidinyl butyrate was added to 0.5 ml of Kpi buffer of pH 8. After addition of 1900 mg of Candida antarctica lipase (SP 435; NOVO) the pH was kept constant at 8 by means of automatic titration. The reaction was stopped by filtration of the reaction mixture, the biocatalyst remaining behind as residue. The biocatalyst was washed with 2 * 10 ml water. The temperature was kept at 40°C. The e.e. of the (R)-N- butyryl-3-pyrrolidinol formed was 91%.

Example IV Enzymatic hydrolysis 0.2 g of racemic N-butyryl-3-pyrrolidinyl butyrate was added to 2 ml of Kpi buffer of pH 8. After addition of 30 mg of enzyme as indicated in the table, the pH was kept constant at 8 by means of automatic titration. The temperature was kept at 40°C. The reaction was stopped by lowering the pH to pH 3. Next, the e.e. of the (R)-N- butyryl-3-pyrrolidinol formed was determined by means of HPLC. The results, without optimization to conversion, are given in the table.

TABLE

Example V

Enzymatic (trans .esterification

20 g of racemic Ν-acetyl-3-pyrrolidinol was added to 500 ml of dichloromethane and 25 ml of butanoic anhydride. After addition of 2 g of Candida antarctica lipase (SP 435; NOVO) the reaction was stopped after 2 hours by filtration of the enzyme. The temperature was kept at 30°C. The e.e. of the (R)-N-acetyl-3-pyrrolidinyl butyrate formed was 95%. After removal of the dichloromethane using a film evaporator, the reaction product, (R)-N-acetyl-3-pyrrolidinyl butyrate, was heated with 6 N hydrochloric acid at 100°C for 17 hours. After the hydrolysis the (R)-3-pyrrolidinol was obtained as hydrochloric acid salt.

Example VI

Enzymatic (trans)esterification

0.5 ml of butyric anhydride and 1 microlitre of water were added to 0.5 g of racemic N-acetyl-3- pyrrolidinol. After addition of 50 mg of Candida antarctica lipase (SP 435; NOVO) the reaction was stopped _- 1 ,3, -

after 60 minutes by filtration of the enzyme. The temperature was kept at 30°C. The e.e. of the (R)-Ν- acetyl-3-pyrrolidinyl butyrate formed was 87%. After hydrolysis by means of 6 N hydrochloric acid the (R)-3- pyrrolidinol was obtained as hydrochloric acid salt.

Example VII Enzymatic hydrolysis

18.3 g of crude racemic N-butyryl-3-pyrrolidinyl butyrate was added to 18 ml Kpi buffer of pH 8. After addition of 57.2 ml 1 N potassium hydroxide solution the pH was 8. After addition of 396 mg Candida antarctica lipase (SP 438; NOVO) the pH was kept constant at 8 by means of automatic titration. The temperature was kept at 35°C. The reaction was stopped by filtration of the reaction mixture, the biocatalyst remaining behind as residue. The biocatalyst was washed with 2 x 10 ml water. The e.e. of the (S)-N-butyryl-3-pyrrolidinyl butyrate was > 99.5%.

Example VIII

0.52 g of racemic N-benzoyl-3-pyrrolidinyl octanoate was added to 20 ml Kpi buffer of pH 8. After addition of 7 mg Fusarium solani pisi lipase the pH was kept constant at 8 by means of automatic titration. The temperature was kept at 35°C. The reaction was stopped by a pH drop to 3, followed by extraction. The e.e. of the (R)-N-benzoyl-3-pyrrolidonol formed was 93% (HPLC). The activity was 9600 nmol/mg/min.

Example IX

0,05 g racemic N-benzoyl-3-pyrrolidinyl octanoate was added to 20 ml Kpi buffer at pH 8. After addition of 1.3 mg Fusarium solani pisi lipase the pH was kept constant at 8 by means of automatic titration. The temperature was kept at 30°C. The reaction was stopped by pH drop to 3, followed by extraction. The e.e. of the (S)- N-benzoyl-3-pyrrolidinyl octanoate was 99% (HPLC). The activity was 1000 nmol/mg/min.

Claims

C L A I M S

Process for the preparation of optically active Ν- substituted-3-pyrrolidinol, wherein a mixture of the enantiomers of a corresponding Ν-substituted-3- acyloxypyrrolidine compound is subjected to an enzymatic enantioselective hydrolysis by means of an enzyme with ester hydrolase activity, characterized in that the N-substituted-3-pyrrolidinol is a compound of the general formula (1):

.C = 0

where R represents H, a linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl group, optionally substituted with one or more halogen atoms, or where R = OR², where R² can have the same meaning as stated in the foregoing for R, and that as N-substituted-3-acyloxypyrrolidine compound an ester is used of the general formula (2):

C = 0 — 10 —

where R is as defined above and where R¹ represents an acid residue.

2. Process according to claim 1, wherein R¹ represents H, a linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl group, optionally substituted with one or more halogen atoms.

3. Process according to claim 1, wherein the substrate concentration in the reaction mixture is higher than 25 wt.%.

4. Process for the preparation of an optically active Ν- substituted-3-acyloxypyrrolidine compound, wherein a mixture of the enantiomers of the corresponding Ν- substituted-3-pyrrolidinol is subjected to an enzymatic enantioselective (trans)esterification in the presence of a suitable enzymatic esterification agent by means of an enzyme with ester hydrolase activity, characterized in that an Ν-substituted-3- pyrrolidinol of formula (1) is applied, in which R is defined as in claim 1, and that the N-substituted-3- acyloxypyrrolidine compound is a compound according to formula (2), in which R and R¹ are defined as in claim 1 or 2.

5. Process according to claim 4, wherein the substrate concentration relative to the total amount of substrate and solvent in the reaction mixture is higher than 50 wt.%.

6. Process according to any one of claims 1-5, characterized in that a lipase or an esterase is used as enzyme.

7. Process according to claim 6, characterized in that an enzyme derived from Candida antarctica is used.

8. Process according to claim 6, characterized in that a cutinase from Fusarium solani pisi is used.

9. Process according to any one of claims 1-8, wherein R = R¹.

10. Process according to any one of claims 1-9, wherein R and R¹ contain 2-5 carbon atoms.

11. Process according to claim 10, wherein R and R¹ both represent ethyl or propyl.

12. Process according to any one of claims 1-11, wherein the reaction temperature is 25-45°C.

13. Process according to any one of claims 1-12, wherein the pH is 6-9.

14. Process according to any one of claims 1-13, wherein the ester present in the reaction mixture after the enzymatic reaction is recovered by means of extraction.

15. Process according to claim 14, wherein the extraction agent used is selected from the group comprising alkanes with 5 or more C atoms and dialkyl ethers with 4 or more C atoms.

16. Process according to claim 14 or 15, wherein the alcohol is isolated by means of extraction out of the reaction mixture remaining after extraction of the ester.

17. Process according to claim 16, wherein an extraction agent is selected from the group comprising chlorine- containing alkanes with 3 or fewer C atoms and alkyl aromatics with 7-10 C atoms.

18. Process for the preparation of optically active 3- pyrrolidinol, wherein an optically active Ν- substituted-3-pyrrolidinol or optically active Ν- substituted-3-acyloxypyrrolidine obtained by the process according to any one of claims 1-17 is converted via hydrolysis into optically active 3- pyrrolidinol.

19. Ν-substituted-3-pyrrolidinol of the general formula (1), wherein R represents a linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl group, optionally substituted with one or more halogen atoms, on the understanding that R ≠ phenyl or methyl.

20. Ν-substituted-3-acyloxypyrrolidine of the general formula (2), wherein R and R¹ each independently represent H, a linear, cyclic or branched alkyl, alkenyl, aryl, alkaryl or aralkyl group, optionally substituted with one or more halogen atoms.

21. Compounds according to claim 20, wherein R = R¹.

22. Compounds according to claim 21, wherein R and R¹ both represent ethyl or propyl.

23. Compounds according to any one of claims 19-21, wherein R and/or R¹ contain 2-5 carbon atoms.

24. The (R)-enantiomer of the compounds according to any one of claims 19-23 with an e.e. __ 95%.

25. The (S)-enantiomer of the compounds according to any one of claims 19-23 with an e.e. ≥ 95%.

25. Compounds according to claim 24 or 25 with an e.e. ≥ 98%.