WO2006012277A2

WO2006012277A2 - Biocatalytic process for preparing enantiomerically enriched pramipexole

Info

Publication number: WO2006012277A2
Application number: PCT/US2005/022417
Authority: WO
Inventors: Rao H. Valivety; Peter C. Michels; David P. Pantaleone; Yuri L. Khmelnitsky
Original assignee: Amr Technology, Inc.
Priority date: 2004-06-30
Filing date: 2005-06-23
Publication date: 2006-02-02
Also published as: WO2006012277A3

Abstract

biocatalytic process for preparing enantiomerically enriched pramipexole and pramipexole precursors are disclosed.

Description

BIOCATALYTIC PROCESS FOR PREPARING ENANTIOMERICALLY

ENRICHED PRAMIPEXOLE

[0001] This application claims the benefit of U.S. Provisional Patent

Application Serial No. 60/584,422, filed June 30, 2004, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] This application relates to biocatalytic processes for preparing enantiomerically enriched pramipexole and pramipexole precursors.

BACKGROUND OF THE INVENTION

[0003] Pramipexole is a dopamine D₃/D₂ agonist presently indicated for the treatment of the signs and symptoms of idiopathic Parkinson's disease. U.S. Patent Nos. 6,653,325 to Svensson, 6,503,920 to Gomez-Mancilla, and 6,492,371 to Roylance, for example. The synthesis of pramipexole is described in U.S. Patent No. 4,886,812 to Griss et al., in European Patent 186 097, and European Patent Application EP-A-85 116 016. Pramipexole has also been described for treating schizophrenia and depression (U.S. Patent Nos. 6,667,329 and 6,255,329 to Maj); as a neuroprotective agent (U.S. Patent No. 5,650,420 to Hall et al.); for the treatment of restless legs syndrome (U.S. Patent Nos. 6,194,445 and 6,001 ,861 to Oertel et al.); in the treatment of addictive disorders (U.S. Patent No. 6,410,579 to Marshall et al.); and in the treatment of CNS disorders, such as, progressive supranuclear palsy ("PSP") and multisystemic atrophy ("MSA") (U.S. Patent Nos. 6,156,749 to Rupniak et al. and 5,925,627 to Baker et al.). [0004] The chemical name of pramipexole is ±2-amino-4,5,6,7-tetrahydro-

6-propylamino-benzothiazole, and its structure is:

[0005] As can be seen, pramipexole has an asymmetric carbon, and thus exists as single enantiomers, and/or in a racemic form. It is commonly known that the pharmacological activity of such racemic compounds is typically connected only or mainly with one stereoisomer thereof. Pramipexole, for instance, is commercially available as an S isomer, of the dihydrochloride monohydrate salt

(S)

(Mirapex , Pfizer, Pharmacia & Upjohn Company, Kalamazoo, MI, USA), and the dopaminergic activity of this isomer is known to be twice as high as that of the R(+) isomer. The structure of commercially available pramipexole is

[0006] It is generally known that where a compound has a chiral center, the produced racemic compound may be resolved into its optical isomers by classical chromatography methods or fractional crystallization. In addition, it is expected that, on an industrial scale, the produced racemic compounds may be resolved into optical isomers by forming a salt with an appropriate optically active acid, resolution of the salts by fractional crystallization, and, if necessary, liberating the free base of the resolved product from the salt. Such a resolution process for producing optically pure pramipexole is disclosed in Schneider et al., J Med. Chem 30:494 (1987). The process uses the diamino derivative of pramipexole as a substrate and L-tartaric acid as a resolution agent. Following resolution, optically active pramipexole is prepared by two-step propylation of the single enantiomer of the diamino-precursor comprising reaction with propionic anhydride followed by reduction of the propionyl intermediate. [0007] Another process for producing (S)-pramipexole is disclosed in published International Application WO 02/22591. This process involves converting pramipexole into its monovalent salts; reacting the monovalent salts with a chiral salt such as a tartaric acid derivative thus forming diastereomers; and selectively separating the diastereomers by fractional crystallization techniques. [0008] Enantiomerically enriched (5)-pramipexole can also be prepared by using chiral agents to carry out enantioselective reductive amination. Published International Application WO 02/22590 discloses a process for producing enantiomerically enriched pramipexole using (S)-2-hydroxypropylamine. [0009] Thus, it is apparent that the prior art processes for preparing enantiomerically enriched pramipexole, as well as chiral precursors of pramipexole, suffer from severe drawbacks, as they are lengthy and economically undesirable.

[0010] The present invention is directed at overcoming these, and other deficiencies in the art.

SUMMARY OF THE INVENTION

[0011] One aspect of the present invention provides a process for preparing (S)-pramipexole which involves reacting a compound of formula I

with an acyl donor in the presence of an enzyme under conditions effective to produce a separable mixture of a compound of formula (i?)-II,

where R is alkyl, aryl, or aralkyl, and a compound of formula (S)-I.

The compound of formula (S)-I is reacted under conditions effective to produce a compound of formula (S)-III, which is (5)-pramipexole.

(5)-pramipexole

The (5)-pramipexole is recovered. [0012] Another aspect of the invention provides a process for preparing

(5)-pramipexole which involves reacting a compound of formula I with an acyl donor in the present of an enzyme under conditions effective to produce a separable mixture of a compound of formula (.S)-II,

where R is alkyl, aryl, or aralkyl, and a compound of formula (R)-I.

The compound of formula (S)-Il is isolated and hydrolyzed under conditions effective to produce a compound of formula (S)-I, which is reacted under conditions effective to produce a compound of formula (S)-III, which compound is (iS)-pramipexole. The (S)-pramipexole is recovered.

[0013] A further aspect of the present invention relates to a process for preparing (S)-pramipexole which involves reacting a compound of formula III

(røc)-pramipexole with an acyl donor in the presence of an enzyme under conditions effective to produce a separable mixture of a compound of formula (S)-III, which compound is (S)-pramipexole, and a compound of formula (R)-W,

(R)-IV where R is alkyl, aryl, or aralkyl. The (S)-pramipexole is isolated from the mixture.

[0014] Another aspect of the present invention relates to a process for preparing (/?)-pramipexole which involves reacting a compound of formula III with an acyl donor in the presence of an enzyme under conditions effective to produce a separable mixture of a compound of formula (/?)-III, which is (R)- pramipexole,

(Λ)-pramipexole and a compound of formula (5)-IV,

where R is alkyl, aryl, or aralkyl. The (7?)-pramipexole is isolated from the mixture.

[0015] Yet another aspect of the present invention relates to a process for preparing (5)-pramipexole which involves reacting a compound of formula II,

where R is alkyl, aryl, or aralkyl, under conditions effective to produce a separable mixture of a compound of formula (R)-U and a compound of formula (S)-I. The compound of formula (S)-I is isolated and reacted under conditions effective to produce a compound of formula (S)-III, which compound is (S)-pramipexole. The (5)-pramipexole is recovered. [0016] Yet a further aspect of the present invention provides a process for producing (S)-pramipexole which involves reacting a compound of formula II with an enzyme under conditions effective to produce a separable mixture of a compound of formula (S)-II and a compound of formula (R)-I. The compound of formula (S)-II is isolated and hydrolyzed under conditions effective to produce a compound of formula (S)-I, which is then reacted under conditions effective to produce a compound of formula (S)-III, which compound is (S)-pramipexole. The (S)-pramipexole is recovered. [0017] Enzymes are highly selective catalysts. Their hallmark is the ability to catalyze reactions with exquisite stereo-, regio-, and chemoselectivity that is unparalleled in conventional synthetic chemistry. Moreover, enzymes are remarkably versatile. They can be tailored to function in organic solvents, operate at extreme pH's and temperatures, and catalyze reactions with compounds that are structurally unrelated to their natural, physiological substrates. [0018] Enzymes are reactive toward a wide range of natural and unnatural substrates, thus enabling the modification of virtually any organic lead compound. Moreover, unlike traditional chemical catalysts, enzymes are highly enantio- and regioselective. Enzymes are also capable of catalyzing many diverse reactions unrelated to their physiological function in nature. For example, peroxidases catalyze the oxidation of phenols by hydrogen peroxide. Peroxidases can also catalyze hydroxylation reactions that are not related to the native function of the enzyme. Other examples are proteases which catalyze the breakdown of polypeptides. In organic solution, some proteases can also acylate sugars, a function unrelated to the native function of these enzymes. [0019] The present invention exploits the unique catalytic properties of enzymes, and provides biocatalytic processes for preparing, recovering, and isolating enantiomerically enriched pramipexole, i.e., (S)-pramipexole and (7?)-pramipexole, and pramipexole precursors. The biocatalytic processes of the present invention are faster and more economically desirable than processes of the prior art. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to biocatalytic processes for preparing enantiomerically enriched pramipexole and pramipexole precursors. 10021] The invention provides a process for preparing (S)-pramipexole which involves reacting a compound of formula 1

where R is, for example, alkyl, aryl, or aralkyl, and a compound of formula (S)-I.

The compound of formula (S)-I is isolated and reacted under conditions effective to produce a compound of formula (S)-III, which is (S)-pramipexole.

The (S)-pramipexole is recovered.

[0022] The enzyme is preferably a hydrolase, such as, for example, a lipase, esterase, or a protease. The hydrolase may, for example, be immobilized on a solid support, such as, for example, Accurel^®, Celite^®, agarose, Amberlite^® IRC-50, Dowex^®-50, Dowex^®-1, biomass support material, chitin, carbon, carrageenan, chitosan, controlled pore glass, glass beads, DEAE-cellulose, ion exchange resin, pig bone, polyamide, polystyrene, polyurethane, Sephadex^® LH- 20, Sephadex^® LH-60, silica gel, sol gel, and zeolites. [0023] According to this aspect of the invention, the enzyme may, for example, be an (Λ)-selective enzyme such as, Chirazyme L- 10 (Alcaligenes sp.), Lipase QL (Alcaligenes sp.), Lipase SL (Burkholderia cepacia), Chirazyme L-I (Burkholdeήa sp.), Chirazyme L-2 (Candida antarctica, B), Lipase L- 10 (Candida lipolytica), Lipase CL (Candida lipolytica), Chirazyme L-3 (Candida rugosa), Lipase CV (Chromobacterium viscosum), Lipase MJ (Mucor javanicus), Lipase M- 10 (Mucor javanicus), Lipase MM (Mucor mieheϊ), Lipase MM, recombinant (Mucor mieheϊ), Chirazyme L-9 (Mucor mieheϊ), Lipase PN (Phycomyces nitens), Lipase-type 250 (porcine pancreas), Lipase Type II - PPL (porcine pancreas), Lipase AH (Pseudomonas cepacia), Lipase LP S

(Pseudomonas cepacia), Lipase PS-CII (Pseudomonas cepacia), Lipase PS-DI (Pseudomonas cepacia), cholesterol esterase (Pseudomonas βuorescens), Lipase Lip-300 (Pseudomonas sp.), Chirazyme L-6 (Pseudomonas sp.), Lipase TL (Pseudomonas stutzeri), Lipase "RA" (Rhizopus arrhizus), Lipase RN (Rhizopus niveus), Newlase F (Rhizopus niveus), A-10FG (Rhizopus oryzae), FAP-15 (Rhizopus oryzae), and Lipoorisi G3x (Rhizopus oryzae). [0024] Preferably, the enzyme is Chirazyme L-I (Burkholderia sp.),

Chirazyme L-2 (Candida antarctica, B), Lipase CV (Chromobacterium viscosum), Lipase MM (Mucor mieheϊ), Lipase MM, recombinant (Mucor mieheϊ), Chirazyme L-9 (Mucor mieheϊ), Lipase-type 250 (porcine pancreas),

Lipase AH (Pseudomonas cepacia), Lipase LP S (Pseudomonas cepacia), Lipase PS-CII (Pseudomonas cepacia), Lipase PS-DI (Pseudomonas cepacia), Lipase Lip-300 (Pseudomonas sp.), Chirazyme L-6 (Pseudomonas sp.), or Lipase TL (Pseudomonas stutzeri). [0025] The invention also provides a process for preparing (S)- pramipexole which involves reacting a compound of formula I with an acyl donor in the presence of an enzyme under conditions effective to produce a separable mixture of a compound of formula (-S)-II,

where R is, for example, alkyl, aryl, or aralkyl, and a compound of formula (R)-I.

The compound of formula (S)-U is isolated and hydrolyzed under conditions effective to produce a compound of formula (S)-I, which is then reacted under conditions effective to produce a compound of formula (S)-III, which compound is (S)-pramipexole. The (S)-pramipexole is recovered.

[0026] The enzyme is preferably a hydrolase, such as, for example, a lipase, esterase, or a protease. The hydrolase may, for example, be immobilized on a solid support as previously described herein. [0027] According to this aspect of the invention, the enzyme may, for example, be an (S)-selective enzyme such as, Amano lipase AP (Aspergillus niger), cholesterol esterase (bovine pancreas), Chirazyme L-5 (Candida antarctica, A), Chirazyme L-5, c.f. (Candida antarctica, A), Lipase GC-4 (Geotήchum candidum), Lipase PR (Penicillium roqueforti), Lipase R- 10 (Penicillium roqueforti), cholesterol esterase (porcine pancreas), Lipase PS-30 (Pseudomonas cepacia), Lipase D (Rhizopus delemar), or Lipase Type I (wheat germ). Preferably, the enzyme is Amano lipase AP (Aspergillus niger), Chirazyme L-5 (Candida antarctica, A), or Chirazyme L-5, c.f. (Candida antarctica, A). [0028] Scheme 1 depicts the stereoselective enzymatic acylation of the racemic mixture of the diamine derivative of pramipexole ("rac-DA") ((±)-2,6- diamino-4,5,6,7-tetrahydrobenzothiazole).

Scheme 1

reductive animation

QΓV^NH,

etc ) (S)-IU

from ^-Selective Acylation (S)-IlI if R= C₂H₅

[0029] As shown in Scheme 1, a compound of formula I, which is a racemic

5 mixture of compounds of formulas (S)-I and (R)-I, is reacted with an acyl donor in the presence of an enantioselective enzyme, under conditions effective to produce a separable mixture of compounds of formulas (R)-U and (S)-I ("(/^-selective acylation"), or of formulas (S)-II and (R)-I ("(.^-selective acylation"), depending on the enzyme. Following (5)-selective acylation, the compound of formula (S)-Il, where R is ethyl, is

10 reduced to produce (S)-pramipexole, whereas the compound of formula (S)-II, where R is other than ethyl, is hydrolyzed to produce a compound of formula (S)-I, which is then reacted under conditions effective to produce (S)-pramipexole. Following (7?)-selective acylation, the compound of formula (S)-I is reacted under conditions effective to produce (5)-pramipexole. As seen in Scheme 1, the compound of formula (S)-I, may for example,

15 undergo reductive amination with propionaldehyde, or reacted with propionic anhydride and reduced, for example, with BH₃ or LiAlH₄, to produce (S)-pramipexole. [0030] The present invention provides a process for preparing (S)-pramipexole which involves reacting a compound of formula 111, which is (røc)-pramipexole,

with an acyl donor in the presence of an enzyme under conditions effective to produce a separable mixture of a compound of formula (S)-III, which is (S)-pramipexole, and a compound of formula (R)-TV,

where R is, for example, alkyl, aryl, or aralkyl. The (S)-pramipexole is isolated from the mixture. [0031] The enzyme is preferably a hydrolase, such as, for example, a lipase, esterase, or a protease. The hydrolase may, for example, be immobilized on a solid support as previously described herein. The enzyme may, for example, be an (R)- selective enzyme as previously described herein.

[0032] In addition, the compound of formula (R)-YV may be hydrolyzed under conditions effective to produce a compound of formula (R)-IIl, which is (/?)-pramipexole.

The (i?)-pramipexole is recovered.

10033] The hydrolysis is preferably carried out in the presence of an aqueous acid, such as, for example, sulfuric, hydrochloric, or hydrobromic acid. The hydrolysis is preferably carried out in the presence of a hydrolase, such as, for example, a lipase, esterase, or protease. The hydrolase may, for example, be immobilized on a solid support as previously described herein.

[0034] The invention provides a process for preparing (/?)-pramipexole which involves reacting a compound of formula III, which is (røc)-pramipexole, with an acyl donor in the presence of an enzyme under conditions effective to produce a separable mixture of a compound of formula (Zi)-IlI, which is (Z?)-pramipexole, and a compound of formula (S)-IV,

where R is, for example, alkyl, aryl, or aralkyl. The (Z?)-pramipexole is isolated. [0035] The enzyme is preferably a hydrolase, such as, for example, a lipase, esterase, or a protease. The hydrolase may, for example, be immobilized on a solid support as previously described herein. According to this aspect of the invention, the enzyme may, for example, be an (S)-selective enzyme as previously described herein. [0036] In addition, the compound of formula (S)-IV may be hydrolyzed under conditions effective to produce a compound of formula (S)-III, which is (S)-pramipexole. The hydrolysis is preferably carried out in the presence of an aqueous acid, as previously described herein. The hydrolysis is preferably carried out in the presence of a hydrolase, such as, for example, a lipase, esterase, or protease. The hydrolase may, for example, be immobilized on a solid support as previously described herein.

[0037] Scheme 2 depicts the stereoselective enzymatic acylation of (racemic)- pramipexole ((±)-2-amino-6-propylamino-4,5,6,7-tetrahydrobenzothiazole).

Scheme 2

C/?;-pramipcxole (-S)-IV

(S^-Selective Acylation

from rø-Selective Acylation rø-pπⁱmipexole

[0038] As shown in Scheme 2, a compound of formula III, which is a racemic mixture of (S)- and (Λ)-pramipexole, is reacted with an acyl donor in the presence of an enantioselective enzyme, under conditions effective to produce a separable mixture of (5)-pramipexole and a compound of formula (R)-YV ("(i?)-selective acylation"), or (R)- pramipexole and a compound of formula (S)-YV ("(5)-selective acylation"), depending on the enzyme. Following either reaction, the pramipexole enantiomer is isolated. In addition, following (5)-selective acylation, and the isolation of the (/?)-pramipexole from the mixture, the compound of formula (S)-YV may be hydrolyzed under conditions effective to produce (5)-pramipexole. Lipase screening has been carried out for the stereoselective acylation of III in dry ethyl acetate at 45 ⁰C at 8-fold excess of additional acylating agent 2,2,2-trifluoroethyl butyrate. A 2.5 mM solution of III was prepared by dissolving 27.0 mg in 50 mL of dry ethyl acetate. To this 0.15 mL of the acylating agent was added and 0.6 mL of this solution was added to each vial of the lipase plate as detailed in Example 3. The selectivity of the enzymes have been determined by analyzing the reaction mixture as described in the analysis section. Lipase N (Rhizopus niveus on Accurel , Amano), FAP-15 {Rhizopus ory∑ae on Accurel , Amano), Lipase "RA" (Rhizopus arrhizus on Accurel , Fluka), Lipase MM (recombinant Mucor miehei (R) (J?) on Accυrel , Fluka), Chirazyme L-9 (Mucor miehei on Accurel , Roche), PGE (calf

® tongue and salivary gland on Accurel , Amano) preferentially acylates the (R)- enantiomer. Acylase I (Aspergillus sp. on Eupergit C, Fluka) acylates the (5)-enantiomer. [0039] The invention provides a process for preparing (<S)-pramipexole which involves reacting a compound of formula II

where R is, for example, alkyl, aryl, or aralkyl, with an enzyme under conditions effective to produce a separable mixture of a compound of formula (R)-W and a compound of formula (S)-I. The compound of formula (S)-I is isolated and reacted under conditions effective to produce a compound of formula (S)-III, which compound is (S)-pramipexole. The (S)-pramipexole is recovered.

[0040] According to this aspect of the invention, the enzyme may, for example, be an (S)-selective enzyme such as, for example, pig liver esterase, and cholesterol esterase from bovine pancreas. [0041] The compound of formula (R)-Il may be hydrolyzed under conditions effective to produce a compound of formula(7?)-I. Hydrolysis may, for example, be carried out in the presence of a hydrolase, such as a lipase, esterase, or protease. The hydrolase may, for example, be pig liver esterase, a liver acetone powder, or penicillin amidase. The hydrolase may be immobilized on a solid support, as previously described herein.

[0042] The invention provides a process for preparing (S)-pramipexole which involves reacting a compound of formula II with an enzyme under conditions effective to produce a separable mixture of a compound of formula (S)-U and a compound of formula (R)-I. The compound of formula (S)-II is isolated and hydrolyzed under conditions effective to produce a compound of formula (S)-I. The compound of formula (S)-I is reacted under conditions effective to produce a compound of formula (.S)-III, which is (S)- pramipexole. The (5)-pramipexole is recovered.

[0043] According to this aspect of the invention, R is preferably, aralkyl. [0044] The enzyme may, for example, be an (i?)-selective enzyme, such as, for example, penicillin amidase, also known as penicillin acylase.

[0045] According to this aspect of the invention, hydrolysis may, for example, be carried out in the presence of a hydrolase, such as a lipase, esterase, or protease. The hydrolase may, for example, be pig liver esterase, a liver acetone powder, or penicillin amidase. The hydrolase may be immobilized on a solid support, as previously described herein.

[0046] Scheme 3 depicts the stereoselective enzymatic amide hydrolysis of (rac)- amide derivatives of pramipexole.

Scheme 3

b C₂H₅ f C₅H_n (Λ)-selective hydrolysis

C C₃H₇ g CH₂CgH₅ d M-C₄H₉ h C₆H₅

[0047] As shown in Scheme 3, a compound of formula II, which is a racemic

15 mixture of compounds of formulas (S)-U and (Λ)-II, is reacted in the presence of an enantioselective enzyme, under conditions effective to produce a separable mixture of compounds of either formulas (K)-II and (S)-I ("(^-selective hydrolysis"), or (S)-II and (R)-I ("(/?)-selective hydrolysis"), depending on the enzyme. Scheme 3 is further described herein in Example 10, below. 0 [0048] Scheme 4 depicts the stereoselective enzymatic hydrolysis of the (rac)- phenylacetamide analogue of pramipexole ((±)-2-amino-6-phenylacetamido-4,5,6,7- tetrahydrobenzothiazole) (R is aralkyl (compound Ig) in Scheme 3). Scheme 4

₂

pramipexole

10

[0049] As shown in Scheme 4, compound 1 g (which is a racemic mixture of compounds 3g and 4g) is reacted in the presence of an (Λ)-selective enzyme, in this case, penicillin amidase, to produce a separable mixture of a compound of formula (R)-I and compound 4 g (which is a compound of formula (S)-H). Compound 4g then undergoes hydrolysis (chemical, or enzymatic, for example, as previously described in Scheme 1) to produce a compound of formula (S)-I, which is reacted under conditions effective to produce (5)-pramipexole. As was previously described in Scheme 1 , the compound of formula (S)-I, may for example, undergo reductive amination with propionaldehyde, or reacted with propionic anhydride and reduced, for example, with BH₃ or LiAlH₄, to produce (5)-pramipexole. The compound of formula (R)-I may undergo chemical racemization to produce a compound of formula 1 (racemic), which may be acylated (for example, as previously described in Scheme 1) to produce additional starting material, i.e., compound 1 g. [0050] Penicillin acylase (Sigma, Cat. No. P-3319 or immobilized PGA-450,

Roche) hydrolyzes preferentially the (/?)-isomer providing unhydrolzyed (4g) with a high ee. The enzyme is active over a wide pH range (5.5 to 10.0) with either buffered (sodium acetate, sodium phosphate, potassium phosphate, Tris, borate, bicarbonate, or bicarbonate-NaOH buffers) or unbuffered solution. The enzyme is active over a temperature range of 20-45 °C and is preferably carried out at room temperature over a wide substrate concentration range (5-175 raM). However a water-miscible co-solvent is needed to increase the enantioselectivity with the present analogue. The co-solvents used are methanol (10-50% v/v), ethanol (60% v/v), DMF (5-40% v/v), ethylene glycol (-60% v/v), ethylene glycol monomethyl ether (-50% v/v), preferably DMF (15-30% v/v). The phenylacetamide analogue 1 g, (300 mg) was dissolved in 3 mL of DMF followed by the addition of 7 mL water. To this, 400 mg of immobilized enzyme PGA- 450 (Roche) or 100 μL penicillin acylase (Sigma) was added and allowed to react for 5 to 6 hr allowing the (/?)-preferential hydrolysis. Scale up studies were carried out with substrate concentrations in the range of 1 to 5% (35 to 175 mM), preferably at 3% (100 mM). With immobilized enzyme hydrolysis, the enzyme was removed by filtration and the filtrate concentrated by rotary evaporation. The unreacted amide was then precipitated by the addition of cold water and (S)-analogue (4 g) was separated by centrifugation. With penicillin acylase solutions, the enzyme was removed by precipitation by the addition of a 4-fold excess of cold acetonitrile or methanol and kept at 4 °C overnight. After centrifugation, the supernatant was concentrated and the unreacted (^-analogue (4g) was extracted with acetone and dried. The hydrolysis of 4g was carried out in 10% (v/v) DMF-water mixture or by suspending the amide in water by acid or enzymatic (PLE or penicillin acylase) hydrolysis. The progress of the reaction and the ee% of the product were determined by analyzing the samples for the unreacted substrate on a Chiralpak-AD-RH column (Chiral Technologies Inc., Exton, PA)and the resultant diamine on a CrownPak CR (+) column as described under analytical procedures.

[0051] Acyl donor compounds of the present invention may, for example, be either non-activated acyl donors or activated acyl donors. Non-activated acyl donors are defined as reagents that contribute an acyl group to the reaction with water or ammonia as the sole leaving group; these include the classes of molecules containing free carbonyl groups (free acids), free amides, carbonates, and carbamates. Activated acyl donors are defined as reagents that contribute an acyl group to the reaction with a more reactive leaving group; these include, but are not limited to, simple esters of acids, trihaloethyl esters, thioethyl esters, oxime esters, vinylic and enol esters (e.g. vinyl acetate and diketene), vinyl carbonates, and anhydrides. [0052] Examples of non-activated acyl donors include, but are not limited to, carboxylic acids such as fumaric acid, maleic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, lauric acid, myristic acid, palmitic acid, stearic acid, glyoxylic acid diethylacetal, acrylic acid, crotonic acid, isocrotonic acid, butenoic acid, pentenoic acid, oleic acid, retinoic acid, linoleic acid, linolenic acid, arachidonic acid, dihomo-γ-linolenic acid, m-5,8,11,14,17-eicosapentaenoic acid, α^'5-4,7,10,13,16,19- docosahexaenoic acid, malonic acid, succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, undecanedioic acid, dodecanedioic acid, 1,12- dodecanedicarboxylic acid, benzoic acid, picolinic acid, nicotinic acid, isonicotinic acid, 2-piperazinecarboxylic acid, 2-pyrrolecarboxylic acid, 3 -pyrrol ecarboxylic acid, 2- furancarboxylic acid, 3-furancarboxylic acid, 2 thiophenecarboxylic acid, 3- thiophenecarboxylic acid, 2-imidazolecarboxylic acid, 4-imidazolecarboxylic acid, phenylacetic acid, 3,5-dibromo-4-hydroxybenzoic acid, 3-(2-furyl)acrylic acid, 3,4- (methylenedioxy)phenyl acetic acid, norbornaneacetic acid, 2-thiopheneacetic acid, 2,6- dimethoxynicotinic acid, 3-indolebutyric acid, 3,4-(methylenedioxy)cinnamic acid, A- formylcinnamic acid, TV-CBZ-isonipecotic acid, TV-CBZ-L-proline acid, TV-CBZ-tyrosine acid, TV-CBZ-alanine acid, Fmoc-sarcosine acid, N-CBZ-glycine acid, TV-CBZ- phenylalanine acid, bromoacetic acid, chloroacetic acid, chlorobenzoic acid, cinnamic acid, formic acid, iodoacetic acid, methacrylic acid, pivalic acid, sorbic acid, 2-ethyl hexanoic acid, salicylic acid, 3-amino-6-thiophen-2-yl-4-trifluoromethylthieno [2,3-b] pyridine carboxylic acid, 2-indolecarboxylic acid, 3-indolecarboxylic acid, 2- indolinecarboxylic acid, 3-indolinecarboxylic acid, 3-(5'-glutathionyl)propionic acid, polyethylene glycol acetic acid, methyl polyethylene glycol acetic acid, TV-protected leucine, TV-protected isoleucine, TV-protected tert-leucine, and TV-protected cycloleucine. [0053] Activated acyl donors such as, for example, vinyl esters, trihaloethyl esters, or vinyl carbonates can also be used for the present invention. Examples of vinyl esters include 3, 3 -diphenyl propionic acid vinyl ester; 3,5-dibromo-4-hydroxybenzoic acid vinyl ester; 3-(2-furyl)acrylic acid vinyl ester; 3,4-(methylenedioxy)phenylacetic acid vinyl ester; norbornaneacetic acid vinyl ester; 2-thiopheneacetic acid vinyl ester; 2,6- dimethoxynicotinic acid vinyl ester; 3-indolebutyric acid vinyl ester; 2-pyrrolecarboxylic acid vinyl ester; 3,4-(methylenedioxy)cinnamic acid vinyl ester; 4-formyl cinnamic acid vinyl ester; TV-CBZ-isonipecotic acid vinyl ester; TV-CBZ-L-proline vinyl ester; TV-CBZ- tyrosine vinyl ester; TV-CBZ-alanine vinyl ester; Fmoc-sarcosine vinyl ester (Fmoc-TV-Me- GIy-OH vinyl ester); TV-CBZ-Glycine vinyl ester; TV-CBZ-phenylalanine vinyl ester; 2- furoic acid vinyl ester; acrylic acid vinyl ester; adipic acid divinyl ester; benzoic acid vinyl ester; bromoacetic acid vinyl ester; butyric acid vinyl ester (vinyl butyrate); caproic acid vinyl ester; chloroacetic acid vinyl ester; chloroformic acid vinyl ester; chlorobenzoic acid vinyl ester; cinnamic acid vinyl ester; crotonic acid vinyl ester; formic acid vinyl ester; iodoacetic acid vinyl ester; lauric acid vinyl ester; methacrylic acid vinyl ester; myristic acid vinyl ester; w-capric acid vinyl ester; rc-caprylic acid vinyl ester; palmitic acid vinyl ester; phenylacetic acid vinyl ester; pivalic acid vinyl ester; propionic acid vinyl ester; sorbic acid vinyl ester; stearic acid vinyl ester; acetic acid vinyl ester; 2- ethylhexanoic acid vinyl ester; vinyl salicylate; trimethylvinyloxycarbonylmethyl ammonium bromide; 3-amino-4,6-dimethylthieno [2,3-b] pyridine-2-carboxylic acid vinyl ester (1 S-27086); 3-amino-6-thiophen-2-yl-4-trifluoromethylthieno [2,3-b] pyridine carboxylic acid vinyl ester (1 S-21501); divinyl succinate; divinyl glutarate; and divinyl subarate.

[0054] The trihaloethyl esters of the present invention can be trifluoroethyl esters or trichloroethyl esters, preferably trifluoroethyl esters. Examples of trifluoroethyl esters include 3,3-diphenyl propionic acid trifluoroethyl ester; 3,6-dioxaheptanoic acid trifluoroethyl ester; oxalic acid trifluoroethyl ester; malonic acid trifluoroethyl ester; (-)- 2-oxo-4-thiazolidine-2-carboxylic acid trifluoroethyl ester; 2-pyrazinecarboxylic acid trifluoroethyl ester; nicotinic acid trifluoroethyl ester; 1 ,4-cyclohexanedicarboxylic acid ditrifluoroethyl ester; terephthalic acid ditrifluoroethyl ester; 4-(dimethylamino)benzoic acid trifluoroethyl ester; 4-(bromomethyl)phenylacetic acid trifluoroethyl ester; benzimidazolepropionic acid trifluoroethyl ester; Fmoc-L-thiazolidine-4-carboxylic acid trifluoroethyl ester; glutaric acid ditrifluoroethyl ester; 2-formylphenoxy acetic acid trifluoroethyl ester; 4-carboxybenzaldehyde trifluoroethyl ester; 4-(dimethylamino)phenyl acetic acid trifluoroethyl ester; isonicotinic acid trifluoroethyl ester; and picolinic acid trifluoroethyl ester. [0055] Examples of vinyl carbonates include butyl vinyl carbonate; l-methyl-3- piperidinemethanol vinyl carbonate; 3,3'-diethoxypropanol vinyl carbonate; 4-tert- butylphenethyl vinyl carbonate; benzyl vinyl carbonate; 4-methyl-5-thiazoleethanol vinyl carbonate; glycidol vinyl carbonate; 1,3-propylene divinyl carbonate; 1,4- cyclohexanedimethanol di(vinyl carbonate); 1,6-hexanediol di(vinyl carbonate); 4- hydroxybenzyl alcohol di(vinyl carbonate); 2,3-O-benzylidenethrietol di(vinylcarbonate); 2,5-furandimethanol di(vinylcarbonate); 2,6-pyridinedimethanol di(vinylcarbonate); acetone oxime vinyl carbonate; l,4-but-2-enediol di(vinylcarbonate); 3- thiophenemethanol vinyl carbonate; 2-methylsulfonylethanol vinyl carbonate; 4-(2- hydroxyethyl)morpholine vinyl carbonate; and 3-methyl-2-norbornanemethanol vinyl carbonate.

[0056] Preferred acyl donors may include, for example, trifluoroethyl butyrate, di-

(2',2',2'-trifluoroethyl) 1,4-cyclohexanedicarboxylate, benzoic acid vinyl ester, butyric acid vinyl ester, caproic acid vinyl ester, lauric acid vinyl ester, butyl vinyl carbonate, 2,6-furandimethanol vinyl carbonate, 1 ,6-hexanediol vinyl carbonate, ethyl acetate, butyl acetate, methyl propionate, ethyl propionate, butyl propionate, hexyl propionate, octyl propionate, vinyl propionate, phenylacetic acid, methyl phenylacetate, and methyl phenoxyacetate. More preferred are, for example, trifluoroethyl butyrate, ethyl acetate, methyl propionate, hexyl propionate, and vinyl propionate.

[0057] Typically, the acyl donor may be in about a 1.5-fold to 20-fold molar excess over the compound of formula I or the compound of formula III. Preferably, the acyl donor may be in a 2-fold molar excess over the compound of formula I or the compound of formula III. [0058] The reaction of the compound of formula I or the compound of formula III and an acyl donor may be carried out in an organic solvent. Possible organic solvents include, but are not limited to, methyl tert-butyl ether ("MTBE"), tetrahydrofuran ("THF"), toluene, pyridine, 1 ,4-dioxane, ethyl acetate, rc-butyl acetate, methylene chloride, benzene, acetonitrile, chloroform, 7V,7V-dimethylformamide ("DMF"), isooctane, and mixtures of these solvents. Preferred organic solvents may include, for example, methyl tert-butyl ether, tetrahydrofuran, toluene, pyridine, 1,4-dioxane, ethyl acetate, w-butyl acetate, methylene chloride, benzene, and acetonitrile. More preferred solvents may include, for example, methyl tert-butyl ether, ethyl acetate, and acetonitrile. The organic solvent may, for example, have a water content of from 0 to 10 volume percent, such as, for example, from 0 to 1 volume percent.

[0059] The reaction of the compound of formula I or the compound of formula III and an acyl donor may, for example, be performed at a temperature of between about 4°C and 95°C such as, for example, between 25°C and 50⁰C. [0060] During the reaction of the compound of formula I or the compound of formula III and an acyl donor, the compound of formula I or the compound of formula III, may, for example, be in a concentration range of about 0.001 M to about 1.0 M. For example, from about 0.005 M to about 0.15 M, and particularly, in a concentration of about 0.05M. [0061] During the reaction of the compound of formula I or the compound of formula 111 and an acyl donor, the acyl donor may, for example, be in a concentration range of about 0.001 M to about 2.0 M. For example, from about 0.01 M to about 0.1 M, and particularly, in a concentration of about 0.1 M. The acyl donor may, for example, be in a concentration of about twice that of the compound of formula I or the compound of formula III.

[0062] During the reaction of the compound of formula I or the compound of formula III and an acyl donor, the enzyme may, for example, be in a concentration of about 1.0 to about 100 mg per mL and more particularly, about 10 mg to about 100 mg per mL, of the reaction mixture.

EXAMPLES

Example 1 - Analytical Methods

[0063] HPLC analyses for both chiral and achiral analyses were performed using a Shimadzu HPLC (Shimadzu Corporation, Kyoto, JP) equipped with LC-IOAT pump, SIL-I OA auto-injector, SPD-MlOA diode array detector, SCL-IOA system controller, DGU- 14A degasser, FCV-IOAL mixer, and Jones Chromatography column heater with controller (Model No. 7955) (Argonaut Technologies, Foster City, CA, USA). Detection was at 254 nm.

® [0064] Achiral analysis was carried out using a Phenomenex C8 (100 x 4.6, 3μ)

® (Phenomenex , Torrance, CA, USA) with an acetonitrile/H₂O (both containing 0.1%

(v/v) trifluoroacetic acid) gradient mobile phase.

[0065] Chiral analysis of the (R) and (S) isomers of the diamine and amides of shorter chain length i.e., acetamide, propionamide, butyramide, and methyl butyramide were analyzed using a chiral crown ether column (CrownPak CR(+), Daicel Chemical Industries Ltd., Tokyo, JP, 4.0 mm x 150 mm; 5 μ). The analysis was carried out isocratically at a flow rate of 1.0 mL/min with water as the mobile phase after adjusting the pH to 2.0 with perchloric acid. The performance and the resulting retention times were highly dependent on the pH. [0066] While in most cases the pH of the mobile phase was adjusted to 2.0, adjustment of the pH to 1.85 was necessary for the analysis of acetamide and the (R)- and (S)-diamine mixtures. The retention times with the mobile phase pH at 2.0 for (R)- diamine, (5)-diamine, propionamide, butyramide, and methyl butyramide were 3.4, 4.1, 5.3, 10.9, and 24.0 minutes, respectively, while the retention times at a pH of 1.85 for the (7?)-diamine, (S)-diamine, and acetamide analogues were 4.8, 6.0, and 3.8 min, respectively.

[0067] The phenyl acetamide analogue of pramipexole and pramipexole itself were analyzed by resolving the compounds on an amylose column, Chiralpak-AD-RH (Chiral Technologies Inc., Exton, PA, 250 mm x 4.6 mm; 5μm). The chiral resolution of the phenylacetamide analogue was carried out isocratically with a 20:80 mixture of borate buffer (20 mM; pH = 9.0):methanol, with a column temperature of 45 °C and a flow rate of 0.5 mL/min. Chiral resolution of pramipexole was carried out with the same mobile phase, but using a flow rate of 1.0 mL and column temperature of 40 °C.

Example 2 - Screening of Acyl Donor and Solvent

[0068] In order to select the acyl donors which do not react spontaneously with the racemic diamine (rac-DA) but could only participate in the enzyme-catalyzed reactions, 13 various acyl donors were tested for the possibility of spontaneous reaction with rac-DA. The acyl donors were selected from the groups of alkyl acetates, vinyl esters, vinyl carbonates, and trifiuoroethyl esters of different carboxylic acids and specifically included vinyl esters of butyric, caproic, lauric and o-chlorobenzoic acids, divinyl ester of adipic acid, 2 ',2 ',2 '-trifiuoroethyl esters of acetic and 3,3- diphenylpropionic acids, di-(2',2',2'-trifluoroethyl)glutarate, butyl vinyl carbonate, acetone oxime vinyl carbonate, 4-(2-hydroxyethyl)morpholine vinyl carbonate and ethyl acetate. Control reactions of rac-DA with acyl donors were set up as follows: a solution of rac-DA (1 mg/mL or ~ 6 mM) in pyridine was mixed with 10-fold molar excess of the donors and the solution was incubated in rotary shaker (200 rpm) at 45 °C over 72 hours. The reactions were sampled after time intervals, worked up by evaporating solvent to dryness and then re-dissolving in methanol with analysis by HPLC. All the acyl donors tested did not show more than 15% conversion of rac-DA to the corresponding acylated product in three days. Due to such insignificant spontaneous acylation of rac-DA, all the studied compounds can be used as acyl donors in the enzymatic reactions of rac-DA acylation. Example 3 - Enzyme (Lipase/Esterase) Screening

[0069] Lipase screening has been carried out to identify enzymes capable of accepting (rac)-diamine ("rac-DA") as a nucleophile and incidentally providing a clue on the stereoselectivity of the enzymes towards the acylation. As the hydrolase catalyzed acylation is carried out in a low water non-aqueous media with temperatures above ambient, sealing of the vials to control the evaporation of solvent and to minimize the water activity changes are very important. Enzyme screening plate consisted of vials (8 x 40 mm Kimble part No. 60832-840) arranged in an 8 x 12 well array fitted to a Beckman tray. The immobilized enzymes were either commercially available or immobilized in- house (by adsorption on Accurel (Accurel Systems International Corp., Sunnyvale, CA, USA)) on a suitable support. The catalyst particles were free flowing powders and individual enzyme powder (~ 10 mg) was added to separate vials with a 1 x 12 well formatted solid dispenser, which functions on a manual spring operated displacement mechanism. The reaction mixture was prepared by dissolving 17 mg (100 μmol) of rac- DA in 50 mL of ethyl acetate (solvent and acyl donor) pre equilibrated over molecular sieves. To this, an additional acylation donor, trifluoroethyl butyrate (0.15 mL), was added and mixed. A 0.6 mL aliquot of this reaction mixture thus prepared was added to each vial of the lipase plate. The vials were sealed with polypropylene plug caps (Marsh Bioproducts/Abgene, Inc., Rochester, NY, USA, part No. C 1000-8) using a bench press, fitted between two metal plates and fixed to the rotating arm arranged to a Fischer Scientific isotemp incubator (Fischer Scientific International, Inc., Hampton, HH, USA). After a 48 hour incubation at 45 ⁰C, 50 μL of the individual reaction mixtures were dried under a stream of nitrogen and re-dissolved in 200 μL of the mobile phase (water adjusted to pH = 1.85 with perchloric acid) and analyzed using HPLC for the products formed as described above. The retention time for the acetamide, (/?)-diamine, (S)-di amine, and butyramide were 3.8 min, 4.8 min, 6.0 min, and 13.8 min, respectively. The % ee was calculated for the unreacted rac-DA. Tables 1 and 2 show the results of enzyme screening for enantioselective preferences. Table 1

Table 2

ND = not determined Example 4 - Lipase Catalyzed Acylation of (røc)-Diamine with Ethyl Acetate

[0070] A lipase plate consisting of selected enzymes shown in Table 3 was prepared for screening of acylation of rac-DA in ethyl acetate, which acts as a solvent as well as an acyl donor. An ~ 2.0 niM rac-DA solution was prepared by dissolving 7.7 mg (45.5 μmol) of the substrate in 22 mL of ethyl acetate pre- equilibrated with molecular sieves. A 0.6 mL of the solution thus prepared was added to each of the vials in the tray and incubated the reaction at 45 °C. A 60 μL aliquot of the reaction mixture was withdrawn at two different time intervals (3 hours and 19 hours) and transferred to a 96 shallow well plate, dried under a stream of nitrogen, re-dissolved in 200 μL of the mobile phase and analyzed on a chiral HPLC. The retention times for the acetamide, (Λ)-diamine and (^-diamine are 3.8 min, 4.8 min, and 6.0 min, respectively. The % optical purity for the unreacted diamine was calculated, and the results are shown in Table 3.

Table 3

Example 5 - Screening of Solvents and Acyl Donors

[0071] Selected acyl donors as described in Example 2 were then tested in the enzymatic acylation of rac-DA catalyzed by a number of lipases in different organic solvents. Selection of organic solvents was based on the literature data and on previous experience with the reactions of acylation catalyzed by lipases in this laboratory. The following solvents were used in the acylation reaction: pyridine, methyl tert-buty\ ether (MTBE), toluene, acetonitrile (MeCN), tetrahydrofuran (THF), and tert-∑any] alcohol (tert-AmOH). Lipase catalysts were selected based on the results of the preceding enzyme screen (see Example 3) and

® included the following catalysts: Lipase AP on Accurel , Chirazyme L-5

CR) (R1 immobilized on Accurel and Celite , Lipase 300 from Pseudomonas sp., Chirazyme L-I from Burkholderia cepacia on Accurel ® and lipase from

Pseudomonas cepacia. Lipase (30 mg) was added to 1 mL of the solvent containing 2 mg (~ 12 μmol) of rac-DA (not totally soluble in the solvents used) and 10 μL of one of the two acyl donors: 2',2',2'-trifluoroethyl butyrate (TFEBut) or vinyl butyrate (VinBut). The reaction mixture was incubated at 45°C in a rotary shaker (200 rpm). Application of the non-chiral HPLC method allowed determination of the reaction conversion [enantiomeric mixture containing the acylated forms of both (S)- and (/?)-DA], but did not allow separate resolution of the acylated stereoisomers.

[0072] Referring now to Table 4, it is seen that the reaction of rac-DA acylation is very solvent-specific and gives best results in MTBE and MeCN. Conversions in both solvents exceeded 50% and are achieved in less than 1 day of the reaction. For this reason, MTBE and MeCN were selected as medium for the reaction of rac-DA acylation in further studies. Table 4 shows percent conversion of the racemic diamine in the reaction of acylation with selected acyl donors catalyzed by several lipases in different organic solvents after 18 hours. Table 4

ND = not determined

Example 6 - Enantiomeric preference of Selected Enzymes

[0073] Acylation reactions were set up as follows: 30 mg lipase was added to 1 mL of methyl tert-bvttyl methyl ether containing 2 mg (~ 12 μmol) of rac-DA (not totally soluble in this solvent) with 10 μL of one of the acyl donors added, vinyl propionate or ethyl propionate. The reaction mixture was incubated at 45 °C in a rotary shaker (200 rpm). For separate determination of the amount of acylated (S)- and (R)-OA in one injection, chiral HPLC analysis was employed as described above. Selection of esters of propionic acid as acyl donors was determined by the fact that the reaction product, propionyl-(5)-DA is an important synthetic intermediate and can be transformed into (S)-pramipexole by chemical reduction in one reaction step as described above.

[0074] The results shown in Table 5 indicate that a number of tested enzymes exhibited the required type of enantioselectivity and thus, can be applied for increasing the relative amount of either (,S)-DA over (R)-OA in the mixture of unreacted substrate or acyl-(iS)-DA over acyl-(/?)-DA in the product mixture. Lipases from Pseudomonas sp. and from Burkholderia cepacia showed high ee_c ,. . values and thus belong to the first group of catalysts, while lipase from

Candida antarctica adsorbed on Celite or Accurel is known for high ee_{R DA} values and belongs to the second group of catalysts. Table 5 provides conversion and enantioselectivity of the diamine acylation in methyl tert-butyl ether catalyzed by selected lipases after 18 hours. Table 5 ihyl propionate EtKyI propionate

^H» ' Lipase (source)

Conversion (% ee (%) Conversion (%) ee (Vo

AP, Aspergillus mger (Amano) ND ND

Chirazyme L-5 on Celite®, 25 23* 53 51 * Candida antarctica (Roche)

Chirazyme L-5 on Accurel®, 66 84* 55 46* Candida antarctica (Roche)

Lipase 300, Pseudomonas sp (Toyobo) 42 31** 61 58**

Chirazyme L-I, Burkholdena cepacia 37 26** 41 20** (Roche)

LP, Pseudomonas cepacia (Amano) ND 43 39**

*ee value corresponds to ee_{R DA}, i.e. stereoselectivity in acylation of (S)-DA **ee value corresponds to ee , i e. stereoselectivity in acylation of (R)-DA

ND = not determined

Example 7 - (R)-Selective Enzymatic Acylation of the Racemic Diamine

[0075] Acylation of the rac-DA catalyzed by lipases from Lipase 300 (Pseudomonas sp.) and Chirazyme L-I (Burkholdena cepacia), which results in preferred formation of (7?)-acyl-DA and enrichment of the reaction system with the target product (.S)-DA was studied in more detail using the reaction set-up described in Example 4. Acylation reactions were set up as follows: 30 mg of either lipase Lipase 300 (Pseudomonas sp.) or Chirazyme L-I (Burkholdena cepacia) was added to 1.0 mL solvent (dry MeCN, MeCN + 1 % water, dry

MTBE, or water-saturated MTBE) containing 2 mg (12 μmol) of rac-DA plus 10 μL of acyl donor (either vinyl propionate, methyl propionate or ethyl propionate). The reaction was incubated at 45 °C in a rotary shaker (200 rpm) followed by chiral HPLC to analyze the conversion and chiral purity of the product. [0076] Both conversion and stereoselectivity of the reaction appeared to be very sensitive to the nature of organic solvent (Table 6). Whereas Pseudomonas Lipase 300 was most reactive and most stereospecific in acetonitrile, Chirazyme L-I showed best results in methyl tert-buty\ ether. Addition of aqueous buffer (acetonitrile containing 1% (v/v) of buffer and methyl tert-buty\ ether saturated with buffer) significantly decreased the acylation activity and stereoselectivity of these enzymes. It is important to note that the level of sensitivity of the reaction of DA acylation to solvent and content of water depends on the enzyme and the nature of acyl donor. Table 6 shows conversion and enantioselectivity of the diamine acylation in methyl tert-butyl ether and acetonitπle catalyzed by (Z?)-specifϊc lipases after 18 hours.

Table 6

ND = not determined

Example 8 - Acylation with Long-chain Acyl Donors / (R)-Specifϊc Lipase

[0077] Acylation reactions were set up as follows: 30 mg Pseudomonas

Lipase 300 was added to 1 mL of methyl tert-butyl methyl ether containing 2 mg (12 μmol) of rac-DA (not totally soluble in this solvent) and 10 μL of the acyl donor. The reaction mixture was incubated at 45 °C in a rotary shaker (200 rpm). For separate determination of the amount of acylated (S)- and (R)-OA in one injection, chiral HPLC analysis was employed. Table 7 shows enantioselectivity of diamine acylation with different acyl donors in methyl tert-butyl ether catalyzed by Pseudomonas Lipase 300 after 18 hours.

Table 7

Example 9 - (.^-Selective Enzymatic Acylation of the Racemic Diamine

[0078] Acylation reactions were set up as follows: 30 mg lipase

(Chirazyme L-5 on Accurel ) was added to 1 mL of methyl tert-buty\ ether containing 2 mg (12 μmol) of rac-DA (not totally soluble in this solvent) and 10 μL of one of the acyl donors shown in Table 8. The reaction mixture was incubated at 45 °C in a rotary shaker (200 rpm). For separate determination of the amount of acylated (S)- and (R)-OA in one injection, chiral HPLC analysis was employed. The amount of acyl (S)-DA and acyl (R)-OA were calculated from equations of mass balance. As seen in Table 8, when less than 50% of racemic DA is acylated in the reaction, the values of ee can reach more than 50% and this value can be further increased by optimization of the reaction conditions. Table 8 shows conversion and stereoselectivity of the diamine acylation in methyl tert-

® butyl ether catalyzed by Chirazyme L-5 on Accurel after 18 hours.

Table 8

Example 10 - Enzymatic Stereoselective Amide Hydrolysis

[0079] ' Various amide substrates 1 a-h were screened with pig liver esterase (PLE) for enzymatic resolution. The hydrolysis reaction was carried out in 50.0 mM Tris-HCl, pH 8.0, at 2.0 mM substrate concentration at 40 ⁰C with pig liver esterase (PLE), ammonium sulfate suspension (Roche). The progress of the reaction was followed by determining the unreacted amides and/or diamines (S)-2 and (R)-2 formed, as measured by chiral HPLC. With all the substrates tested (Table 9), PLE was identified to hydrolyze the amides in an (5)-preferential manner. Results are summarized in Table 9 for a variety of amide substrates. Table 9

IU = I μmol/min based on the conversion to the single (S)-isomer Comparison to acetamide analogue Ia With 20% (v/v) DMSO N/A = not applicable

[0080] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED:

1. A process for preparing (S)-pramipexole comprising:

a) reacting a compound of formula I

with an acyl donor in the presence of an enzyme under conditions effective to produce a separable mixture comprising: a compound of formula (R)-Il

wherein R is selected from the group consisting of alkyl, aryl, or aralkyl; and a compound of formula (S)-I

b) reacting the compound of formula (S)-I under conditions effective to produce a compound of formula (S)-III, which is (S)-pramipexole

c) recovering said (S)-pramipexole.

2. The process according to claim 1, wherein the enzyme is a hydrolase.

3. The process according to claim 2, wherein the hydrolase is immobilized on a solid support.

4. The process according to claim 2, wherein the hydrolase is selected from the group consisting of lipases, esterases, and proteases.

5. The process according to claim 1 , wherein the enzyme is selected from the group consisting of Chirazyme L-IO (Alcaligenes sp.), Lipase QL (Alcaligenes Sp.),

Lipase SL (Burkholdeήa cepacia), Chirazyme L-I (Burkholdeήa sp.), Chirazyme L-2 (Candida antarctica, B), Lipase L-IO (Candida lipolytica), Lipase CL (Candida lipolytica), Chirazyme L-3 (Candida rugosa), Lipase CV (Chromobacterium viscosum), Lipase MJ (Mucor javanicus), Lipase M- 10 (Mucor javanicus), Lipase MM (Mucor mieheϊ), Lipase MM, recombinant (Mucor mieheϊ), Chirazyme L-9 (Mucor miehei), Lipase PN (Phycomyces nitens), Lipase-type 250 (porcine pancreas), Lipase Type II - PPL (porcine pancreas), Lipase AH (Pseudomonas cepacia), Lipase LP S (Pseudomonas cepacia), Lipase PS-CII (Pseudomonas cepacia), Lipase PS-DI (Pseudomonas cepacia), cholesterol esterase (Pseudomonas fluorescens), Lipase Lip-300 (Pseudomonas sp.), Chirazyme L-6 (Pseudomonas sp.), Lipase TL (Pseudomonas stutzeri), Lipase "RA"

(Rhizopus arrhizus), Lipase RN (Rhizopus niveus), Newlase F (Rhizopus niveus), A-10FG (Rhizopus oryzae), FAP-15 (Rhizopus oryzae), and Lipoorisi G3x (Rhizopus oryzae).

6. The process according to claim 5, wherein the enzyme is selected from the group consisting of Chirazyme L-I (Burkholderia sp.), Chirazyme L-2 (Candida antarctica, B), Lipase CV (Chromobacterium viscosum), Lipase MM (Mucor miehei), Lipase MM, recombinant (Mucor miehei), Chirazyme L-9 (Mucor miehei), Lipase-type 250 (porcine pancreas), Lipase AH (Pseudomonas cepacia), Lipase LP S (Pseudomonas cepacia), Lipase PS-CII (Pseudomonas cepacia), Lipase PS-DI (Pseudomonas cepacia), Lipase Lip-300 (Pseudomonas sp.), Chirazyme L-6 (Pseudomonas sp.), and Lipase TL (Pseudomonas stutzeri).

7. The process according to claim 1 , wherein the acyl donor is selected from the group consisting of trifluoroethyl butyrate, di-(2',2',2'-trifluoroethyl) 1,4- cyclohexanedicarboxylate, benzoic acid vinyl ester, butyric acid vinyl ester, caproic acid vinyl ester, lauric acid vinyl ester, butyl vinyl carbonate, 2,6-furandimethanol vinyl carbonate, 1 ,6-hexanediol vinyl carbonate, ethyl acetate, butyl acetate, methyl propionate, ethyl propionate, butyl propionate, hexyl propionate, octyl propionate, vinyl propionate, phenylacetic acid, methyl phenyl acetate, and methyl phenoxyacetate.

8. The process according to claim 7, wherein the acyl donor is selected from the group consisting of trifluoroethyl butyrate, ethyl acetate, methyl propionate, hexyl propionate, and vinyl propionate.

9. The process according to claim 1 , wherein the acyl donor is in a 1.5-fold to 20-fold molar excess over the compound of formula I.

10. The process according to claim 1, wherein said reacting a compound of formula I and an acyl donor is carried out in an organic solvent.

11. The process according to claim 10, wherein the organic solvent is selected from the group consisting of methyl tert-buty\ ether, tetrahydrofuran, toluene, pyridine,

1 ,4-dioxane, ethyl acetate, «-butyl acetate, methylene chloride, benzene, and acetonitrile.

12. The process according to claim 11, wherein the organic solvent is selected from the group consisting of methyl tert-butyl ether, ethyl acetate, and acetonitrile.

13. The process according to claim 10, wherein the organic solvent has water content from 0 to 1 volume %.

14. The process according to claim 1, wherein said reacting a compound of formula I and an acyl donor is performed at a temperature of between 25 ⁰C and 50 ⁰C.

15. The process according to claim 1, wherein the compound of formula I is, during said reacting a compound of formula I and an acyl donor, in a concentration range of about 0.005 M to about 0.15M.

16. The process according to claim 1 , wherein the acyl donor is, during said reacting a compound of formula I and an acyl donor, in a concentration range of about 0.01 M to about 0.1 M.

17. The process according to claim 1 , wherein said reacting a compound of formula I and an acyl donor is carried out with an enzyme concentration of about 10 mg to about 100 mg per mL of the reaction mixture.

18. A process for preparing (S)-pramipexole comprising:

a) reacting a compound of formula I

with an acyl donor in the presence of an enzyme under conditions effective to produce a separable mixture comprising: a compound of formula (S)-U

wherein R is selected from the group consisting of alkyl, aryl, and aralkyl, and a compound of formula (R)-I

b) hydrolyzing the compound of formula (S)-U under conditions effective to produce a compound of formula (S)-I

c) reacting the compound of formula (S)-I under conditions effective to produce a compound of formula (5)-III, which is (S)-pramipexole

d) recovering said (S)-pramipexole.

19. The process according to claim 18, wherein the enzyme is a hydrolase.

20. The process according to claim 19, wherein the hydrolase is immobilized on a solid support.

21. The process according to claim 19, wherein the hydrolase is selected from the group consisting of lipases, esterases, and proteases.

22. The process according to claim 18, wherein the enzyme is selected from the group consisting of Amano lipase AP (Aspergillus niger), cholesterol esterase (bovine pancreas), Chirazyme L-5 {Candida antarctica, A), Chirazyme L-5, c.f. (Candida antarctica, A), Lipase GC-4 (Geotrichum candidum), Lipase PR (Penicillium roqueforti), Lipase R- 10 (Penicillium roqueforti), cholesterol esterase (porcine pancreas), Lipase PS- 30 (Pseudomonas cepacia), Lipase D (Rhizopus delemar), and Lipase Type I (wheat germ).

23. The process according to claim 22, wherein the enzyme is selected from the group consisting of Amano lipase AP (Aspergillus niger), Chirazyme L-5 (Candida antarctica, A), and Chirazyme L-5, c.f. (Candida antarctica, A).

24. The process according to claim 18, wherein the acyl donor is selected from the group consisting of trifluoroethyl butyrate, di-(2',2',2^l-trifluoroethyl) 1,4- cyclohexanedicarboxylate, benzoic acid vinyl ester, butyric acid vinyl ester, caproic acid vinyl ester, lauric acid vinyl ester, butyl vinyl carbonate, 2,6-furandimethanol vinyl carbonate, 1,6-hexanediol vinyl carbonate, ethyl acetate, butyl acetate, methyl propionate, ethyl propionate, butyl propionate, hexyl propionate, octyl propionate, vinyl propionate, phenylacetic acid, methyl phenylacetate, and methyl phenoxyacetate.

25. The process according to claim 24, wherein the acyl donor is selected from the group consisting of trifluoroethyl butyrate, ethyl acetate, methyl propionate, hexyl propionate, and vinyl propionate.

26. The process according to claim 18, wherein the acyl donor is in a 1.5-fold to 20-fold molar excess over the compound of formula I.

27. The process according to claim 18, wherein said reacting a compound of formula I and an acyl donor is carried out in an organic solvent.

28. The process according to claim 27, wherein the organic solvent is selected from the group consisting of methyl tert-buty] ether, tetrahydrofuran, toluene, pyridine,

1 ,4-dioxane, ethyl acetate, rc-butyl acetate, methylene chloride, benzene, and acetonitrile.

29. The process according to claim 28, wherein the organic solvent is selected from the group consisting of methyl ter/-butyl ether, ethyl acetate, and acetonitrile.

30. The process according to claim 27, wherein the organic solvent has water content from 0 to 1 volume %.

31. The process according to claim 18, wherein said reacting a compound of formula I and an acyl donor is performed at a temperature of between 25 ⁰C and 50 ⁰C.

32. The process according to claim 18, wherein the compound of formula I is, during said reacting a compound of formula I and an acyl donor, in a concentration range of about 0.005 M to about 0.15M.

33. The process according to claim 18, wherein the acyl donor is, during said reacting a compound of formula I and an acyl donor, in a concentration range of about 0.01 M to about 0.1 M.

34. The process according to claim 18, wherein said reacting a compound of formula I and an acyl donor is carried out with an enzyme concentration of about 10 mg to about 100 mg per mL of the reaction mixture.

35. A process for preparing (>S)-pramiρexole comprising: a) reacting a compound of formula III

(røc)-pramipexole

with an acyl donor in the presence of an enzyme under conditions effective to produce a separable mixture comprising: a compound of formula (-S)-III, which is (S)-pramipexole,

and a compound of formula (R)-TV

wherein R is selected from the group consisting of alkyl, aryl, and aralkyl; and

b) isolating said (.S)-pramipexole.

36. The process according to claim 35, wherein the enzyme is a hydrolase.

37. The process according to claim 36, wherein the hydrolase is immobilized on a solid support.

38. The process according to claim 36, wherein the hydrolase is selected from the group consisting of lipases, esterases, and proteases.

39. The process according to claim 35, wherein the enzyme is selected from the group consisting of Chirazyme L-IO (Alcaligenes sp.), Lipase QL (Alcaligenes sp.),

Lipase SL (Burkholderia cepacia), Chirazyme L-I {Burkholderia sp.), Chirazyme L-2 (Candida antarctica, B), Lipase L-IO {Candida lipolytica), Lipase CL {Candida lipolytica), Chirazyme L-3 {Candida rugosa), Lipase CV {Chromobacterium viscosum), Lipase MJ {Mucor javanicus), Lipase M- 10 {Mucor javanicus), Lipase MM {Mucor miehei), Lipase MM, recombinant {Mucor miehei), Chirazyme L-9 {Mucor miehei),

Lipase PN {Phycomyces nitens), Lipase-type 250 (porcine pancreas), Lipase Type II-PPL (porcine pancreas), Lipase AH {Pseudomonas cepacia), Lipase LP S {Pseudomonas cepacia), Lipase PS-CII {Pseudomonas cepacia), Lipase PS-DI {Pseudomonas cepacia), cholesterol esterase {Pseudomonas βuorescens), Lipase Lip-300 {Pseudomonas sp.), Chirazyme L-6 {Pseudomonas sp.), Lipase TL (Pseudomonas .stutzeri), Lipase "RA"

(Rhizopus arrhizus), Lipase RN {Rhizopus niveus), Newlase F {Rhizopus niveus), A-10FG {Rhizopus oryzae), FAP-15 {Rhizopus oryzae), and Lipoorisi G3x {Rhizopus oryzae).

40. The process according to claim 39, wherein the enzyme is selected from the group consisting of Chirazyme L-I {Burkholderia sp.), Chirazyme L-2 (Candida antarctica, B), Lipase CV {Chromobacterium viscosum), Lipase MM {Mucor miehei), Lipase MM, recombinant {Mucor miehei), Chirazyme L-9 {Mucor miehei), Lipase-type 250 (porcine pancreas), Lipase AH {Pseudomonas cepacia), Lipase LP S {Pseudomonas cepacia), Lipase PS-CII {Pseudomonas cepacia), Lipase PS-DI {Pseudomonas cepacia), Lipase Lip-300 {Pseudomonas sp.), Chirazyme L-6 {Pseudomonas sp.), and Lipase TL (Pseudomonas stutzeri).

41. The process according to claim 35, wherein the acyl donor is selected from the group consisting of trifluoroethyl butyrate, di-(2',2',2'-trifluoroethyl) 1,4- cyclohexanedicarboxylate, benzoic acid vinyl ester, butyric acid vinyl ester, caproic acid vinyl ester, lauric acid vinyl ester, butyl vinyl carbonate, 2,6-furandimethanol vinyl carbonate, 1 ,6-hexanediol vinyl carbonate, ethyl acetate, butyl acetate, methyl propionate, ethyl propionate, butyl propionate, hexyl propionate, octyl propionate, vinyl propionate, phenylacetic acid, methyl phenylacetate, and methyl phenoxyacetate.

42. The process according to claim 41, wherein the acyl donor is selected from the group consisting of trifluoroethyl butyrate, ethyl acetate, methyl propionate, hexyl propionate, and vinyl propionate.

43. The process according to claim 35, wherein the acyl donor is in a 1.5-fold to 20-fold molar excess over the compound of formula III.

44. The process according to claim 35, wherein said reacting a compound of formula III and an acyl donor is carried out in an organic solvent.

45. The process according to claim 44, wherein the organic solvent is selected from the group consisting of methyl tert-butyl ether, tetrahydrofuran, toluene, pyridine,

46. The process according to claim 45, wherein the organic solvent is selected from the group consisting of methyl /er/-butyl ether, ethyl acetate, and acetonitrile.

47. The process according to claim 44, wherein the organic solvent has water content from 0 to 1 volume %.

48. The process according to claim 35, wherein said reacting a compound of formula III and an acyl donor is performed at a temperature of between 25 ⁰C and 50⁰C.

49. The process according to claim 35, wherein the compound of formula III is, during said reacting a compound of formula IH and an acyl donor, in a concentration range of about 0.005 M to about 0.15M.

50. The process according to claim 35, wherein the acyl donor is, during said reacting a compound of formula III and an acyl donor, in a concentration range of about 0.01 M to about 0.1 M.

51. The process according to claim 35, wherein said reacting a compound of formula III and an acyl donor is carried out with an enzyme concentration of about 10 mg to about 100 mg per mL of the reaction mixture.

52. The process according to claim 35, further comprising after said isolating: c) hydrolyzing the compound of formula (R)-YV under conditions effective to produce a compound of formula (R)-III, which is (R)-ρramipexole

d) recovering said (R)-pramipexole.

53. The process according to claim 52, wherein said hydrolyzing is carried out in the presence of an aqueous acid.

54. The process according to claim 52, wherein said hydrolyzing is carried out in the presence of a hydrolase.

55. The process according to claim 54, wherein the hydrolase is immobilized on a solid support.

56. The process according to claim 54, wherein the hydrolase is selected from the group consisting of lipases, esterases, and proteases.

57. A process for preparing (R)-pramipexole comprising: a) reacting a compound of formula III

with an acyl donor in the presence of an enzyme under conditions effective to produce a separable mixture comprising: a compound of formula (R)-III, which is (R)-pramipexole,

and a compound of formula (S)-W

b) isolating said (R)-pramipexole.

58. The process according to claim 57, wherein the enzyme is a hydrolase.

59. The process according to claim 58, wherein the hydrolase is immobilized on a solid support.

60. The process according to claim 58, wherein the hydrolase is selected from the group consisting of lipases, esterases, and proteases.

61. The process according to claim 57, wherein the enzyme is selected from the group consisting of Amano lipase AP (Aspergillus niger), cholesterol esterase (bovine pancreas), Chirazyme L-5 (Candida antarctica, A), Chirazyme L-5, c.f. (Candida antarctica, A), Lipase GC-4 (Geotήchum candidum), Lipase PR (Penicillium roqueforti), Lipase R- 10 (Penicillium roqueforti), cholesterol esterase (porcine pancreas), Lipase PS- 30 (Pseudomonas cepacia), Lipase D (Rhizopus delemar), and Lipase Type I (wheat germ).

62. The process according to claim 61 , wherein the enzyme is selected from the group consisting of Amano lipase AP (Aspergillus niger), Chirazyme L-5 (Candida antarctica, A), and Chirazyme L-5, c.f. (Candida antarctica, A).

63. The process according to claim 57, wherein the acyl donor is selected from the group consisting of trifluoroethyl butyrate, di-(2',2',2'-trifluoroethyl) 1,4- cyclohexanedicarboxylate, benzoic acid vinyl ester, butyric acid vinyl ester, caproic acid vinyl ester, lauric acid vinyl ester, butyl vinyl carbonate, 2,6-furandimethanol vinyl carbonate, 1 ,6-hexanediol vinyl carbonate, ethyl acetate, butyl acetate, methyl propionate, ethyl propionate, butyl propionate, hexyl propionate, octyl propionate, vinyl propionate, phenylacetic acid, methyl phenyl acetate, and methyl phenoxyacetate.

64. The process according to claim 63, wherein the acyl donor is selected from the group consisting of trifluoroethyl butyrate, ethyl acetate, methyl propionate, hexyl propionate, and vinyl propionate.

65. The process according to claim 57, wherein the acyl donor is in a 1.5-fold to 20-fold molar excess over the compound of formula 111.

66. The process according to claim 57, wherein said reacting a compound of formula III and an acyl donor is carried out in an organic solvent.

67. The process according to claim 66, wherein the organic solvent is selected from the group consisting of methyl tert-buty\ ether, tetrahydrofuran, toluene, pyridine, 1,4-dioxane, ethyl acetate, w-butyl acetate, methylene chloride, benzene, and acetonitrile.

68. The process according to claim 67, wherein the organic solvent is selected from the group consisting of methyl tert-buty\ ether, ethyl acetate, and acetonitrile.

69. The process according to claim 66, wherein the organic solvent has water content from 0 to 1 volume %.

70. The process according to claim 57, wherein said reacting a compound of formula III and an acyl donor is performed at a temperature of between 25 ⁰C and 50 ⁰C.

71. The process according to claim 57, wherein the compound of formula III is, during said reacting a compound of formula III and an acyl donor, in a concentration range of about 0.005 M to about 0.15M.

72. The process according to claim 57, wherein the acyl donor is, during said reacting a compound of formula III and an acyl donor, in a concentration range of about 0.01 M to about 0.1 M.

73. The process according to claim 57, wherein said reacting a compound of formula III and an acyl donor is carried out with an enzyme concentration of about 10 mg to about 100 mg per mL of the reaction mixture.

74. The process according to claim 57, further comprising after said isolating: c) hydrolyzing the compound of formula (S)-TV under conditions effective to produce a compound of formula (S)-III, which is (S)-pramipexole,

d) recovering said (S)-pramipexole.

75. The process according to claim 74, wherein said hydrolyzing is carried out in the presence of an aqueous acid.

76. The process according to claim 74, wherein said hydrolyzing is carried out in the presence of a hydrolase.

77. The process according to claim 76, wherein the hydrolase is immobilized on a solid support.

78. The process according to claim 76, wherein the hydrolase is selected from the group consisting of lipases, esterases, and proteases.

79. A process for preparing (S)-pramipexole comprising: a) reacting a compound of formula II

wherein R is selected from the group consisting of alkyl, aryl, and aralkyl, with an enzyme under conditions effective to produce a separable mixture comprising: a compound of formula (R)-U

and a compound of formula (S)-I

b) reacting the compound of formula (S)-I under conditions effective to produce a compound of formula (S)-Ul, which is (5)-pramipexole,

c) recovering said (5)-pramipexole.

80. The process according to claim 79, wherein the enzyme is pig liver esterase.

81. The process according to claim 79, further comprising hydrolyzing the compound of formula (R)-U under conditions effective to produce a compound of formula (R)-I

82. The process according to claim 81, wherein said hydrolyzing is carried out in the presence of a hydrolase.

83. The process according to claim 82, wherein the hydrolase is immobilized on a solid support.

84. The process according to claim 82, wherein the hydrolase is selected from the group consisting of lipases, esterases, and proteases.

85. The process according to claim 82, wherein the hydrolase is selected from the group consisting of pig liver esterase, liver acetone powders, and penicillin amidase.

86. A process for preparing (-S)-pramipexole comprising: a) reacting a compound of formula II

wherein R is selected from the group consisting of alkyl, aryl, and aralkyl, with an enzyme under conditions effective to produce a separable mixture comprising: a compound of formula (S)-Il

and a compound of formula (R)-I

b) hydrolyzing the compound of formula (S)-W under conditions effective to produce a compound of formula (S)-I

c) reacting the compound of formula (S)-I under conditions effective to produce a compound of formula (S)-III, which is (5)-pramipexole

(S)-pramipexole

d) recovering said S-pramipexole.

87. The process according to claim 86, wherein the enzyme is penicillin amidase.

88. The process according to claim 86, wherein said hydrolyzing is carried out in the presence of a hydrolase.

89. The process according to claim 88, wherein the hydrolase is immobilized on a solid support.

90. The process according to claim 88, wherein the hydrolase is selected from the group consisting of lipases, esterases, and proteases.

91. The process according to claim 88, wherein the hydrolase is selected from the group consisting of pig liver esterase, liver acetone powders, and penicillin amidase.

92. The process according to claim 86, wherein R is aralkyl.

93. The process according to claim 92, wherein the enzyme is penicillin amidase.