(19) TZZ Z_¥_T (11) EP 2 250 143 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07B 57/00 (2006.01) C07C 269/04 (2006.01) 20.04.2016 Bulletin 2016/16 C07C 329/06 (2006.01) C07D 207/46 (2006.01) C12P 41/00 (2006.01) C07C 271/22 (2006.01) (2006.01) (2006.01) (21) Application number: 09704177.6 A61K 31/325 C12P 11/00 (22) Date of filing: 23.01.2009 (86) International application number: PCT/US2009/031873 (87) International publication number: WO 2009/094569 (30.07.2009 Gazette 2009/31) (54) METHOD FOR THE ENZYMATIC KINETIC RESOLUTION OF ACYLOXYALKYL THIOCARBONATES USED FOR THE SYNTHESIS OF ACYLOXYALKYL CARBAMATES VERFAHREN ZUR ENZYMATISCHEN KINETISCHEN RACEMATSPALTUNG VON FÜR DIE SYNTHESE VON ACYLOXYALKYLCARBAMATEN VERWENDETEN ACYLOXYALKYLTHIOCARBONATEN RESOLUTION CINETIQUE ENZYMATIQUE DE THIOCARBONATES D’ACYLOXYALKYLE UTILISES DANS LA SYNTHESE DE CARBAMATES D’ACYLOXYALKYLE (84) Designated Contracting States: • PENG, Ge AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Mountain View HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL California 94040 (US) PT RO SE SI SK TR • RAILLARD, Stephen, P. Mountain View, California 94040 (US) (30) Priority: 25.01.2008 US 23813 P 25.01.2008 US 23808 P (74) Representative: Wytenburg, Wilhelmus Johannes 11.12.2008 US 121859 P et al Mewburn Ellis LLP (43) Date of publication of application: City Tower 17.11.2010 Bulletin 2010/46 40 Basinghall Street London EC2V 5DE (GB) (73) Proprietor: XenoPort, Inc. Santa Clara, CA 95051 (US) (56) References cited: US-B2- 6 927 036 US-B2- 7 109 239 (72) Inventors: US-B2- 7 227 028 • GALLOP, Mark, A. Palo Alto, California 94301-3003 (US) • U. T. BORNSCHEUER, R. J. KAZLAUSKAS: •YAO,Fenmei "Hydrolases in Organic Synthesis. Regio- and Mountain View, California 94043 (US) Stereoselective Biotransformations. 2nd • LUDWIKOW, Maria, J. edition." WILEY-VCH, November 2005 (2005-11), Cupertino, California 95014 (US) pages 61-183, XP002538624 Weinheim ISBN: 3-527-31029-0 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 250 143 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 250 143 B1 Description Field 5 [0001] Methods provided by the present disclosure relate to the enzymatic resolution of acyloxyalkyl thiocarbonates useful in the synthesis of acyloxyalkyl carbamate prodrugs. Background 10 [0002] The oral bioavailability of certain drugs can be improved by conversion to prodrugs. Certain prodrugs are derivatives of the parent drug in which a functional group is "masked" by a promoiety. Following administration to a patient the prodrug is metabolized to release the parent drug. The 1-(acyloxy)-alkyl group is an example of a promoiety that has been used to functionalize amine-containing drugs such as pregabalin and baclofen. [0003] Pregabalin ((3S)-(aminomethyl)-5-methyl-hexanoic acid) is an FDA approved drug that is marketed for the 15 treatment of, for example, post herpetic neuralgia, peripheral diabetic neuropathy, fibromyalgia, and epilepsy. Pregabalin is not absorbed from the lower gastrointestinal tract and exhibits a short half life in vivo, and therefore frequent dosing is required to maintain therapeutic levels in the body when orally administered. (3S)-{[1-Isobutanoyloxyethoxy]carbon- ylaminomethyl}-5-methyl-hexanoic acid, (3S)-{[1-isobutanoyloxyisobutoxy]carbonylaminomethyl}-5-methyl-hexanoic acid, and (3S)-{[1-benzoyloxyethoxy]carbonylaminomethyl}-5-methyl-hexanoic acid are examples of 1-(acyloxy)-alkyl 20 carbamate prodrugs of pregabalin, which exhibit high bioavailability as pregabalin when dosed either orally or directly into the colon of a mammal (Gallop et al., US 6,972,341 and US 7,186,855; and Yao and Gallop, U.S. Provisional Application Nos. 61/023,808 filed January 25, 2008 and 61/023,813 filed January 25, 2008). [0004] The 1-(acyloxy)-alkyl promoiety has also been used to provide prodrugs of baclofen, 6( )-4-amino-3-(4-chlo- rophenyl)butanoic acid. Gallop et al., US 7,109,239 and US 7,300,956 disclose 1-(acyloxy)-alkyl carbamate prodrugs 25 of R-baclofen such as (3R)-4-{[(1S)-2-methyl-1-(2-methylpropanoyloxy)propoxy]carbonylamino}-3-(4-chlorophenyl)bu- tanoic acid. Baclofen is an analog of gamma-aminobutyric acid (GABA) that selectively activates GABAB receptors, resulting in neuronal hyperpolarization. Baclofen is an FDA approved drug that is marketed for the treatment of spasticity and muscle relaxation. More recent studies have indicated that the R-isomer of baclofen is effective for treating gastro- esophageal reflux disease (GERD). Baclofen and R-baclofen, like pregabalin, have poor colonic absorption and a short 30 half life in vivo, and when orally administered frequent dosing is required to maintain therapeutic levels in the body. [0005] The (acyloxy)alkylcarbamate functionality has been widely used to prepare prodrugs for therapeutics containing amine groups (Gogate et al., International Journal of Pharmaceutics 1987, 40, 235-248; Alexander et al., J. Med. Chem. 1988, 31, 318-322; Sun et al., Bioorganic & Medicinal Chemistry Letters 2001, 11, 1875-1879; Alexander et al., J. Med. Chem. 1991, 34, 78-81; and Gallop et al., US 6,972,341). Methods of synthesizing 1-(acyloxy)-alkyl carbamate prodrugs 35 are disclosed in Gallop et al., US 6,818,787, US 6,927,036, US 6,972,341, US 7,186,855, US 7,025,351, US 7,109,239, and US 7,227,028; Raillard et al., US 7,232,924; Gallop and Bhat, WO 2005/010011; Raillard et al., U.S. Provisional Application No. 61/087,056 filed August 7, 2008 and 61/087,038 filed August 7, 2008); and in Alexander, US 4,760,057, US 4,916,230, and US 5,684,018. One method, as outlined in Figure 1, involves an acyloxyalkylthiocarbonate interme- diate (Sun et al., Bioorganic & Medicinal Chemistry Letters 2001, 11, 1875-1879; and Gallop et al., US 7,026, 351 and 40 US 7,227,028). [0006] A deficiency common to such methods for synthesizing acyloxyalkyl derivatives is that, except when the R2 substituent is hydrogen, the prodrugs are generated as racemates or diastereomeric mixtures. The presence of an additional chiral center in the promoiety may lead to differences in the physical properties and in the pharmacokinetics of the prodrug. Complexities associated with the introduction of an uncontrolled stereocenter in acyloxyalkyl promoieties 45 have led others to focus prodrug design efforts around the achiral acyloxymethyl moiety (R 2 is hydrogen). Further, many (acyloxy)alkylcarbamate prodrugs generate formaldehyde as a toxic metabolite during hydrolysis in vivo. In comparison with acetaldehyde, formaldehyde shows greater acute mammalian toxicity and mutagenicity, and its oxidative metabolite formate is associated with specific ocular toxicity in humans. Furthermore, because the thiocarbonates do not have acidic or basic functional groups, they are not readily resolved by classical chemical methods. 50 [0007] Thus, improved methods of synthesizing enantiomerically enriched acyloxyalkyl thiocarbonates are desirable. [0008] Bornscheuer et al., "Chapter 5: Lipases and Esterases", in Hydrolases in Organic Synthesis: Regio- and Ster- eoselective Biotransformations, 2nd edition, 2005, pp. 61-183 describes the availability, structure, and properties of lipases and esterase (Section 5.1), a survey of enantioselective lipase-catalyzed reactions (Section 5.2), chemo- and regioselective lipase-catalyzed reactions (Section 5.3), and reactions catalyzed by esterases (Section 5.4). 55 SUMMARY OF THE INVENTION [0009] A first aspect of the invention is a method of enantiomerically enriching an enantiomeric mixture of a compound 2 EP 2 250 143 B1 of Formula (I): 5 10 comprising: reacting the enantiomeric mixture with an enzyme that is an esterase or a lipase to provide an enantiomerically enrichedmixture having atleast 90% enantiomericexcess of one enantiomerof thecompound of Formula( I),wherein: 15 R1 is chosen from methyl, n-propyl, isopropyl, and phenyl; R2 is chosen from methyl, n-propyl, and isopropyl; and R3 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl. 20 [0010] In one embodiment, R3 is methyl. [0011] In one embodiment, the enzyme isCandida rugosa lipase, Candida cylindracea lipase, Candida antarctica lipase B, porcine liver esterase, or Candida antarctica lipase A. [0012] In one embodiment, the enantiomerically enriched mixture has an enantiomeric excess of theR isomer; and the enzyme is Candida rugosa lipase. 25 [0013] In one embodiment, the enantiomerically enriched mixture has an enantiomeric excess of theR isomer; and the enzyme is Candida cylindracea lipase. [0014] In one embodiment, the enantiomerically enriched mixture has an enantiomeric excess of theR isomer; and the enzyme is Candida antarctica lipase B. [0015] In one embodiment, the enantiomerically enriched mixture has an enantiomeric excess of the S isomer; and 30 the enzyme is porcine liver esterase. [0016] In one embodiment, the enantiomerically enriched mixture has an enantiomeric excess of the S isomer; and the enzyme is Candida antarctica lipase A. [0017] In one embodiment, the enantiomerically enriched mixture has an enantiomeric excess of the S isomer; and the enzyme is Candida antarctica lipase B. 35 [0018] In one embodiment: the enzyme is Candida antarctica lipase B; R1 is isopropyl; R2 is methyl; 40 R3 is methyl; and the enantiomerically enriched mixture has an enantiomeric excess of the R enantiomer of the compound of Formula (I). [0019] In one embodiment: 45 the enzyme is Candida antarctica lipase A; R1 is isopropyl; R2 is isopropyl; R3 is methyl; and 50 the enantiomerically enriched mixture has an enantiomeric excess of the S enantiomer of the compound of Formula ( I).