CA2170059C

CA2170059C - Resolution of the racemates of primary and secondary amines by enzyme-catalyzed acylation

Info

Publication number: CA2170059C
Application number: CA002170059A
Authority: CA
Inventors: Friedhelm Balkenhohl; Bernhard Hauer; Wolfgang Ladner; Uwe Pressler; Christoph Nubling
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 1993-09-25
Filing date: 1994-09-16
Publication date: 2009-01-27
Anticipated expiration: 2014-09-16
Also published as: WO1995008636A1; US5728876A; EP0720655B1; JP3594602B2; CA2170059A1; DE4332738A1; DK0720655T3; EP0720655A1; JPH09503658A; PT720655E; ATE187774T1; ES2141254T3; DE59409010D1

Abstract

A process for producing optically active primary and secondary amines from the corresponding racemates is characterised in that (a) a racemic amine is enantioselectively acylated in the presence of a hydrolase with an ester whose acid component bears a fluorine, nitrogen, oxygen or sulphur atom at the proximity of the carbonyl carbon atom;
(b) the mixture of optically active amine and optically active acylated amine is separated so that an enantiomer of amine is produced;
(c) if desired the other enantiomer of the amine is extracted from the acylated amine by amide cleavage.

Description

~ 0050/44319 .2174059 Resolution of the racemates of primary and secondary amines by enzyme-catalyzed acylation The present invention relates to a novel process for resolving the racemates of primary and secondary amines by reacting with an ester in the presence of a hydrolase and subsequently separating one amine which has been enantioselectively acylated from the other, unreacted, enantiomer of the amine.
Resolution of the racemates of amines by enzyme-catalyzed reac-tion with esters is known. Kitaguchi et al. (J. Amer. Chem. Soc.
111 (1989) 3094-3095) describe the resolution of racemates of amines with trifluoroethyl butyrate with catalysis by subtilisin.
The enantioselectivity of this reaction is, however, greatly de-pendent on the solvent. Even with the most suitable of the sol-vents described (3-methyl-3-pentanol) only moderate selectivity is achieved.

2 describes a process for the chiral enrichment of asymmetric primary amines, in which the amines are reacted with ethyl acetate or ethyl butyrate with catalysis by subtilisin. The enantiomeric excesses achieved thereby are, however, unsatisfac-tory; in addition, long reaction times of up to several weeks are required.

Gotor et al. (J. Chem. Soc. Chem. Commun. (1988) 957-958) des-cribe the enantioselective acylation of 2-amino-l-butanol with ethyl acetate with catalysis by pig pancreatic lipase (PPL). In this case, the ester used (ethyl acetate) is also employed as a solvent. No satisfactory results are obtained on use of other solvents or other enzymes.

Brieva et al. (J. Chem. Soc. Chem. Commun. (1990) 1386-1387) describe the enantioselective synthesis of amides from racemic primary amines by reaction with 2-chloropropionate with catalysis by subtilisin in hexane or Candida cylindracea lipase in 3-methyl-3-pentanol.

Quiros et al. (Tetrahedron: Asymmetry 4 (1993) 1105-1112) describe the lipase-catalyzed synthesis of optically active amides from racemic a-halo-substituted ethyl propionates and pri-mary amines. However, the enantioselectivity achieved with this reaction is unsatisfactory.

Asens~o et al. (Tetrahedron Letters 32 (1991) 4197-4198) describe the lipase-catalyzed enantioselective acylation of secondary amines. However, this reaction takes place enantioselectively only with one amine and even there only with moderate success.
other amines show absolutely no enantioselectively.

The processes hitherto disclosed for enzyme-catalyzed racemate resolution either have too little enantioselectivity or can be carried out only under very specific conditions (solvent, en-zyme). In addition, they require long reaction times and enormous amounts of enzyme for the catalysis so that a process based thereon is uneconomic.

It is an object of the present invention to provide a process for the enzyme-catalyzed resolution of racemates of primary and sec-ondary amines which ensures high enantioselectivity, can be employed in a wide range of reaction conditions and moreover makes do with minimal amounts of catalyst.

More specifically, the invention as broadly disclosed is a process for resolving the racemates of primary and secondary amines by reacting with an ester with specific catalysis by a hydrolase and subsequently separating one amine which has been enantioselectively acylated from the other, unreacted, enantiomer of the amine, which process functions particularly well when the acid component of the ester has an electron-rich heteroatom selected from the group comprising fluorine, nitrogen, oxygen and sulphur atoms in the vicinity of the carbonyl carbon atom.

The invention as claimed is however restricted to a process for resolving racemates of primary and secondary amines by reacting with an ester in the presence of a protease or lipase and subsequently separating one amine which has been enantioselectively acylated from the other, unreacted, enantiomer of the amine, wherein the acid component of the ester has an oxygen atom in the a, p or 7 position relative to the carbonyl carbon.

2a The invention as claimed is also directed to a process for preparing acylated primary and secondary amines by reacting the amines with an ester in the presence of a protease or lipase, wherein the acid component of the ester has an oxygen atom in the a, R or y position relative to the carbonyl carbon.

Esters suitable for the process according to the invention are those which have an electron-rich heteroatom in the vicinity of the carbonyl carbon in the acid component of the ester.

The heteroatom must have at least one free pair of electrons. It can be a fluorine, nitrogen, oxygen or sulphur atom. In the invention as claimed, the heteroatom is exclusively oxygen.

It should be located in the vicinity of the carbonyl carbon. This means that the heteroatom is bonded to a carbon atom in the position alpha, beta or gamma to the carbonyl carbon. Preferred acid components of the ester are those in which the heteroatom is bonded to the C-alpha atom. Oxygen is the preferred heteroatom.
The heteroatom may be linked to other groups, eg. alkyl groups.
If the heteroatom is oxygen, for example, an ether moiety is present.

The alcohol component of the ester is not so crucial for the pro-cess according to the invention. It is possible to use for this purpose branched and unbranched C1-Clo-alcohols, which may also be substituted.

Particularly suitable esters are those having the structure \
x 0 1 e R2-CH-(CH2)n-C
\

where R1 is C1-Clo-alkyl, R2 is C1-Clo-alkyl, H, R3 is H, C1-Cio-alkyl, or phenyl which is unsubstituted or sub-stituted by NH2, OH, C1_4-alkoxy or halogen, X is 0, S, NR4, R4 is H, C1-Clo-alkyl, or phenyl which is unsubstituted or sub-stituted by NH2, OH, C1_4-alkoxy or halogen, n is 0, 1 or 2.

Among these, the C1_4-alkyl esters of C1_4-alkoxyacetic acids, such as ethyl methoxyacetate, are preferred.

A large number of enzymes can be employed as hydrolases in the process according to the invention. Proteases and, in particular, lipases are preferably used. Especially suitable lipases are microbial lipases which can be isolated, for example, from yeasts or bacteria. Particularly suitable lipases are those from 0050/44319 217O 05g Pseudomonas, eg. Amano P or the lipase from Pseudomonas spec. DSM
8246.

Furthermore the lipases Chirazymes L1 to L8, which are commercially available (Boehringer Mannheim), can be advantageously used in the process according to the invention.
The enzyme can be used in native or immobilized form.

Suitable solvents are in general organic solvents. The reaction takes place especially well in ethers, for example in MTBE or THF, or in hydrocarbons such as hexane, cyclohexane, toluene or halohydrocarbons such as methylene chloride.

The primary and secondary amines used can also be amino alcohols.
The reaction of the ester with the racemic amine or amino alcohol with enzyme catalysis is normally carried out at room tempera-ture. The reaction times depend on the substrate and are from 1 to 48 hours. As a rule, longer reaction times are required for secondary amines/amino alcohols than for primary amines/amino alcohols. The lower reactivity of secondary amines may also be compensated by increasing the amount of catalyst compared with the primary amines.
From 1 to 3 mol of ester are added per mole of substrate to be reacted. From 1 to 3 mol of ester are added even when racemic substrates are used.

The amount of enzyine to be added depends on the nature of the hydrolase and the activity of the enzyme preparation. The optimal amount of enzyme for the reaction can easily be established by simple preliminary tests. As a rule, 1000 units of lipase are added per mmol of amine or amino alcohol.
The course of the reaction can easily be followed by conventional methods, for example gas chromatography. In the case of racemate resolution, it is sensible to terminate the reaction when 50% of the racemic amine or amino alcohol has reacted. This usually takes place by removing the catalyst from the reaction medium, for example by filtering off the enzyme.

The enantioselective reaction of the racemic substrate with the ester results in the correspondingly acylated product (amide) of one enantiomer, whereas the other enantiomer remains unchanged.
The resulting mixture of amine and amide can easily be separated by conventional methods. Examples of very suitable methods for separating the mixture of amine and amide are extraction or distillation.

The process according to the invention is suitable for acylating 5 all primary and secondary amines. It can also be used to resolve the racemates of virtually all primary and secondary amines. It takes place particularly well in the case of primary arylalkyl-amines, for example those of the following structures:

X X X

N \
X

X
where X is any conventional aromatic substituent, especially halogen, nitro, cyano, C1-C4-alkyl, C1-C4-alkoxy or C1-C4-alkyl-thio.

The process according to the invention is furthermore suitable for the enantioselective acylation of amino alcohols of the general formula R5 (CH2)n R6 where ~ 0050/44319 R5, R6 = independently of one another H, branched and unbranched C1-Clo-alkyl, C1-C4-alkoxycarbonyl, phenyl, phenyl-C1-C4-alkyl, it being possible for the phenyl groups to be substituted by halogen, nitro, cyano, C1-C4-alkyl, C1-C4-alkoxy and C1-C4-alkylthio. R5 and R6 may also be closed to a mono-, bi- or tricyclic system by a carbon chain which may be interrupted by oxygen, sulfur or nitrogen and in turn substituted R7 = H, C1-Clo-alkyl, C1-C4-alkoxycarbonyl R8 = H, C1-Clo-alkyl n = 0 or 1.

When the carbon atoms substituted by OR7 or NHR8 are stereogenic centers, the process according to the invention relates both to the syn and to the anti isomers.

Examples of amino alcohols of the above general structure are:
2-amino-l-butanol; ephedrine; pseudoephedrine; norephedrine;
norpseudoephedrine; tert-leucinol; phenylglycidol; 1,2-diphenyl-aminoethanol; cis- and trans-2-aminocyclopentanol; cis- and trans-l-amino-2-hydroxyindane; cis- and trans-2-aminocyclo-hexanol, statine, 2-hydroxy-3-amino-phenylpropionic acid.
Preferred amino alcohols are cis- and trans-l-amino-2-hydroxy-indane.

The invention is also suitable for preparing optically active primary and secondary amines from the corresponding racemates by a) enantioselectively acylating a racemic amine or a racemic amino alcohol with an ester whose acid component has a fluorine, nitrogen, oxygen or sulfur atom in the vicinity of the carbonyl carbon atom, in the presence of a hydrolase, b) separating the mixture of optically active amine and optical-ly active acylated amine and obtaining one enantiomer of the amine, c) if required obtaining the other enantiomer of the amine or amino alcohol from the acylated amine by amide cleavage.

The process according to the invention can be made even more eco-nomic if, after removal of the required enantiomer, the remaining unwanted enantiomer is racemized and reused in the process. This recycling makes it possible to obtain overall more than 50% of the required enantiomer from the racemic amine.

The processes according to the invention not only are suitable as processes for producing optically active primary and secondary amines and ainino alcohols but can also form part of complicated multistage chemical syntheses, for example in the preparation of pharmaceutical active ingredients or crop protection agents. The following examples illustrate the invention.
Example 1: General method for the lipase-catalyzed acylation of amines 10 minol of the primary or secondary amine are dissolved in MTBE
(methyl tert-butyl ether) to give an approximately 10% strength solution. 11 mmol of ethyl methoxyacetate are added to the solu-tion, and the reaction is started by adding 100 mg of lipase (about 1000 U/mg, Pseudomonas spec. DSM 8246). When the reaction is complete (12-48 h depending on the amine), the enzyme is fil-tered off and the solution is concentrated under reduced pres-sure. The methoxyacetamides are obtained in a yield of more than 90 percent.

Amines employed:

Example 2: Method for racemate resolution The primary or secondary amine is dissolved in MTBE (about 10% by weight). 1 mole of ethyl methoxyacetate is added per 1 mole of racemic amine and then Pseudomonas lipase (DSM 8246) is added and the suspension is stirred at room temperature. About 10,000 units of lipase (10 mg) are added per mmol of amine. After 50% reaction has been reached (checked by gas chromatography), which takes 1-48 h depending on the amine, the enzyme is filtered off. The mixture of amine and acylated amine (amide) is separated by dis-tillation or extraction.

Exampl,e 3: Racemate resolution 20 g of (1) were reacted with 1 equivalent of (2) in the presence of 2 g of Pseudomonas lipase in MTBE at room temperature as in Example 2. 50% reaction was reached after 22 h.

11 Lipase + / 0 MTBE UO.

(1) (2) 0 a HN \/
NH2 =
=
+
(S) (R) (3) (4) The yield of (3) was 45% (ee > 99%), the yield of (4) was 45% (ee > 90%).
Example 4: Racemate resolution Various racemate resolutions were carried out as in Example 2.
Various amines were employed with diverse reaction conditions.
The details are to be found in the table.
Table + / 0\OR
1 equivalent II
HN
NH2 =
+
(S) (R) No. Racemic R MTBE Lipase Time Reac- ee ee amine (ml) (g;U/mg) (h) tion (amine) (amide) (9) by GC ($) ($) ($) 1 20 Et 180 2; 1000 7 47 72 57 > 99 93 2 5 Et 45 0.5;1000 3 43.6 22 56.1 95 96 3 5 Me 45 0.5;1000 23 38.5 23 51.4 79 97 4 2.5 Et 20 0.5;1000 1 39.6 3 50.1 48 59 > 99 87 5 2.5 Et 20 0.5;1000 1 41 6 2.5 Et 25 0.125;1000 27 56.1 87 97 7 2.5 Et 25 0.5;1000 21 60.1 93 98 8 2.5 Et 10 0.25;1000 22 50.6 82 98 9 2.5 Et 25 0.25;1000 21 55.7 87 99 10*) 2.5 Et 25 0.25;1000 4.5 40 50 97 11**) 2.5 Et 25 0.25;1000 21 56.1 92 98 12 2.5 Et 25 1; 200 2 56.1 67 > 99 13 2.5 Me 25 0.5; 200 4 54.3 69 99 14 2.5 Bu 25 0.5; 200 4 54.2 68 > 99 15***) 2.5 Et 25 0.5; 200 3 54.8 57 > 99 16 ) Amine 1 Et 20 0.5;1000 15.5 56 48 52 17 S) Amine 2 Et 20 0.5;1000 3 53.6 70 75 18 5) Amine 3 Et 25 0.25;1000 2 53.9 83 > 99 19 S) Amine 4 Et 25 0.5;1000 1.5 54.6 97 > 99 *) as Experiment No. 9 but reaction at 500C

**) The reaction mixture was shaken not stirred ***} Ethyl butoxyacetate was used as ester in place of the meth-oxyacetate.

) 20.6 mmol of each Amine 1:

Amine 2:

Amine 3:

Amine 4:

Example 5: Resolution of racemic amino alcohols a) to a suspension of 150 mg (1 mmol) of trans-l-amino-2-hydroxyindane and 350 mg (3 mmol) of ethyl methoxyacetate in 10 ml of methyl-tert.-butyl ether were added 9.6 mg of Chira-zyme L2, and the mixture was stirred at room temperature. 49%
conversion was reached after 41 h. After adding 3 ml of etha-nol, the enzyme was filtered off, the filtrate was evaporated to dryness and the residue was taken up in 10 ml of 1N HC1.
Extraction (CH2C12), drying (MgSO4) of the organic phase and concentration resulted in the hydroxy amide in the form of white crystals.

Crude yield: 100 mg (42%) [a]D~= -53.9 (c = 1.65; CH2C12), ee > 95%.

b) 150 mg (1 mmol) of cis-l-amino-2-hydroxyindane were reacted with 350 mg (3 mmol) of ethyl methoxyacetate and 50 mg of Chirazyme L1 as in Example 5a). 50% conversion was reached after 48 h. The enantiomeric excess of the hydroxy amide obtained by workup similar to that above was 71%.

Claims

WHAT IS CLAIMED IS:

1. A process for preparing acylated primary and secondary amines by reacting the amines with an ester in the presence of a protease or lipase, wherein the acid component of the ester has an oxygen atom in the .alpha., .beta. or .gamma.
position relative to the carbonyl carbon.

2. A process for resolving racemates of primary and secondary amines by reacting with an ester in the presence of a protease or lipase and subsequently separating one amine which has been enantioselectively acylated from the other, unreacted, enantiomer of the amine, wherein the acid component of the ester has an oxygen atom in the .alpha., .beta. or .gamma. position relative to the carbonyl carbon.

3. A process for preparing optically active primary and second ary amines from the corresponding racemates, which comprises:
a) enantioselectively acylating a racemic amine with an ester whose acid component has an oxygen atom in the .alpha., .beta. or .gamma. position relative to the carbonyl carbon, in the presence of a protease or lipase, b) separating the mixture of optically active amine and optically active acylated amine and obtaining one enantiomer of the amine, c) if required obtaining the other enantiomer of the amine from the acylated amine by amide cleavage.

4. A process as claimed in claim 3, wherein step b) or c) is followed by another step in which the unwanted enantiomer is racemized and then returned to the process for resolving the racemate.

5. A process for preparing optically active compounds, which comprises a process as claimed in any one of claims 2 to 4.

6. A process as claimed in claim 2 or 3, wherein the primary or secondary amine used is an amino alcohol.

7. A process as claimed in claim 6, wherein the amino alcohol is cis- or trans-1-amino-2-hydroxyindane.

8. A process as defined in claim 6, wherein the amino alcohol has the formula:

where:

R5 and R6 independently of one another are H, branched and unbranched C1-C10-alkyl, C1-C4-alkoxycarbonyl, phenyl, phenyl-C1-C4-alkyl, it being possible for the phenyl groups to be substituted by halogen, nitro, cyano, C1-C4-alkyl, C1-C4-alkoxy and C1-C4-alkylthio, where R5 and R6 may also be closed to a mono-, bi- or tricyclic system by a carbon chain which may be interrupted by oxygen, sulfur or nitrogen, R7 is H, C1-C10-alkyl or C1-C4-alkoxycarbonyl, R8 is H or C1-C10-alkyl, and n is 0 or 1.

9. A process as claimed in any one of claims 1 to 8, wherein the ester is ethyl methoxyacetate.