Review

Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates Ana Caroline Lustosa de Melo Carvalho 1 , Thiago de Sousa Fonseca 1 , Marcos Carlos de Mattos 1, *, Maria da Conceição Ferreira de Oliveira 1 , Telma Leda Gomes de Lemos 1 , Francesco Molinari 2 , Diego Romano 2 and Immacolata Serra 2 Received: 28 October 2015; Accepted: 04 November 2015; Published: 11 December 2015 Academic Editor: Vladimír Kˇren 1

2

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Laboratório de Biotecnologia e Síntese Orgânica (LABS), Department of Organic and Inorganic Chemistry, Federal University of Ceará, Campus do Pici, Postal box 6044, 60455-970 Fortaleza, Ceará, Brazil; [email protected] (A.C.L.M.C.); [email protected] (T.S.F.); [email protected] (M.C.F.O.); [email protected] (T.L.G.L.) Department of Food, Environmental and Nutritional Sciences (DEFENS), University of Milan, via Mangiagalli 25, 20133 Milan, Italy; [email protected] (F.M.); [email protected] (D.R.); [email protected] (I.S.) Correspondance: [email protected]; Tel.: +55-85-3366-9144; Fax: +55-85-3366-9978

Abstract: Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modified. Further improvements and new applications have been achieved in the syntheses of biologically active compounds catalyzed by lipases. This review critically reports and discusses examples from recent literature (2007 to mid-2015), concerning the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates in which the key step involves the action of a lipase. Keywords: biocatalysis; lipases; kinetic resolution; enantioselectivity; pharmaceuticals

1. Introduction Among the applications of lipases, the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates using these enzymes is a subject of continuing interest. It is noteworthy that most of the drugs are chiral, and it is important to know which stereoisomer has the desired biological activity. Thus, only the active stereoisomer is administered, thereby preventing the patient receiving a dose of stereoisomer with unnecessary activity. Such practice avoids unnecessary consumption of the substance and minimizes side effects. Biocatalysis offers an alternative approach to conventional chemical processes for the production of enantiomerically pure chiral drugs. Lipases are highlighted as being among the most frequently used enzymes for the production of drugs and their intermediates, a subject covered in an elegant review in 2006 [1]. The use of lipases is mainly due to the characteristics of the regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modified [1]. Some reviews on the preparation of APIs via biocatalysis were also published, but involved various types of enzymes and not only lipases [2–6]. Int. J. Mol. Sci. 2015, 16, 29682–29716; doi:10.3390/ijms161226191

www.mdpi.com/journal/ijms

On this topic, we will present representative examples of recent reports in the literature (2007 to mid-2015) concerning the lipase-catalyzed preparation of APIs and their intermediates through hydrolytic (Section 2) and esterification (Section 3) approaches. Additionally, examples involving both approaches as2015, complementary methods are discussed (Section 4). Int. J. Mol. Sci. 16, 29682–29716 2. HydrolyticOn Approach this topic, we will present representative examples of recent reports in the literature (2007 to mid-2015) concerning lipase-catalyzed preparationby of lipases APIs andfor their intermediates through Hydrolysis of racemic or the prochiral esters catalyzed preparing an enantiomerically hydrolytic (Section 2) and esterification (Section 3) approaches. Additionally, examples involving pure drug intermediate is a well-established method in organic chemistry; nevertheless, new both approaches as complementary methods are discussed (Section 4). applications, new enzymes, and improved techniques for bettering the efficiency of already known 2. Hydrolytic Approach lipase-catalyzed hydrolysis are continuously proposed.

2.1.

Hydrolysis of racemic or prochiral esters catalyzed by lipases for preparing an enantiomerically pure drug intermediate is aSide well-established method in organic chemistry; nevertheless, new Key Intermediates of Paclitaxel Chain applications, new enzymes, and improved techniques for bettering the efficiency of already known An interesting example ofarethe application of lipases for preparing a series of useful lipase-catalyzed hydrolysis continuously proposed.

chiral intermediates was proposed for the chemoenzymatic synthesis of analogues of Paclitaxel (trade 2.1. Key Intermediates of Paclitaxel Side Chain names: Taxol or Onxal). This compound is a polyoxygenated diterpenoid isolated from the bark of An interesting example of the application of lipases for preparing a series of useful chiral Taxus brevifolia, which has been extensively used in anti-cancer therapy. A number of compounds intermediates was proposed for the chemoenzymatic synthesis of analogues of Paclitaxel (trade from the names: class ofTaxol arylazetidiones, the amino acid side chain of Paclitaxel, as well as a or Onxal). Thisprecursors compound isof a polyoxygenated diterpenoid isolated from the bark new generation of taxanoides side chains, were prepared in enantiomerically pure form by kinetic of Taxus brevifolia, which has been extensively used in anti-cancer therapy. A number of compounds from the class of arylazetidiones, precursors of the amino acid side chain of Paclitaxel, as well as a resolution, via hydrolysis, of the corresponding esters in the presence of lipases. A screening was new generation of taxanoides side chains, were prepared in enantiomerically pure form by kinetic performed with 14 lipases, and four of them (PS (Burkholderia cepacia), PS-C (B. cepacia immobilized resolution, via hydrolysis, of the corresponding esters in the presence of lipases. A screening was on ceramics), AS (Aspergillus niger), ABL (Arthrobacter sp.)) showed Among these performed with 14 lipases, and and four of them (PS (Burkholderia cepacia), PS-Cpositive (B. cepaciaresults. immobilized lipases, Arthrobacter wasniger), the only one (Arthrobacter that hydrolyzed all tested substrates. ABL was used on ceramics), sp. AS (ABL) (Aspergillus and ABL sp.)) showed positive results. Among these lipases, sp. (ABL) was the only one that hydrolyzed substrates. ABL was7.0, in the on the resolution ofArthrobacter four arylazetidiones derivatives (Scheme 1) all intested buffer solution pH used on the resolution of four arylazetidiones derivatives (Scheme 1) in buffer solution pH 7.0, in the presence of co-solvents such as acetonitrile (MeCN), dimethylformamide (DMF) or dimethylsulfoxide presence of co-solvents such as acetonitrile (MeCN), dimethylformamide (DMF) or dimethylsulfoxide (DMSO), (DMSO), and resulted in enantiomeric excess of both alcohols and acetates of >99% and conversions and resulted in enantiomeric excess of both alcohols and acetates of >99% and conversions close to 50% close[7]. to 50% [7].

Scheme 1. Kinetic enzymatic hydrolysis racemic arylazetidiones, precursors of amino side acid side Scheme 1. Kinetic enzymatic hydrolysis ofofracemic arylazetidiones, precursors of acid amino chain of Paclitaxel [7]. chain of Paclitaxel [7].

The compound (2R,3S)-3-phenylisoserineis isalso alsoa akey keyintermediate intermediate of side The compound (2R,3S)-3-phenylisoserine ofthe thePaclitaxel Paclitaxel side chain, chain, and it has been already produced by biocatalytical methods [8]. The racemate of and it hasethyl been already produced by biocatalytical methods [8]. The racemate of ethyl 3-amino-33-amino-3-phenyl-2-hydroxy-propionate (3-phenylisoserine ethyl ester) was synthesized and phenyl-2-hydroxy-propionate (3-phenylisoserine ethyl lipases ester) were wasscreened synthesized andresult resolved via resolved via lipase-mediated kinetic hydrolysis. Various and the best (c 50%, E kinetic > 200) was obtained with lipase from B. cepacia immobilized diatomaceous earth (PS lipase-mediated hydrolysis. Various lipases were screenedon and the best result (c IM). 50%, E > 200) After optimizing the reaction (diisopropyl ether (DIPE) 0.5 eq. H2 O as solvent, 50 ˝ C), was obtained with lipase fromconditions B. cepacia immobilized on with diatomaceous earth (PS IM). After 3 h, the (2R,3S)-3-amino-3-phenyl-2-hydroxy-propionate was obtained with 100% ee, c 50% and optimizing the reaction conditions (diisopropyl ether (DIPE) with 0.5 eq. H2O as solvent, 50 °C), 3 h, E > 200. Subsequent hydrolysis of this latter compound led to (2R,3S)-3-phenylisoserine (Scheme 2a). the (2R,3S)-3-amino-3-phenyl-2-hydroxy-propionate wastoobtained One advantage of this strategy is that it was not necessary protect thewith amino100% group ee, [9].c 50% and E > 200. Subsequent hydrolysis of this latter compound led to (2R,3S)-3-phenylisoserine (Scheme 2a). One advantage of this strategy is that it was not necessary to protect the amino group [9]. Another strategy to produce the (2R,3S)-3-phenylisoserine involved the enantioselective ring-cleavage of (3R*,4S*)-β-lactam (R=H) and its 3-acetoxy derivative (R=Ac) using CAL-B (lipase B from Candida antarctica produced by the submerged fermentation of a genetically modified Aspergillus oryzae 29683 and absorbed on a macroporous resin) as an enzyme at 60 °C (Scheme 2b). The kinetic hydrolyses were performed on gram-scale (1.0 g of substrate) after optimizing the reaction conditions for each starting material. The enantioselective ring-cleavage of the (3R*,4S*)-β-lactams (R=H) in tert-butylmethyl

Int. J. Mol. Sci. 2015, 16, 29682–29716

Another strategy to produce the (2R,3S)-3-phenylisoserine involved the enantioselective ring-cleavage of (3R*,4S*)-β-lactam (R=H) and its 3-acetoxy derivative (R=Ac) using CAL-B (lipase B from Candida antarctica produced by the submerged fermentation of a genetically modified Aspergillus oryzae and absorbed on a macroporous resin) as an enzyme at 60 ˝ C (Scheme 2b). The kinetic hydrolyses were performed on gram-scale (1.0 g of substrate) after optimizing the reaction conditions for each starting material. The enantioselective ring-cleavage of the (3R*,4S*)-β-lactams (R=H) in tert-butylmethyl ether (TBME) (with 0.5 eq. of water) yielded, after 18 h, the (3S,4R)-β-lactam Int. J. Mol. Sci. 2015, 16, page–page (>99% ee, 48% yield) and (2R,3S)-3-phenylisoserine (98% ee, 48% yield). These two compounds were also formed with high enantioselectivity (>98% ee) and good yields (43% and 49%, respectively) enantioselectivity (>98%was ee) performed and goodwith yields (43% and 49%, respectively) reaction was when the reaction the (3R*,4S*)-3-acetoxy-β-lactam (R=Ac).when In thisthe case, the performed withwas the DIPE (3R*,4S*)-3-acetoxy-β-lactam In this case, the was DIPE solvent (1.0 eq. H2 O) and, after 50 (R=Ac). h, the starting material wassolvent 100% converted into(1.0 theeq. H2O) enantiopure products (>98% ee) [10]. and, after 50 h, the starting material was 100% converted into the enantiopure products (>98% ee) [10].

Scheme Scheme 2. Synthesis of (2R,3S)-3-phenylisoserine, Paclitaxel chain, via 2. Synthesis of (2R,3S)-3-phenylisoserine,key keyintermediates intermediates ofofPaclitaxel sideside chain, enzymaticofhydrolysis of theethyl (a) racemic ethyl 3-amino-3-phenyl-2-hydroxypropionate [7] or enzymaticviahydrolysis the (a) racemic 3-amino-3-phenyl-2-hydroxypropionate [7] or (b) β-lactams [10]. (b) β-lactams [10].

2.2. Key Intermediate of Crizotinib

2.2. Key Intermediate of Crizotinib

An extensive screening among commercial lipases and esterases was carried out by researchers An extensive screening among commercial lipases and esterases was carried out by researchers at Agouron Pharmaceuticals for for thethe preparation (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol from at Agouron Pharmaceuticals preparation of of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol from its corresponding racemic ester ester (Scheme 3); this chiral intermediate be linked linkedwith withanan ether bond its corresponding racemic (Scheme 3); this chiral intermediate can can be ether bond 2-aminopyridine to different 2-aminopyridine compounds potently inhibit inhibit auto-phosphorylation of human to different compounds thatthat potently auto-phosphorylation of human heptocyte growth factor receptor. In addition, it is an intermediate on the preparation of the heptocyte growth factor receptor. In addition, it is an intermediate on the preparation of the potent potent antitumor compound Crizotinib [11]. Highly enantioselective hydrolysis (E > 100) was antitumor compound Crizotinib [11]. Highly enantioselective hydrolysis (E > 100) was observed with observed with different commercial lipases (CAL-B and Rhizopus delemar lipase, among the others), differentand commercial lipases (CAL-B and Rhizopus among the Then, others), and provided provided the (S)-ester and (R)-alcohol with eedelemar ranginglipase, from 80% to 97%. these two the (S)-ester and (R)-alcohol with ee ranging from 80% to 97%. Then, these two compounds compounds were not separated, and the mixture was subjected to mesylation reaction (conversionwere not of and the (R)-alcohol intowas its mesyl derivative), followed reaction by treatment with potassium to separated, the mixture subjected to mesylation (conversion of theacetate (R)-alcohol into exclusively yield the (S)-ester. Hydrolysis of this compound furnished the desired product its mesyl derivative), followed by treatment with potassium acetate to exclusively yield the (S)-ester. (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol in high yield [12]. Hydrolysis of this compound furnished the desired product (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol in high yield [12].

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different commercial lipases (CAL-B and Rhizopus delemar lipase, among the others), and provided the (S)-ester and (R)-alcohol with ee ranging from 80% to 97%. Then, these two compounds were not separated, and the mixture was subjected to mesylation reaction (conversion of the (R)-alcohol into its mesyl derivative), followed by treatment with potassium acetate to exclusively yield the (S)-ester. Hydrolysis this16, compound furnished the desired product (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol in Int. J. Mol. Sci.of2015, 29682–29716 high yield [12].

Int. J. Mol. Sci. 2015, 16, page–page Scheme Scheme 3. 3. Synthesis Synthesis of of (S)-1-(2,6-dichloro-3 (S)-1-(2,6-dichloro-3 fluorophenyl)ethanol fluorophenyl)ethanol via via kinetic kinetic enzymatic enzymatic hydrolysis hydrolysis of of the corresponding racemic ester [12].

2.3. Pregabalin Analogues the and corresponding racemic ester [12].

Pregabalin ((S)-3-(aminomethyl)-5-methylhexanoic acid, trade name Lyrica®) is a drug used in 2.3. Pregabalin and Analogues anticonvulsant therapy. Pregabalin and its analogues were obtained by hydrolysis of γ-nitro esters, ((S)-3-(aminomethyl)-5-methylhexanoic tradeHydrolysis name Lyricar drug used followed byPregabalin hydrogenation of the nitro group on3 Raneyacid, nickel. of) is thea γ-nitro esters can in anticonvulsant therapy. Pregabalin and its analogues were obtained by hydrolysis of γ-nitro esters, be effectively catalyzed by lipase Novozym 435, as seen in Scheme 4a, affording (S)-γ-nitro acids with followed by hydrogenation of the nitro group on Raney nickel. Hydrolysis of the γ-nitro esters can be moderate conversion (18%–24%) and ee ranging between 87% and 94% [13]. Additionally, effectively catalyzed by lipase Novozym 435, as seen in Scheme 4a, affording (S)-γ-nitro acids 2-carboxyethyl-3-cyano-5-methylhexanoic ethylbetween ester87%(CNDE) was Additionally, enantioselectively with moderate conversion (18%–24%) andacid ee ranging and 94% [13]. 2-carboxyethyl-3-cyano-5-methylhexanoic acidofethyl ester (CNDE) was enantioselectively hydrolyzed hydrolyzed as the key step in the preparation Pregabalin (Scheme 4b). This biotransformation was as the key step in the preparation of Pregabalin (Scheme 4b). This biotransformation was achieved achieved by using Thermomyces lanuginosus lipase (TLL), the enzyme contained in commercial by using Thermomyces lanuginosus lipase (TLL), the enzyme contained in commercial Lipolaser Lipolase® and Lipozyme rTL IM®. Recombinant lipase from T. lanuginosus DSM 10635 showed and Lipozyme TL IM . Recombinant lipase from T. lanuginosus DSM 10635 showed excellent excellent(S)-enantioselectivity (S)-enantioselectivity to CNDE (E > 200), but with low catalytic activity. The enzyme was to CNDE (E > 200), but with low catalytic activity. The enzyme was evolved by evolvedprotein by protein engineering, allowingof (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic the preparation of (3S)-2-carboxyethyl-3-cyano-5engineering, allowing the preparation acid with 42%acid conversion and 98% ee, starting from 255ee, g/L of substrate [14]. methylhexanoic with 42% conversion and 98% starting from 255 g/L of substrate [14].

SchemeScheme 4. Synthesis of Pregabalin through kinetic enzymatic of (a) rac-γ4. Synthesis of Pregabalinintermediates intermediates through kinetic enzymatic hydrolysishydrolysis of (a) rac-γ-nitro esters [13]and and (b) (b) rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl acid ester [14]. nitro esters [13] rac-2-carboxyethyl-3-cyano-5-methylhexanoic ethyl ester [14].

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Scheme 4. Synthesis of Pregabalin Int. J. Mol. Sci. 2015, 16, 29682–29716

intermediates through kinetic enzymatic hydrolysis of (a) rac-γnitro esters [13] and (b) rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester [14].

Scheme 5. Kinetic enzymatic hydrolysis of racemic methyl-1,4-benzodioxan-2-carboxylate, affording Scheme 5. Kinetic enzymatic hydrolysis of racemic methyl-1,4-benzodioxan-2-carboxylate, affording intermediates used in the synthesis of (S)-Prosimpal, (S)-Piperoxan, (S,S)-Dibozane and [15]. intermediates used in the synthesis of (S)-Prosimpal, (S)-Piperoxan, (S,S)-Dibozane and (S)-Doxazosin (S)-Doxazosin [15].

2.4. Prosimpal, Piperoxan, Dibozane and Doxazosin 2.4. Prosimpal, Piperoxan, Dibozane and Doxazosin

The drugs (S)-Prosimpal, (S)-Piperoxan and (S,S)-Dibozane are α-adrenergic receptor The drugs (S)-Prosimpal, (S)-Piperoxan and (S,S)-Dibozane are α-adrenergic receptor antagonists, antagonists, and (S)-Doxazosin is a drug used in the treatment of hypertension. All (S)-isomers are and (S)-Doxazosin is a drug used in the treatment of hypertension. All (S)-isomers are more effective more effective than the corresponding (R)-isomers. The aforementioned drugs were synthesized by than the corresponding (R)-isomers. The aforementioned drugs were synthesized by hydrolytic hydrolytic kinetic resolution of the racemic intermediate methyl-1,4-benzodioxan-2-carboxylate in kinetic resolution of the racemic intermediate methyl-1,4-benzodioxan-2-carboxylate in the presence the presence lipase from whole cells of wild species of Arthrobacter (ABL), as seen in Scheme 5. The of lipase of from whole cells of wild species of Arthrobacter (ABL), as seen in Scheme 5. The reactions were performed in two different conditions: (i) 0.1 M phosphate buffer with 20% DIPE as a co-solvent 4 and (ii) 0.1 M phosphate buffer with 20% of n-butanol as a co-solvent. In the first reaction condition, after 2 h of reaction, the corresponding (S)-carboxylic acid (79% ee) and the (R)-methyl ester (>99% ee) were obtained with c 55% and E-value of 33. In the second reaction condition, after 4 h, (S)-carboxylic acid (>99% ee) and (R)-methyl ester (73% ee) were obtained with c 42% and E > 200. The syntheses of the pharmaceuticals (S)-Prosimpal, (S)-Piperoxan and (S,S)-Dibozane were performed by using the (R)-methyl ester (>99% ee) obtained from the first reaction condition, while the (S)-carboxylic acid (>99% ee) obtained in the second condition was used in the synthesis of (S)-Doxazosin [15]. 2.5. Key Intermediate of Ezetimibe Ezetimibe is a drug used in the reduction of cholesterol and blood lipids. The synthesis of this drug requires the enantiopure 3-[5-(4-fluorophenyl)-5(S)-hydroxypentanoyl]-4(S)-4-phenyl-1, 3-oxazolidin-2-one ((S)-FOP alcohol) as a key intermediate. Kinetic resolution of the diastereoisomeric mixture of FOP acetates was assessed using several commercial lipases; the most efficient lipase was from Candida rugosa (CRL) and the best reaction conditions were buffer solution (pH 7.0) containing 30% of DIPE as a co-solvent, at 40 ˝ C, and an enzyme:substrate (w/w) ratio of 2.5:1. In such conditions, after reaching 50% conversion, it was possible to obtain (S)-FOP acetate with diastereomeric excess (de) 98.5% (Scheme 6). The same results were obtained when the method was applied to scale-up, starting from 1 g of the diastereoisomeric mixture of FOP acetates [16].

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mixture of FOP acetates was assessed using several commercial lipases; the most efficient lipase was from Candida rugosa (CRL) and the best reaction conditions were buffer solution (pH 7.0) containing 30% of DIPE as a co-solvent, at 40 °C, and an enzyme:substrate (w/w) ratio of 2.5:1. In such conditions, after reaching 50% conversion, it was possible to obtain (S)-FOP acetate with diastereomeric excess Int. Mol. Sci.(Scheme 2015, 16, 29682–29716 (de)J. 98.5% 6). The same results were obtained when the method was applied to scale-up, starting from 1 g of the diastereoisomeric mixture of FOP acetates [16].

Scheme 6. 6. Kinetic mixture of of FOP FOP acetates acetates to to produce produce the the Scheme Kinetic enzymatic enzymatic hydrolysis hydrolysis of of diastereoisomeric diastereoisomeric mixture (S)-FOP alcohol used in the synthesis of the drug Ezetimibe [16]. (S)-FOP alcohol used in the synthesis of the drug Ezetimibe [16].

2.6. Key Intermediate of Hepatitis C Virus Protease Inhibitor 2.6. Key Intermediate of Hepatitis C Virus Protease Inhibitor The chiral compound trans-alkyl (1R,2S)-1-(tert-butoxycarbonylamino)-2-vinylcyclopropanecarboxylate The chiral compound trans-alkyl (1R,2S)-1-(tert-butoxycarbonylamino)-2-vinylcyclopropanecarboxylate is an important intermediate for the synthesis of certain Hepatitis C virus protease inhibitors. Initially, is an important intermediate for the synthesis of certain Hepatitis C virus protease inhibitors. rac-dialkyl 2-vinyl-1,1-cyclopropanedicarboxylate was submitted to a kinetic resolution in the Initially, rac-dialkyl 2-vinyl-1,1-cyclopropanedicarboxylate was submitted to a kinetic resolution presence of various hydrolytic enzymes including lipases (lipase OF from Candida rugosa; lipase from in the presence of various hydrolytic enzymes including lipases (lipase OF from Candida rugosa; lipase Mucor miehei; lipase OF from C. rugosa (formerly C. cylindracea); and lipase N from Rhizopus niveus). from Mucor miehei; lipase OF from C. rugosa (formerly C. cylindracea); and lipase N from Rhizopus niveus). The reactions were performed in phosphate buffer, pH 7.0, at 28 °C. As results, the remaining dialkyl The reactions were performed in phosphate buffer, pH 7.0, at 28 ˝ C. As results, the remaining dialkyl (2R)-2-vinylcyclopropane-1,1-dicarboxylate and the hydrolysis product, the 1-alcoxycarbonyl-2(2R)-2-vinylcyclopropane-1,1-dicarboxylate and the hydrolysis product, the 1-alcoxycarbonylvinylcyclopropane-1-carboxylic acid, were obtained with diastereomeric excess values (de) >90% in 2-vinylcyclopropane-1-carboxylic acid, were obtained with diastereomeric excess values (de) >90% favor of the cis-isomer (ester/vinyl group), and enantiomeric excess values (ee) ranging from 70% to in favor of the cis-isomer (ester/vinyl group), and enantiomeric excess values (ee) ranging from 70% >99% in favor of the cis-(1S,2S) enantiomer (Scheme 7). After a few chemical steps, it was possible to to >99% in favor of the cis-(1S,2S) enantiomer (Scheme 7). After a few chemical steps, it was possible obtain the key chiral intermediate for the synthesis of the virus protease inhibitors of Hepatitis C, to obtain the key chiral intermediate for the synthesis of the virus protease inhibitors of Hepatitis C, trans-alkyl (1R,2S)-1-(tert-butoxycarbonylamino)-2-vinylcyclopropanecarboxylate [17]. trans-alkyl (1R,2S)-1-(tert-butoxycarbonylamino)-2-vinylcyclopropanecarboxylate [17].

Int. J. Mol. Sci. 2015, 16, page–page

5 Scheme Scheme 7. 7. Kinetic Kinetic enzymatic enzymatic hydrolysis hydrolysis of of dialkyl dialkyl 2-vinyl-1,1-cyclopropanedicarboxylate 2-vinyl-1,1-cyclopropanedicarboxylate to to produce produce the cis-(1S,2S)-1-alcoxycarbonyl-2-vinylcyclopropane-1-carboxylic acid used to obtain the cis-(1S,2S)-1-alcoxycarbonyl-2-vinylcyclopropane-1-carboxylic acid used to obtain the the key key intermediate in the the synthesis synthesis of of the the virus virus protease protease inhibitors inhibitors of of Hepatitis Hepatitis C C [17]. [17]. intermediate in

2.7. 2.7. Naproxen Naproxen In been reported concerning thethe application of In the the last last few few years, years,aanumber numberofofexamples exampleshave have been reported concerning application immobilized lipases for achieving highly efficient stereoselective hydrolysis of esters of pharmaceutical interest. of immobilized lipases for achieving highly efficient stereoselective hydrolysis of esters of A classic example of the use of immobilized lipase in hydrolysis is the obtainment of (S)pharmaceutical interest. Naproxen ((S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid) from racemic Naproxen methyl ester. A classic example of the use of immobilized lipase in hydrolysis is the obtainment of (S)-Naproxen belongs to a class of non-steroidal anti-inflammatory drugs and their activity is 28-fold (S)-Naproxen ((S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid) from racemic Naproxen methyl ester. higher than the corresponding (R)-enantiomer. This drug was prepared from enzymatic hydrolysis (S)-Naproxen belongs to a class of non-steroidal anti-inflammatory drugs and their activity is 28-fold of racemic Naproxen methyl ester. The kinetic resolution was carried out in the presence of lipase higher than the corresponding (R)-enantiomer. This drug was prepared from enzymatic hydrolysis of from Candida rugosa immobilized on Amberlite XAD7 (CRL type VII). The best results were obtained racemic Naproxen methyl ester. The kinetic resolution was carried out in the presence of lipase from at 45 °C in an aqueous phase/isooctane biphasic batch system, at pH 6.0, a lipase load of 800 U/mL Candida rugosa immobilized on Amberlite XAD7 (CRL type VII). The best results were obtained at and˝ a substrate concentration of 10 mg/mL. Under these conditions, a conversion of 49% and an 45 C in an aqueous phase/isooctane biphasic batch system, at pH 6.0, a lipase load of 800 U/mL and E-value of 174.2 were reached (Scheme 8) [18]. The same biotransformation was improved by using nanoparticles as additives for the encapsulation of the enzyme via the sol–gel method. The encapsulated lipase showed outstanding 29687 enantioselectivity, with an E-value ranging from 265 to 371 and >98% ee depending on the sol–gel encapsulation process employed [19].

A classic example of the use of immobilized lipase in hydrolysis is the obtainment of (S)Naproxen ((S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid) from racemic Naproxen methyl ester. (S)-Naproxen belongs to a class of non-steroidal anti-inflammatory drugs and their activity is 28-fold higher than the corresponding (R)-enantiomer. This drug was prepared from enzymatic hydrolysis of methyl ester. The kinetic resolution was carried out in the presence of lipase Int.racemic J. Mol. Sci.Naproxen 2015, 16, 29682–29716 from Candida rugosa immobilized on Amberlite XAD7 (CRL type VII). The best results were obtained at 45 °C in an aqueous phase/isooctane biphasic batch system, at pH 6.0, a lipase load of 800 U/mL a substrate concentration of 10 mg/mL. UnderUnder these conditions, a conversion of 49% of and49% an E-value and a substrate concentration of 10 mg/mL. these conditions, a conversion and an of 174.2 were reached (Scheme 8) [18]. E-value of 174.2 were reached (Scheme 8) [18]. The same same biotransformation biotransformation was was improved improved by additives for for the the The by using using nanoparticles nanoparticles as as additives encapsulation of the enzyme via the sol–gel method. The encapsulated lipase showed outstanding encapsulation of the enzyme via the sol–gel method. The encapsulated lipase showed outstanding enantioselectivity, with ee depending depending on on the the sol–gel sol–gel enantioselectivity, with an an E-value E-value ranging ranging from from 265 265 to to 371 371 and and >98% >98% ee encapsulation process encapsulation process employed employed [19]. [19].

Scheme 8. Synthesis of (S)-Naproxen through kinetic enzymatic hydrolysis of the racemic Naproxen Scheme 8. Synthesis of (S)-Naproxen through kinetic enzymatic hydrolysis of the racemic Naproxen methyl ester [18]. methyl ester [18].

2.8. Key Intermediate of Prostaglandins, Prostacyclins and Thromboxane 2.8. Key Intermediate of Prostaglandins, Prostacyclins and Thromboxane The compound (1S,4R)-4-hydroxycyclopent-2-enyl acetate is an important intermediate in the The compound (1S,4R)-4-hydroxycyclopent-2-enyl acetate is an important intermediate in the synthesis of cyclopentenoid molecules with important biological activity, such as prostaglandins, synthesis of cyclopentenoid molecules with important biological activity, such as prostaglandins, prostacyclins and thromboxane. Enzymatic hydrolysis of meso-cyclopent-2-en-1,4-diacetate may give prostacyclins and thromboxane. Enzymatic hydrolysis of meso-cyclopent-2-en-1,4-diacetate may give access to (1S,4R)-4-hydroxycyclopent-2-enyl acetate, taking advantage of the so-called meso-trick. access to (1S,4R)-4-hydroxycyclopent-2-enyl acetate, taking advantage of the so-called meso-trick. Lipases from Pseudomonas fluorescens (PFL) and Candida rugosa (CRL) were immobilized by physical Lipases from Pseudomonas fluorescens (PFL) and Candida rugosa (CRL) were immobilized by physical adsorption or by chemical functionalization on core-shell superparamagnetic nanoparticles, and their adsorption or by chemical functionalization on core-shell superparamagnetic nanoparticles, and their performances were compared with the ones of the free enzymes. The biotransformations were performances were compared with the ones of the free enzymes. The biotransformations were performed in a two-liquid-phase system composed with 80% hexane and 20% water under mild performed in a two-liquid-phase system composed with 80% hexane and 20% water under mild end-over-end rotation. CRL was poorly enantioselective, while free and immobilized PFL afforded end-over-end rotation. CRL was poorly enantioselective, while free and immobilized PFL afforded enantiopure (1S,4R)-4-hydroxycyclopent-2-enyl acetate (Scheme 9). The re-use of the differently enantiopure (1S,4R)-4-hydroxycyclopent-2-enyl acetate (Scheme 9). The re-use of the differently nanoparticle-immobilized PFL showed that the activity of physically adsorbed PFL decreased more nanoparticle-immobilized PFL showed that the activity of physically adsorbed PFL decreased more than 50% after two cycles, whereas the chemically immobilized enzyme was much more stable [20]. than 50% after two cycles, whereas the chemically immobilized enzyme was much more stable [20]. Int. J. Mol. Sci. 2015, 16, page–page

6 Scheme 9. Desymmetrization of meso-cyclopent-2-en-1,4-diacetate by enzymatic hydrolysis [20]. Scheme 9. Desymmetrization of meso-cyclopent-2-en-1,4-diacetate by enzymatic hydrolysis [20].

2.9. Ketoprofen 2.9. Ketoprofen Only the (S)-enantiomer of Ketoprofen (2-(3-benzoylphenyl)propionic acid) is therapeutically Only the (S)-enantiomer of Ketoprofen (2-(3-benzoylphenyl)propionic acid) is therapeutically relevant as a nonsteroidal anti-inflammatory drug (NSAID). Kinetic resolution of racemic Ketoprofen relevant as a nonsteroidal anti-inflammatory drug (NSAID). Kinetic resolution of racemic Ketoprofen vinyl ester was obtained by enzymatic hydrolysis using a lipase from Aspergillus terreus immobilized vinyl ester was obtained by enzymatic hydrolysis using a lipase from Aspergillus terreus immobilized on modified alginate and cyclodextrin hollow spheres [21]. Under optimized conditions (enzyme on modified alginate and cyclodextrin hollow spheres [21]. Under optimized conditions (enzyme immobilized on Alg-g-PEG/α-CD hollow spheres used in acetone/water 80/20, pH 7.4, at 30 °C), the immobilized on Alg-g-PEG/α-CD hollow spheres used in acetone/water 80/20, pH 7.4, at 30 ˝ C), biotransformation was enantioselective (E-value of 129), furnishing (R)-Ketoprofen (96% ee at 46% the biotransformation was enantioselective (E-value of 129), furnishing (R)-Ketoprofen (96% ee at conversion). Under the same conditions, free A. terreus lipase gave only 16% conversion with low 46% conversion). Under the same conditions, free A. terreus lipase gave only 16% conversion with enantioselectivity (E-value of 11.4), as seen in Scheme 10a and Table 1. Notably, immobilized low enantioselectivity (E-value of 11.4), as seen in Scheme 10a and Table 1. Notably, immobilized A. terreus lipase was continuously used up to 20 cycles with minimal loss of activity, whereas the free A. terreus lipase was continuously used up to 20 cycles with minimal loss of activity, whereas the free enzyme cannot be recycled. enzyme cannot be recycled. A new approach to resolve racemic Ketoprofen vinyl ester was developed after comparison of the performances of different lipases (from Mucor javanicus, Rhizomucor miehei, Candida rugosa and Pseudomonas cepacia) employed both as free enzymes or immobilized in micro-emulsion-based organogels 29688 (MBGs) [22]. The reactions were conducted in the presence of DIPE at 30 °C. The bioconversion with free lipases generally provided low conversions (maximum 12%) compared with the immobilized lipases (maximum yield 49%). Immobilized Mucor javanicus lipase (MJL) exhibited the highest

immobilized on Alg-g-PEG/α-CD hollow spheres used in acetone/water 80/20, pH 7.4, at 30 °C), the biotransformation was enantioselective (E-value of 129), furnishing (R)-Ketoprofen (96% ee at 46% conversion). Under the same conditions, free A. terreus lipase gave only 16% conversion with low enantioselectivity (E-value of 11.4), as seen in Scheme 10a and Table 1. Notably, immobilized Int. J. Mol. Sci. 2015, 16, 29682–29716 A. terreus lipase was continuously used up to 20 cycles with minimal loss of activity, whereas the free enzyme cannot be recycled. new approachtotoresolve resolveracemic racemicKetoprofen Ketoprofenvinyl vinylester esterwas wasdeveloped developed after after comparison comparison of AA new approach of the performances of different lipases (from Mucor javanicus, Rhizomucor miehei, Candida rugosa the performances of different lipases (from Mucor javanicus, Rhizomucor miehei, Candida rugosa and and Pseudomonas employed bothenzymes as free enzymes or immobilized in micro-emulsion-based Pseudomonas cepacia)cepacia) employed both as free or immobilized in micro-emulsion-based organogels organogels (MBGs) [22]. were The conducted reactions were conducted the presence DIPE at 30 ˝ C. The (MBGs) [22]. The reactions in the presence in of DIPE at 30 °C.ofThe bioconversion with bioconversion with free lipases generally provided(maximum low conversions with free lipases generally provided low conversions 12%) (maximum compared 12%) with compared the immobilized the immobilized yield 49%).Mucor Immobilized Mucor javanicus (MJL) the exhibited lipases (maximumlipases yield (maximum 49%). Immobilized javanicus lipase (MJL)lipase exhibited highest the highest enantioselectivity (E > 200), whereas immobilized Rhizomucor miehei lipase (RML) a enantioselectivity (E > 200), whereas immobilized Rhizomucor miehei lipase (RML) displayed displayed a moderate (35), but proved to betotolerant to organic various organic solvents as DIPE, moderate E-value (35), E-value but proved to be tolerant various solvents such assuch DIPE, TBME, TBME, tetrahydrofuran (THF), 2-MeTHF, 1,4-dioxane, acetone and MeCN. Moreover, immobilized tetrahydrofuran (THF), 2-MeTHF, 1,4-dioxane, acetone and MeCN. Moreover, immobilized RML RML was very thermostable up to 50 ˝ C. It is noteworthy that immobilized RML and MJL showed was very thermostable up to 50 °C. It is noteworthy that immobilized RML and MJL showed high high activity over 30 cycles and maintained the same initial enantioselectivity. RML immobilized activity over 30 cycles and maintained the same initial enantioselectivity. RML immobilized in microin micro-emulsion-based organogels was employed for a 5-g-scale kinetic resolution of Ketoprofen emulsion-based organogels was employed for a 5-g-scale kinetic resolution of Ketoprofen vinyl ester, vinyl ester, furnishing the desired product (91% ee) in 47% yield after 72 h (Scheme 10b and Table 1). furnishing the desired product (91% ee) in 47% yield after 72 h (Scheme 10b and Table 1). The authors The authors highlight the advantages of biotransformations catalyzed by immobilized enzymes in highlight biotransformations catalyzed byenzymatic immobilized enzymes in the form the formthe of advantages MBGs; theseofadvantages include an improved efficiency (conversion and of MBGs; these advantages include an enzymatic efficiency (conversion and enantioselectivity), excellent reusability, lowimproved enzyme loading, and enhanced resistance to organic enantioselectivity), excellent reusability, low enzyme loading, and enhanced resistance to organic solvents. Long-term resistance to high concentrations of organic solvents is a crucial feature in solvents. Long-termreactions, resistance to high concentrations of organic solventswater-soluble is a crucial feature in lipase-catalyzed lipase-catalyzed especially when working with poorly substrates. Studies reactions, especially when working with poorly water-soluble substrates. Studies on the effect on the effect of interfacial composition on lipase activity in two-liquid phase systems revealed that of interfacial in the two-liquid phase systems revealed substrate substrate composition inaccessibilityon is alipase majoractivity reason for low activity of lipase in the absencethat of organic inaccessibility is a major reason for the low activity of lipase in the absence of organic solvents [23]. solvents [23].

Scheme 10. Kinetic enzymatic hydrolysis of racemic Ketoprofen vinyl ester, a member of the class of Scheme 10. Kinetic enzymatic hydrolysis of racemic Ketoprofen vinyl ester, a member of the class nonsteroidal anti-inflammatory drugs (NSAIDs), using lipases immobilized on (a) Alg-g-PEG/α-CD [21] of nonsteroidal anti-inflammatory drugs (NSAIDs), using lipases immobilized on (a) Alg-g-PEG/ or α-CD in (b)[21] micro-emulsion-based organogelsorganogels (MBGs) [22]. or in (b) micro-emulsion-based (MBGs) [22]. Table 1. Enzymes and conditions used on the kinetic enzymatic hydrolysis of rac-Ketoprofen vinyl ester by methods a and b. Method a b

Enzyme

Conditions

lipase from A. terreus immobilized7on Alg-g-PEG/α-CD, hollow spheres MJL MBGs or RML MBGs

acetone/water (80/20, v/v), pH 7.4, 30 ˝ C, 4 days DIPE, 30 ˝ C, 72 h

Reference [21] [22]

2.10. Dihydropyridine Derivatives The 1,4-Dihydropyridines (1,4-DHPs) are recognized as pharmacophores widely used in clinics for the treatment of cardiovascular diseases. A series of racemic 1,4-DHPs were prepared through several chemical steps including a multicomponent Hantzsch process and a Vilsmeir-Haack reaction. These 1,4-DHPs were subjected to enzymatic kinetic resolution via hydrolysis in the presence of lipases in water saturated with organic solvent [24]. Reactions were carried out until conversions next to 50% and several lipases (such as from Candida antarctica A (CAL-A, NZL-101), porcine pancreatic lipase (PPL), from C. antarctica B (CAL-B, Novozym 435) and from C. rugosa (CRL, type VII)) were assayed. The best results were obtained in the presence of CRL or CAL-B depending on the aryl group and the organic solvent. For reaction systems containing CRL and EtOAc as a solvent, the highest 29689

several chemical steps including a multicomponent Hantzsch process and a Vilsmeir-Haack reaction. These 1,4-DHPs were subjected to enzymatic kinetic resolution via hydrolysis in the presence of lipases in water saturated with organic solvent [24]. Reactions were carried out until conversions next to 50% and several lipases (such as from Candida antarctica A (CAL-A, NZL-101), porcine pancreatic lipase (PPL), from antarctica B (CAL-B, Novozym 435) and from C. rugosa (CRL, type VII)) were Int. J. Mol. Sci. 2015, 16, C. 29682–29716 assayed. The best results were obtained in the presence of CRL or CAL-B depending on the aryl group and the organic solvent. For reaction systems containing CRL and EtOAc as a solvent, the highest enantioselectivity values enantioselectivity values (E) (E) were were obtained obtained with with 1,4-DHPs 1,4-DHPs containing containing aryl aryl groups groupsas as2-NO 2-NO22–C –C66H H44,, naphtyl and 2-Cl–5-NO –C H with E-values of 103, 170 and >200, respectively. The ee values of the naphtyl and 2-Cl–5-NO22–C66H33with E-values of 103, 170 and >200, respectively. The ee values of the remaining ester while the the ee ee of of the the carboxylic carboxylic acid acid product product ranged ranged remaining ester were were in in the the range range of of 84% 84% to to >99% >99% while from 94% to 98%. It is noteworthy that some of the carboxylic acid products had the enantiomeric from 94% to 98%. It is noteworthy that some of the carboxylic acid products had the enantiomeric excess increased Applying this this methodology, methodology, ee ee values values of of the the carboxylic carboxylic acid acid excess increased by by crystallization. crystallization. Applying products containing containing aryl aryl groups groups (i.e., (i.e., 3-NO 3-NO22–C –C66HH4;4 Ph; ; Ph;and and4-NO 4-NO enhanced from 88% 2 –C 64H 4 ) were products 2–C 6H ) were enhanced from 88% to to 97%; 60% to 92%; and 69% to 95%, respectively. In the presence of CAL-B and EtOAc as a solvent, 97%; 60% to 92%; and 69% to 95%, respectively. In the presence of CAL-B and EtOAc as a solvent, the the best result obtained a 1,4-DHP-containing aryl group as3O–C 3-CH an 3 O–C 6H 4 with best result was was obtained with with a 1,4-DHP-containing aryl group such assuch 3-CH 6H 4 with an E-value E-value of 63, c 34%, 49% ee to the remaining ester and 95% ee to the carboxylic acid product. In order of 63, c 34%, 49% ee to the remaining ester and 95% ee to the carboxylic acid product. In order to obtain to obtain the remaining substrate with high ee, of hydrolysis 1,4-DHPsaryl containing aryl as groups such the remaining substrate with high ee, hydrolysis 1,4-DHPsof containing groups such 4-Br–C 6H4 as 4-Br–C H4 and with CAL-B TBME as a solvent affording 57% 3 O–C6 H 4 was and 3-CH63O–C 6H4 3-CH was carried out withcarried CAL-Bout and TBME as a and solvent affording 57% and 54% molar and 54% molar conversion, respectively. In this case, remaining substrates (aryl = 4-Br–C H4 ) 6and conversion, respectively. In this case, remaining substrates (aryl = 4-Br–C 6H4) and (aryl = 3-CH63O–C H4) (aryl = 3-CH O–C H ) were obtained with high ee, 97% and 94%, respectively (Scheme 11). 3 with 6 high 4 were obtained ee, 97% and 94%, respectively (Scheme 11).

Scheme Scheme 11. 11. Kinetic Kinetic enzymatic enzymatic hydrolysis hydrolysis of of racemic racemic 1,4-DHPs 1,4-DHPs to to obtain obtain the the chiral chiral pharmacophores pharmacophores used in clinics for the treatment of cardiovascular diseases [24]. used in clinics for the treatment of cardiovascular diseases [24].

3. Esterification Approach 3. Esterification Approach As known to most of the researchers working in this area, lipases can efficiently catalyze the As known to most of the researchers working in this area, lipases can efficiently catalyze stereoselective acylation of alcohols when the reaction is performed under low water activity the stereoselective acylation of alcohols when the reaction is performed under low water activity conditions. This opportunity can be exploited for achieving the highly regio- or stereoselective conditions. This opportunity can be exploited for achieving the highly regio- or stereoselective preparation of esters with pharmaceutical relevance under mild and controlled conditions. Examples preparation of esters with pharmaceutical relevance under mild and controlled conditions. Examples span from structurally simple drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), to span from structurally simple drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), to multifunctional complex molecules, such as immunosuppressive agents (i.e., Rapamycin or multifunctional complex molecules, such as immunosuppressive agents (i.e., Rapamycin or Pimecrolimus), Pimecrolimus), where regioselectivity is a major issue. where regioselectivity is a major issue. 3.1. Rapamycin

8 A brilliant example of regioselectivity observed in lipase-catalyzed acylation is the synthesis of the proline analogue of Rapamycin dihydroxyesters, where the chemoenzymatic approach allowed the preparation of the desired molecule in only two steps [25]. Identification of a suitable activated ester of the 2,2-bis(hydroxymethyl) propionic acid side chain was crucial for performing the lipase-catalyzed acylation; vinyl esters provided the highest activity and the best yield, allowing for preparative synthesis. Immobilized lipase PS-C “Amano” II was the best biocatalyst, displaying optimal activity in anhydrous TBME (Scheme 12).

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the proline analogue of Rapamycin dihydroxyesters, where the chemoenzymatic approach allowed the preparation of the desired molecule in only two steps [25]. Identification of a suitable activated ester of the 2,2-bis(hydroxymethyl) propionic acid side chain was crucial for performing the lipase-catalyzed acylation; vinyl esters provided the highest activity and the best yield, allowing for preparative synthesis. Immobilized lipase PS-C “Amano” II was the best biocatalyst, displaying optimal activity Int. J. Mol. Sci. 2015, 16, 29682–29716 in anhydrous TBME (Scheme 12).

Scheme 12. Chemoenzymatic synthesis of proline analogue of Rapamycin dihydroxyester [25].

3.2. Pimecrolimus Pimecrolimus (32-epi-chloro-derivative of Ascomycyn) is a macrolide which has anti-inflammatory, Scheme 12. Chemoenzymatic synthesis analogueofofRapamycin Rapamycin dihydroxyester Scheme 12. Chemoenzymatic synthesisof ofproline proline analogue dihydroxyester [25]. [25]. antiproliferative and immunosuppressive properties, used in the topical treatment of inflammatory 3.2. Pimecrolimus skin diseases. This substance is present as an active ingredient in the drug marketed as Elidel®. The 3.2. Pimecrolimus first chemoenzymatic synthesis of Pimecrolimus was performed from Ascomycin, a substance Pimecrolimus (32-epi-chloro-derivative macrolidewhich which anti-inflammatory, Pimecrolimus (32-epi-chloro-derivativeof ofAscomycyn) Ascomycyn) isisaamacrolide hashas anti-inflammatory, isolatedantiproliferative from the fermentation broth of Streptomyces hygroscopicus. Ascomycin has two hydroxyl antiproliferative immunosuppressive properties, used ininthe treatment of inflammatory andand immunosuppressive properties, used thetopical topical treatment of inflammatory ®. The skin diseases. This substance is present as active ingredientininthe thedrug drug marketed marketed Elidel diseases. substance is present as an an active as and Elidelr groups skin attached toThis secondary carbons, one in theingredient cyclohexane moiety (C-32)as the other in The first chemoenzymatic synthesis of Pimecrolimus wasperformed performed from Ascomycin, a substance first chemoenzymatic synthesis of Pimecrolimus was from Ascomycin, a substance macrolactam cycle (C-24). In this synthesis, it was necessary to acetylate the hydroxyl specifically isolated from fermentationbroth broth of of Streptomyces Streptomyces hygroscopicus. hashas twotwo hydroxyl from thethe fermentation hygroscopicus.Ascomycin Ascomycin hydroxyl locatedisolated in cyclohexane The strategy wasinbased on the regioselectivity lipase Candida groups attachedmoiety. to secondary carbons, one the cyclohexane moiety (C-32) andofthe other from in groups attached to secondary carbons, one in the cyclohexane moiety (C-32) and the other in cycle (C-24).435) In this synthesis, it was necessary to acetylate thehydroxyl hydroxyl specifically antarctica Bmacrolactam (CAL-B, Novozym with regioselective acylation of the group in question, macrolactam cycle (C-24). In this synthesis, it was necessary to acetylate the hydroxyl specifically located in cyclohexane moiety. The strategy was based on the regioselectivity of lipase from 94% yield in the presence of vinyl acetate leading to the theregioselectivity corresponding acetate with located in cyclohexane moiety.and The toluene, strategy was based on of lipase from Candida Candida antarctica B (CAL-B, Novozym 435) with regioselective acylation of the hydroxyl group (Scheme 13). Then, the 32-monoacetate derivative was subjected to silylation with trimethylsilyl antarctica B (CAL-B, Novozym withacetate regioselective acylation of the hydroxyl group inacetate question, in question, in the presence435) of vinyl and toluene, leading to the corresponding in the presence of vinyl acetate and toluene, leading to the corresponding acetate with 94% trifluoromethanesulfonate (TBMSOTf), leading to a 24-silyloxy-32-monoacetate The latter with 94% yield (Scheme 13). Then, the 32-monoacetate derivative was subjected derivative. to silylationyield (Scheme 13). Then, the 32-monoacetate derivative was subjected to silylation with trimethylsilyl with trimethylsilyl trifluoromethanesulfonate to apresence 24-silyloxy-32-monoacetate was subjected to a regioselective alcoholysis (TBMSOTf), (n-octanol)leading in the of a CAL-B-producing trifluoromethanesulfonate (TBMSOTf), to a 24-silyloxy-32-monoacetate derivative. Theoflatter32 with derivative. The latter was subjected leading to a regioselective alcoholysis (n-octanol) in the presence 24-silyloxy-32-hydroxy derivative. After the steps of introducing the chlorine atom at position a CAL-B-producing 24-silyloxy-32-hydroxy derivative. After the steps of introducing the chlorine was subjected to a regioselective alcoholysis (n-octanol) in the presence of a CAL-B-producing polymer-bound and deprotection of theand silyl group atofposition 24, Pimecrolimus atom attriphenyl position 32phosphine with polymer-bound triphenyl deprotection theatsilyl group atwith 24-silyloxy-32-hydroxy derivative. After the steps ofphosphine introducing the chlorine atom position 32 was obtained with an overall yield of 29% [26,27]. position 24, Pimecrolimus was obtained with an overall yield of 29% [26,27]. polymer-bound triphenyl phosphine and deprotection of the silyl group at position 24, Pimecrolimus was obtained with an overall yield of 29% [26,27].

Scheme 13. Synthesis of Pimecrolimus via regioselective enzymatic acylation of Ascomycin [26,27].

Scheme 13.Scheme Synthesis of Pimecrolimus viavia regioselective enzymatic acylation of Ascomycin [26,27]. 13. Synthesis of Pimecrolimus regioselective enzymatic acylation of Ascomycin [26,27]. 9

9 29691

Int. J. Mol. Sci. 2015, 16, page–page Int. J. Mol. Sci. 2015, 16, 29682–29716

3.3. Loxoprofen Int. J. Mol. Sci. 2015, 16, page–page A considerable number of biocatalytic routes have been developed so far for the production of 3.3. Loxoprofen 3.3. Loxoprofen (S)-profens; nevertheless, more efficient and sustainable processes are needed due to the commercial A the production production of A considerable considerable number number of of biocatalytic biocatalytic routes routes have have been been developed developed so so far far for for the of importance of these drugs. more Loxoprofen, an (S)-2-(4-(R)-2-oxo–cyclopentylmethyl)phenyl propionic (S)-profens; nevertheless, efficient and sustainable processes are needed due to the commercial (S)-profens; nevertheless, more efficient and sustainable processes are needed due to the commercial acid,importance is a NSAI drug belongingLoxoprofen, to the group of propionic acid derivatives with anti-inflammatory, an (S)-2-(4-(R)-2-oxo–cyclopentylmethyl)phenyl (S)-2-(4-(R)-2-oxo–cyclopentylmethyl)phenyl propionic importance of of these these drugs. drugs. Loxoprofen, an propionic analgesic and antipyretic activities. In fact, Loxoprofen is a pro-drug because its active form is the acid, is a NSAI drug belonging to the group of propionic acid derivatives with anti-inflammatory, acid, is a NSAI drug belonging to the group of propionic acid derivatives with anti-inflammatory, corresponding trans-alcohol metabolite. The preparation Loxoprofen involved theform enzymatic analgesic and antipyretic activities. activities. fact, Loxoprofen a pro-drug because its active is analgesic and antipyretic InInfact, Loxoprofen is ais of pro-drug because its active form is the the corresponding trans-alcohol metabolite. preparation Loxoprofeninvolved involved the the enzymatic enzymatic kinetic resolution oftrans-alcohol the racemicmetabolite. alcohol 2-(p-{[(p-methoxy phenyl)methoxy]methyl}phenyl)propanol corresponding TheThe preparation ofofLoxoprofen kinetic resolution of the racemic alcohol 2-(p-{[(p-methoxy phenyl)methoxy]methyl}phenyl)propanol via transesterification reaction the 2-(p-{[(p-methoxy presence of lipase from Burkholderia cepacia (lipase-PS), kinetic resolution of the racemicin alcohol phenyl)methoxy]methyl}phenyl)propanol via reaction lipase from cepacia molecular via transesterification transesterification reaction in presence the presence ofacetate lipaseBurkholderia from Burkholderia cepacia (lipase-PS), molecular sieves 4 Å, in DIPE asina the solvent andofvinyl as an acyl donor(lipase-PS), (Scheme 14). After 12 h, sieves 4 Å, in DIPE as a solvent and vinyl acetate as an acyl donor (Scheme 14). After 12 h, was molecular sieves 4 Å, in DIPE as a solvent and vinyl acetate as an acyl donor (Scheme 14). After h, the it was possible to obtain the corresponding (S)-acetate in 98% enantiomeric excess (ee)it12 and possible to obtain the corresponding (S)-acetate in 98% enantiomeric excess (ee) and the (R)-alcohol it was possible the corresponding (S)-acetate in 98%with enantiomeric excess (ee) and the (R)-alcohol in 94% to ee.obtain The synthesis of Loxoprofen followed (S)-acetate having the desired in 94% ee. The of Loxoprofen with (S)-acetate having the desired configuration of (R)-alcohol in synthesis 94% ee. The synthesis offollowed Loxoprofen followed with (S)-acetate having the desired configuration of the drug [28]. the drug [28]. of the drug [28]. configuration

Scheme Enzymatickinetic kinetic resolution resolution of of the phenyl) Scheme 14. 14. Enzymatic the racemic racemic alcohol alcohol2-(p-{[(p-methoxy 2-(p-{[(p-methoxy phenyl) Scheme 14. Enzymatic kinetic resolution of the racemic alcohol 2-(p-{[(p-methoxy phenyl) methoxy]methyl}phenyl)propanol producethe the(S)-acetate (S)-acetate used of the drug Loxoprofen [28]. [28]. methoxy]methyl}phenyl)propanol toto produce usedininthe thesynthesis synthesis of the drug Loxoprofen

methoxy]methyl}phenyl)propanol to produce the (S)-acetate used in the synthesis of the drug Loxoprofen [28]. 3.4. Flurbiprofen

3.4. Flurbiprofen

(S)-Flurbiprofen is a NSAI drug, whereas its enantiomer was found to inhibit tumor growth in 3.4. Flurbiprofen (S)-Flurbiprofen is a NSAI drug, whereas its enantiomer was found to inhibit tumor growth in animal models. The kinetic resolution of racemic Flurbiprofen was performed using dry mycelia of animal models. The kinetic resolution racemic Flurbiprofen was performed dry mycelia (S)-Flurbiprofen is as a NSAI drug, of whereas itsoptimizing enantiomerthe was found to inhibitusing tumor growth in of Aspergillus oryzae, MIM biocatalysts [29]. After reaction conditions (organic solvent, animal models. Theas kinetic resolution of After racemic Flurbiprofen was performed using dry mycelia Aspergillus biocatalysts [29]. optimizing theobtained reaction conditions (organic solvent, type oforyzae, alcoholMIM and temperature), (R)-Flurbiprofen ester was in varied conversion and of Aspergillus oryzae, MIM as biocatalysts [29]. After optimizing the reaction conditions (organic type enantioselectivity of alcohol and (62%–92% temperature), ester varied conversion ee), as(R)-Flurbiprofen seen in Scheme 15. Laterwas on, obtained the kineticin resolution of the same and solvent, type of alcohol and temperature), (R)-Flurbiprofen ester was obtained in varied conversion enantioselectivity (62%–92% ee),inasflow-reactor, seen in Scheme Later on, the kinetic resolution offorthe racemic drug was performed which15. is considered an interesting approach thesame and enantioselectivity (62%–92% ee), as seen in Scheme 15. Later on, the kinetic resolution of the development the lipase-catalyzed preparation of enantiopure molecules [30,31]. It approach is also worth racemic drug wasofperformed in flow-reactor, which is considered an interesting for the same racemicthat drug was automated performed in flow-reactor, which is considered an interesting approach for mentioning these systems help continuous bioprocesses, provide easy reaction development of the lipase-catalyzed preparation of enantiopure molecules [30,31]. It is also worth the development of the parameters lipase-catalyzed preparation of enantiopure [30,31]. It is also optimization such as residence time), and allowmolecules for multistep reactions and mentioning that (including these automated systems help continuous bioprocesses, provide easy reaction worth mentioning that these automated systems help continuous bioprocesses, provide easy reaction inline recovery and purification of the products [32,33]. Thus, as a proof of concept, direct optimization (including parameters such asas residence time), and for reactions and optimization such residence time),(Novozym andallow allow435) formultistep multistep reactions esterification (including in organic parameters solvent using commercial enzyme [30] and the whole inline recovery and purification of the products Thus, proofof of concept, direct and inline recovery and purification of the products[32,33]. [32,33]. Thus, as asbatch aa proof concept, direct microorganism (A. oryzae, MIM) [31] were compared with the classical method. The protocol esterification in organic solvent commercial enzyme(Novozym (Novozym 435) the whole esterification organic solventusing using commercial enzyme [30][30] andand theyielded whole inflow reactorin showed a significant reduction of the reaction time (from 6 h 435) to 15–60 min) and microorganism (A.(A. oryzae, MIM) [31] withthe theclassical classical batch method. The protocol microorganism oryzae, MIM) [31]were were compared compared method. The>98%) protocol both the (S)-Flurbiprofen and (R)-Flurbiprofen butyl with ester with ≥90% eebatch (chemical purity by inflow reactor showed a significant reduction of reaction time (from 6 h to 15–60 min) and yielded inflow reactor showed a significant reduction of the reaction time (from 6 h to 15–60 min) and yielded modulating the reaction conditions (temperature and residence time), as seen in Scheme 15. the (S)-Flurbiprofen and(R)-Flurbiprofen (R)-Flurbiprofen butyl ě90% ee ee (chemical purity >98%) by by bothboth the (S)-Flurbiprofen and butylester esterwith with ≥90% (chemical purity >98%) modulating reaction conditions(temperature (temperature and as as seen in Scheme 15. 15. modulating the the reaction conditions andresidence residencetime), time), seen in Scheme

Scheme 15. Enzymatic kinetic resolution of racemic Flurbiprofen, a non-steroidal anti-inflammatory drug, using classical batch method [29] and inflow reactors [30,31].

Scheme 15. Enzymatic kinetic resolution Flurbiprofen,a anon-steroidal non-steroidal anti-inflammatory Scheme 15. Enzymatic kinetic resolutionof ofracemic racemic Flurbiprofen, anti-inflammatory 3.5. Ibuprofen drug,drug, using classical batch method reactors[30,31]. [30,31]. using classical batch method[29] [29]and and inflow inflow reactors Ibuprofen is another NSAI agent with anti-inflammatory activity and wide commercial interest. A racemic mixture of Ibuprofen was resolved in the presence of commercially available lipases OF 3.5. Ibuprofen and CRL type VII from Candida rugosa immobilized onto magnetic beads. Immobilization was Ibuprofen via is another NSAI agent anti-inflammatory activity and wide commercial interest. performed a glutaraldehyde or with N-3-(3-dimethylaminopropyl)-N´-ethylcarbodiimide (EDC)/N29692 A racemic mixture of Ibuprofen was resolved in the presence of commercially available lipases OF 10

and CRL type VII from Candida rugosa immobilized onto magnetic beads. Immobilization was performed via a glutaraldehyde or N-3-(3-dimethylaminopropyl)-N´-ethylcarbodiimide (EDC)/N-

Int. J. Mol. Sci. 2015, 16, 29682–29716

3.5. Ibuprofen Ibuprofen is another NSAI agent with anti-inflammatory activity and wide commercial interest. A racemic mixture of Ibuprofen was resolved in the presence of commercially available lipases OF and CRL type VII from Candida rugosa immobilized onto magnetic beads. Immobilization was Int. J. Mol. Sci. 2015, 16, page–page performed via 16, a glutaraldehyde or N-3-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC)/ Int. J. Mol. Sci. 2015, page–page N-hydroxysulfosuccinimide sodium salt(sulfo-NHS) (sulfo-NHS)cross-linking cross-linkingreaction. reaction. The The immobilized immobilized enzymes enzymes hydroxysulfosuccinimide sodium salt were evaluated for the the preparation preparation of Ibuprofen Ibuprofen propyl ester. All Allreaction. reactions provided the (S)-ester (S)-ester as hydroxysulfosuccinimide sodium salt (sulfo-NHS) cross-linking The immobilized enzymes were evaluated for of propyl ester. reactions provided the as the product, and the best result (E-value of 19, 83% ee and c 42%) was achieved in the presence of were evaluated preparation of Ibuprofen propyl Allwas reactions provided the (S)-ester as the product, andfor thethe best result (E-value of 19, 83% ee andester. c 42%) achieved in the presence of the the immobilized lipase from C. rugosa OF, as well as in the presence of additives such as Na SO and product, and thefrom best result (E-value of well 19, 83% ee the andpresence c 42%) was the presence the 44 and immobilized lipase C. rugosa OF, as as in of achieved additivesinsuch as Na22SOof molecular sieves 44 Å Åfrom (Scheme 16). The study of reuse reuse demonstrated continued stability and catalytic immobilized lipase C. rugosa OF,study as well as in demonstrated the presence ofcontinued additivesstability such asand Na2catalytic SO4 and molecular sieves (Scheme 16). The of activity after five cycles [34]. molecular sieves Å (Scheme 16). The study of reuse demonstrated continued stability and catalytic activity after five 4reaction reaction cycles [34].

activity after five reaction cycles [34].

Scheme 16. Kinetic enzymatic esterification of rac-Ibuprofen, a nonsteroidal drug with anti-inflammatory Scheme 16. Kinetic enzymatic esterification of rac-Ibuprofen, a nonsteroidal drug with anti-inflammatory Scheme [34]. 16. Kinetic enzymatic esterification of rac-Ibuprofen, a nonsteroidal drug with anti-inflammatory activity activity [34]. activity [34].

3.6. Ketorolac 3.6. Ketorolac 3.6. Ketorolac Ketorolac (rac-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2-a]pyrrole-1-carboxylic acid) is a potent (rac-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2-a]pyrrole-1-carboxylic is potent Ketorolac (rac-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2-a]pyrrole-1-carboxylic acid) is aa used potent NSAIKetorolac drug belonging to the class of the heterocyclic acetic acid derivatives, and itacid) is widely as NSAI drug belonging to the class of the heterocyclic acetic acid derivatives, and it is widely used as an NSAI drug belonging to the class of the heterocyclic acetic acid derivatives, and it is widely used as an analgesic. The microwave (MW)-assisted kinetic resolution of this compound was investigated analgesic. The microwave (MW)-assisted kinetic resolution of this compound was investigated using an analgesic. The microwavelipases, (MW)-assisted resolution of this compound was investigated using different immobilized such as kinetic Novozym 435, Lipozyme TL IM, Lipozyme RM IM, different immobilized lipases, such Novozym 435,Among Lipozyme TL Novozym IM,TL Lipozyme IM, lipase using different immobilized lipases, as Novozym 435, Lipozyme IM, Lipozyme RM IM, lipase Amano AS, and lipase AYSassuch Amano [35]. them, 435 RM catalyzed the Amano AS, and lipase AYS Amano [35]. Among them, Novozym 435 catalyzed the enantioselective lipase Amano AS, and of lipase AYS Amano them, the enantioselective acylation rac-Ketorolac in 3 h,[35]. at 50 Among °C and 300 rpm,Novozym with c 50%435 and catalyzed high ee values acylation rac-Ketorolac in h, at 50 ˝ C and rpm, with c 17). 50% and high values (>99%) both enantioselective acylation of 3and rac-Ketorolac in(S)-acid 3300 h, at 50 °C and 300 In rpm, withee c the 50% and high eefor values (>99%) forofboth the (R)-ester remaining (Scheme addition, reaction was found the (R)-ester and remaining (Scheme 17). addition, to was follow the (>99%) forthe both the (R)-ester and remaining (S)-acid (Scheme 17). Inreaction addition,was thefound reaction found to follow Ping-Pong bi-bi(S)-acid mechanism, and to beIninhibited bythe n-octanol. Ping-Pong bi-bi mechanism, to be inhibited to follow the Ping-Pong bi-biand mechanism, and toby ben-octanol. inhibited by n-octanol.

Scheme 17. Enzymatic kinetic resolution of rac-Ketorolac, a potent nonsteroidal anti-inflammatory drug [35]. Scheme 17. Enzymatic kinetic resolution of rac-Ketorolac, a potent nonsteroidal anti-inflammatory drug [35]. Scheme 17. Enzymatic kinetic resolution of rac-Ketorolac, a potent nonsteroidal anti-inflammatory 3.7. Chloramphenicol and Thiamphenicol drug [35].

3.7. Chloramphenicol and Thiamphenicol Another pharmacological field where the use of lipases has been particularly active in the recent Another pharmacological field where use of lipases has been particularly active recent 3.7. Chloramphenicol and Thiamphenicol past is the elaboration of antibiotics. Thethe compound (1R,2R)-(−)-Chloramphenicol is in anthe effective past is the elaboration of antibiotics. The compound (1R,2R)-(−)-Chloramphenicol is an effective antibiotic against a wide range of Gram-positive and Gram-negative bacteria. This drug can be Another pharmacological field where the use of lipases has been particularly active in the recent antibiotic against a wide Gram-positive Gram-negative This this drugproblem can be administered in capsule orrange liquid of forms, and it has aand bitter taste. One waybacteria. to overcome past is the elaboration of antibiotics. The compound (1R,2R)-(´)-Chloramphenicol is an effective administered or liquid forms, and has a palatable. bitter taste.Several One way to overcome this problem is to use estersinofcapsule chloramphenicol, which areit more chloramphenicol esters were antibiotic against a wide range of Gram-positive and Gram-negative bacteria. This drug can be is to use esters of chloramphenicol, which are more palatable. Several chloramphenicol esters were prepared regioselectively in the presence of lipases. Then (−)-Chloramphenicol was subjected to administered in capsule or liquid forms, and it has a bitter taste. One way to overcome this problem prepared regioselectively in presence the presence of lipases. Then (−)-Chloramphenicol was subjected to esterification reaction in the of a series of vinyl esters containing alkyl chains of varying is to use esters of chloramphenicol, which are more palatable. Several chloramphenicol esters were esterification reaction the presence of vinylof esters containing alkyl The chains of efficient varying sizes (one to 15 carboninatoms), as well of as ainseries the presence a number of lipases. more prepared regioselectively in the presence of lipases. Then (´)-Chloramphenicol was subjected to sizes (one to from 15 carbon atoms), as well as in theNovozym presence of a number lipases. preparations The more efficient lipases were Candida antarctica B (CAL-B, 435) and twoofdifferent from esterification reaction in the presence of a series of vinyl esters containing alkyl chains of varying lipases were cepacia, from Candida antarctica (CAL-B, Novozym 435) and differentcepacia) preparations from Pseudomonas one from AmanoB(PSL-C Amano, also known as two Burkholderia and another sizes (one to 15 carbon atoms), as well as in the presence of a number of lipases. The more efficient ® Pseudomonas cepacia, one from Amano (PSL-C Amano, also known as Burkholderia cepacia) and another from Sigma-Aldrich (PSLC-I). With CAL-B, the best reaction conditions were in the presence of lipases were from Candida antarctica B (CAL-B, Novozym 435) and two different preparations from from Sigma-Aldrich the best reaction conditions in the presence of MeCN as a solvent at®20(PSLC-I). °C; with With PSL-CCAL-B, I and PSL-C Amano, the ideal solvent were was 1,4-dioxane at 30 °C. Pseudomonas cepacia, one from Amano (PSL-C Amano, also known as Burkholderia cepacia) and another MeCN as amonoesters solvent at 20 with PSL-C I and PSL-ConAmano, the ideal solvent wasobtained 1,4-dioxane °C. Generally, of°C; chloramphenicol acylated the primary hydroxyl were withatc30 >99% Generally, monoesters chloramphenicol acylatedtimes on thebetween primary3hydroxyl were obtained c >99% (isolated yields betweenof75% and 91%) for reaction and 10 h (Scheme 18 andwith Table 2). It (isolated yields between andof91%) reaction times between 3 and 10 hwith (Scheme 18 palmitate and Table 2). It is noteworthy that the 75% study the for reuse of29693 CAL-B in esterification vinyl was is noteworthy the it study the reuse CAL-B in reaction, esterification vinyl palmitate was performed. As that a result, was of observed thatofafter 3 h of there with was complete conversion performed. As a result, it was observed after h of reaction, was complete conversion during 10 cycles of reuse of the enzymethat [36]. The3 same strategy there was used on the synthesis of during 10 cycles of reuse the enzyme [36]. The same strategy was used on the synthesis of Thiamphenicol esters from of (1R,2R)-(−)-Thiamphenicol, an antibiotic analogue of chloramphenicol

Int. J. Mol. Sci. 2015, 16, 29682–29716

from Sigma-Aldrichr (PSLC-I). With CAL-B, the best reaction conditions were in the presence of MeCN as a solvent at 20 ˝ C; with PSL-C I and PSL-C Amano, the ideal solvent was 1,4-dioxane at 30 ˝ C. Generally, monoesters of chloramphenicol acylated on the primary hydroxyl were obtained with c >99% (isolated yields between 75% and 91%) for reaction times between 3 and 10 h (Scheme 18 and Table 2). It is noteworthy that the study of the reuse of CAL-B in esterification with vinyl palmitate was performed. As a result, it was observed that after 3 h of reaction, there was complete conversion during 10 cycles of reuse of the enzyme [36]. The same strategy was used on the synthesis of Thiamphenicol esters from (1R,2R)-(´)-Thiamphenicol, an antibiotic analogue of chloramphenicol with veterinary applications. The esters were regioselectively prepared via acylation reaction in the Int. J. Mol. Sci. 2015, 16, page–page presence of lipases and vinyl esters with variable lengths. The most effective lipase was CAL-B in ˝ C. Thiamphenicol esters (acylated at the primary hydroxyl group) were regioselectively MeCN, at 20 at 20 °C. Thiamphenicol esters (acylated at the primary hydroxyl group) were regioselectively produced with c >99% and isolated yields yields between between 94% 94% and and 98%, 98%, as as seen seen in in Scheme Scheme 18 18 and and Table Table 2. 2. produced with c >99% and isolated A study of the reuse of lipase from C. antarctica type B (CAL-B, Novozym 435) was conducted for the A study of the reuse of lipase from C. antarctica type B (CAL-B, Novozym 435) was conducted for the acylation of with vinyl vinyl decanoate. decanoate. The The corresponding corresponding 3’-monoester 3’-monoester was was obtained obtained acylation of (´)-Thiamphenicol (−)-Thiamphenicol with in 96% yield and the enzyme was reused five times without any loss of the activity [37]. in 96% yield and the enzyme was reused five times without any loss of the activity [37].

Scheme 18. Regioselective enzymatic acylation of (−)-Chloramphenicol [36] and (−)-Thiamphenicol [37]. Scheme 18. Regioselective enzymatic acylation of (´)-Chloramphenicol [36] and (´)-Thiamphenicol [37]. Table 2. Optimized conditions for the regioselective enzymatic acylation of (−)-Chloramphenicol and Table 2. Optimized conditions for the regioselective enzymatic acylation of (´)-Chloramphenicol and (−)-Thiamphenicol. (´)-Thiamphenicol. Compound

Enzyme Conditions 3‘-Monoester Yield (%) Enzyme Conditions 3‘-Monoester Yield (%) CAL-B MeCN, 20 °C (−)-Chloramphenicol 75–91 PSL-C I or PSL-C 1,4-dioxane, 30˝°C CAL-BAmano MeCN, 20 C (´)-Chloramphenicol 75–91 ˝ PSL-C CAL-B I or PSL-C Amano 1,4-dioxane, 30 C MeCN, 20 °C (−)-Thiamphenicol 94–98 Compound

(´)-Thiamphenicol

CAL-B

MeCN, 20 ˝ C

94–98

Ref. Ref.

36

[36]

37

[37]

3.8. Key Intermediates of Modified Cephalosporins 3.8. Key Intermediates of Modified Cephalosporins Some modified Cephalosporins and the anti-inflammatory agent N-[(9H-fluoren-9Some modified Cephalosporins and anti-inflammatory agent ylethoxy)carbonyl]L-leucine tert-butyl ester havethe a chiral fragment resulting from N-[(9H-fluoren-9the incorporation ylethoxy)carbonyl]L -leucine tert-butyl ester have a chiral fragment resulting from the incorporation of 1-(9H-fluoren-9-yl)ethanol in their structures. The first chemoenzymatic synthesis of this chiral of 1-(9H-fluoren-9-yl)ethanol inpresence their structures. first chemoenzymatic synthesis of this chiral fragment was performed in the of lipases.The Initially, racemic 1-(9H-fluoren-9-yl)ethanol was fragment was performed in the presence of lipases. Initially, racemic 1-(9H-fluoren-9-yl)ethanol chemically obtained, and then it was submitted to a kinetic resolution via acetylation reaction. Fifteen was chemically obtained, then it by wasstudying submitted to a kinetic resolutionimportant via acetylation commercial lipases were and evaluated parameters considered in thereaction. kinetic Fifteen commercial lipases were evaluated by ratio studying parameters considered in the resolution process, such as the substrate/lipase and solvent. The best results important were obtained in kinetic resolution process, such as the substrate/lipase ratio and solvent. The best results were the kinetic resolution with an enzymatic aggregate of lipase from Candida antarctica A (CAL-A CLEA, obtainedlipase in the resolution enzymatic ofconditions, lipase fromboth Candida antarctica Amano A).kinetic It is worth notingwith that an depending on aggregate the reaction (S)- and (R)-1A (CAL-A CLEA,ethanol Amano (obtained lipase A). from It is worth noting that on the reaction (9H-fluoren-9-yl) the hydrolysis of depending the corresponding acetate)conditions, could be both (S)- with and >99% (R)-1-(9H-fluoren-9-yl) ethanol from the hydrolysis of the corresponding prepared ee. In the first condition (i),(obtained after 24 h of reaction, with an enzyme/substrate (w/w) acetate) couldand be TBME prepared >99%it ee. the firsttocondition (i),(S)-alcohol after 24 h and of reaction, with an ratio of 10% as awith solvent, wasInpossible obtain the (R)-acetate with enzyme/substrate (w/w) ratio of 10% and TBME as a solvent, it was possible to obtain the (S)-alcohol enantiomeric excess (ee) >99% and 93%, respectively (E-value of 145, c 52%), as seen in Scheme 19. In andsecond (R)-acetate with enantiomeric excess (ee) >99% and 93%, respectively (E-valuewith of 145, c 52%), as the condition (ii), a dramatic decrease in the reaction rate was observed a decreasing seen in Scheme 19.ratio In the a dramatic decrease(68% in the rate was (>99% observed enzyme/substrate tosecond 1.25%. condition After nine(ii), days, the (S)-alcohol ee)reaction and (R)-acetate ee) with a decreasing enzyme/substrate ratio to 1.25%. After nine days, the (S)-alcohol (68% ee) and were obtained with c 41% and E > 200 [38]. (R)-acetate (>99% ee) were obtained with c 41% and E > 200 [38].

29694

prepared with >99% ee. In the first condition (i), after 24 h of reaction, with an enzyme/substrate (w/w) ratio of 10% and TBME as a solvent, it was possible to obtain the (S)-alcohol and (R)-acetate with enantiomeric excess (ee) >99% and 93%, respectively (E-value of 145, c 52%), as seen in Scheme 19. In the second condition (ii), a dramatic decrease in the reaction rate was observed with a decreasing enzyme/substrate to 1.25%. After nine days, the (S)-alcohol (68% ee) and (R)-acetate (>99% ee) Int. J. Mol. Sci. 2015, 16,ratio 29682–29716 were obtained with c 41% and E > 200 [38].

Scheme 19. intermediates in in Scheme 19. Kinetic Kineticenzymatic enzymaticacylation acylationofofrac-1-(9H-fluoren-9-yl)ethanol, rac-1-(9H-fluoren-9-yl)ethanol, affording affording intermediates the synthesis of modified Cephalosporins and the anti-inflammatory agent N-[(9H-fluoren-9the synthesis of modified Cephalosporins and the anti-inflammatory agent N-[(9H-fluoren-9-leucine tert-butyl tert-butyl ester ester [38]. [38]. ylethoxy)carbonyl]-LL-leucine ylethoxy)carbonyl]-

3.9. Key Intermediate of Duloxetin 3.9. Key Intermediate of Duloxetin BASF AG patented the chemoenzymatic synthesis of (S)-3-(methylamino)-1-(thiophen-2BASF AG patented the chemoenzymatic synthesis of (S)-3-(methylamino)-1-(thiophen-2Int. J. Mol. Sci. 2015, 16, page–page yl)propan-1-ol, a key intermediate in the synthesis of the serotonin-norepinephrine reuptake yl)propan-1-ol, key intermediate in the synthesis of the serotonin-norepinephrine reuptake inhibitor Int. J. Mol. Sci. 2015,a16, page–page 12 Duloxetine ((+)-(S)-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)propylamine oxalate) [39]. Chemical inhibitor Duloxetine ((+)-(S)-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)propylamine oxalate) [39]. reduction of 3-chloro-1-(thiophen-2-yl)propan-1-one gives access to the corresponding rac-alcohol, inhibitor Duloxetine ((+)-(S)-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)propylamine oxalate) [39]. Chemical reduction of 3-chloro-1-(thiophen-2-yl)propan-1-one gives access to the corresponding rac-alcohol, which was enantioselectively acylated withwith succinic anhydride using lipase from Burkholderia plantarii or Chemical reduction of 3-chloro-1-(thiophen-2-yl)propan-1-one gives access to the corresponding rac-alcohol, which was enantioselectively acylated succinic anhydride using lipase from Burkholderia from Pseudomonas sp., furnishing the corresponding (R)-semiester, leaving the unreacted (S)-alcohol. which was enantioselectively acylated with succinic anhydride using lipase from Burkholderia plantarii or from Pseudomonas sp., furnishing the corresponding (R)-semiester, leaving the unreacted The reaction between the between (S)-alcohol methylamine afforded(R)-semiester, the enantiomerically pure desired plantarii or from Pseudomonas sp., furnishing the corresponding the unreacted (S)-alcohol. The reaction theand (S)-alcohol and methylamine affordedleaving the enantiomerically product (Scheme 20). (Scheme (S)-alcohol. The reaction between pure desired product 20). the (S)-alcohol and methylamine afforded the enantiomerically pure desired product (Scheme 20).

Scheme 20. Kinetic enzymatic acylation of the rac-3-chloro-1-(thiophen-2-yl)propan-1-ol used in the Scheme 20. Kinetic enzymatic acylation of the rac-3-chloro-1-(thiophen-2-yl)propan-1-ol used in the Scheme 20. Kinetic enzymatic acylation of the rac-3-chloro-1-(thiophen-2-yl)propan-1-ol used in the chemoenzymatic synthesis of (S)-3-(methylamino)-1-(thiophen-2-yl)propan-1-ol, a key intermediate chemoenzymatic synthesis of (S)-3-(methylamino)-1-(thiophen-2-yl)propan-1-ol, a key intermediate chemoenzymatic of (S)-3-(methylamino)-1-(thiophen-2-yl)propan-1-ol, a key intermediate in the synthesis ofsynthesis the serotonin-norepinephrine reuptake inhibitor Duloxetine [39]. in the synthesis of the serotonin-norepinephrine reuptake inhibitor Duloxetine [39]. in the synthesis of the serotonin-norepinephrine reuptake inhibitor Duloxetine [39].

3.10. Nebracetam 3.10. Nebracetam 3.10. Nebracetam Nebracetam, a nootropic drug from the racetam family, is used as an anti-depressant (M1 Nebracetam, nootropic drug from the racetamsynthesis family, is as anti-depressant (M1 Nebracetam, aa nootropic the racetam family, isofused used as an an anti-depressant (M1 acetylcholine receptor agonist).drug The from first asymmetric (S)-Nebracetam was efficiently acetylcholine receptor agonist). The first asymmetric synthesis of (S)-Nebracetam was efficiently conducted acetylcholine receptor agonist). The (5S)-1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one first asymmetric synthesis of (S)-Nebracetam waswhich efficiently conducted from the intermediate was from thebyintermediate (5S)-1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one which was obtained by conducted from the intermediate (5S)-1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one which was obtained kinetic resolution of the racemic hydroxylactam (1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one). kinetic resolution of the racemic The obtained by kinetic resolution of the racemic hydroxylactam (1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one). The acetylation was performed inhydroxylactam the presence of(1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one). lipase from Burkholderia cepacia (lipase PS-D), vinyl acetylation was performed in the presence of lipase from Burkholderia cepacia (lipase PS-D), vinyl The acetylation was performed in theaspresence of lipase from Burkholderia cepacia and (lipase vinyl acetate as an acyl donor, 1,4-dioxane the organic solvent at room temperature 48 hPS-D), of reaction acetate an Accordingly, acyl donor, donor, 1,4-dioxane as solvent room and h ofexcellent reaction acetate as21). an acyl 1,4-dioxane as the the organic organic solvent at(S)-alcohol room temperature temperature and 48 48with h of reaction (Schemeas the corresponding (R)-acetate andat were obtained (Scheme Accordingly, the (R)-acetate (S)-alcohol were obtained with excellent (Scheme 21). 21). Accordingly, the corresponding corresponding (R)-acetate and (S)-alcohol were obtained withsteps, excellent enantioselectivity and conversion (>99% ee, c 49% and E >and 200). Then, after several chemical the enantioselectivity and conversion (>99% ee, c 49% and E > 200). Then, after several chemical steps, enantioselectivity and conversion (>99% ee, c 49%[40]. and E > 200). Then, after several chemical steps, the (5S)-alcohol was converted into (S)-Nebracetam the (5S)-alcohol converted (S)-Nebracetam (5S)-alcohol waswas converted intointo (S)-Nebracetam [40].[40].

Scheme 21. Kinetic resolution of the racemic hydroxylactam to produce the (5S)-1-benzyl-5-hydroxyScheme 21. Kinetic resolution to produce the[40]. (5S)-1-benzyl-5-hydroxy1,5-dihydropyrrol-2-one used of in the racemic synthesishydroxylactam of the drug (S)-Nebracetam Scheme 21. Kinetic resolution of the racemic hydroxylactam to produce the (5S)-1-benzyl-5-hydroxy-1, 1,5-dihydropyrrol-2-one used in the synthesis of the drug (S)-Nebracetam [40]. 5-dihydropyrrol-2-one used in the synthesis of the drug (S)-Nebracetam [40].

3.11. Naphthofurandione derivative 3.11. Naphthofurandione derivative The naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione, with The naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione, with antitumor activity, is a secondary metabolite isolated from the bark of Tabebuia impetiginosa 29695 antitumor activity, is a secondary metabolite isolated from the bark of Tabebuia impetiginosa (Bignoniaceae). Naphthofurandione was prepared in racemic form through several chemical steps (Bignoniaceae). Naphthofurandione was prepared in racemic formthe through several chemical steps from commercially available 1,5-dihydroxynaphthalene. Then, rac-naphthofurandione was from commercially available 1,5-dihydroxynaphthalene. Then,(Scheme the rac-naphthofurandione was subjected to enzymatic kinetic resolution via acetylation reaction 22). Among the evaluated subjected to enzymatic kinetic resolution via acetylation reaction (Scheme 22). Among the evaluated lipases, Pseudomonas cepacia, (PSL-CI, 3 h of reaction) and lipase from Candida antarctica B, (Novozym 435,

Int. J. Mol. Sci. 2015, 16, 29682–29716 Scheme 21. Kinetic resolution of the racemic hydroxylactam to produce the (5S)-1-benzyl-5-hydroxy-

1,5-dihydropyrrol-2-one used in the synthesis of the drug (S)-Nebracetam [40].

3.11. Naphthofurandione Derivative 3.11. Naphthofurandione derivative The naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione, with The naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione, with antitumor activity, is a secondary metabolite isolated from the bark of Tabebuia impetiginosa antitumor activity, is a secondary metabolite isolated from the bark of Tabebuia impetiginosa (Bignoniaceae). Naphthofurandione was prepared in racemic form through several chemical steps (Bignoniaceae). Naphthofurandione was prepared in racemic form through several chemical steps from commercially available 1,5-dihydroxynaphthalene. Then, the rac-naphthofurandione was from commercially available 1,5-dihydroxynaphthalene. Then, the rac-naphthofurandione was subjected to enzymatic kinetic resolution via acetylation reaction (Scheme 22). Among the evaluated subjected to enzymatic kinetic resolution via acetylation reaction (Scheme 22). Among the evaluated lipases, Pseudomonas cepacia, (PSL-CI, 3 h of reaction) and lipase from Candida antarctica B, (Novozym lipases, Pseudomonas cepacia, (PSL-CI, 3 h of reaction) and lipase from Candida antarctica B, (Novozym 435, 435, 2 h of reaction) were the most efficient. The best reaction conditions were vinyl acetate as the acyl 2 h of reaction) were the most efficient. The best reaction conditions were vinyl acetate as the acyl donor, THF as the solvent, 250 rpm, at 30 ˝ C, leading to (S)-alcohol and the corresponding (R)-acetate donor, THF as the solvent, 250 rpm, at 30 °C, leading to (S)-alcohol and the corresponding (R)-acetate with 99% ee for both and E > 200 [41]. with 99% ee for both and E > 200 [41].

Scheme 22. Synthesis of the naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furanScheme 22. Synthesis of the naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b] 4,9-dione via kinetic enzymatic acylation of the corresponding racemic alcohol [41]. furan-4,9-dione via kinetic enzymatic acylation of the corresponding racemic alcohol [41].

Int. J. Mol. Sci. 2015, 16, page–page

13

3.12. GABOB GABOB and and carnitine Carnitine 3.12. The compounds acidacid (GABOB) and (R)-carnitine are two important The compounds(R)-γ-Amino-β-hydroxybutyric (R)-γ-Amino-β-hydroxybutyric (GABOB) and (R)-carnitine are two bioactive molecules that play that an important role in mammalian systems. The key of the important bioactive molecules play an important role in mammalian systems. The step key step of chemoenzymatic syntheses of these two compounds involved the lipase-mediated resolution of the chemoenzymatic syntheses of these two compounds involved the lipase-mediated resolution of the racemic 3-hydroxy-4-tosyloxybutanenitrile in organic solvents (Scheme 23). Lipases (free or the racemic 3-hydroxy-4-tosyloxybutanenitrile in organic solvents (Scheme 23). Lipases (free or immobilized forms) forms) from fromPseudomonas Pseudomonascepacia cepacia(PS), (PS),P.P.fluorescens fluorescens(AK), (AK), Candida rugosa (CRL AYS), immobilized Candida rugosa (CRL AYS), C. C. antarctica (CAL-B) and Mucor miehei (Lipozyme) were used as biocatalysts, and yielded the antarctica (CAL-B) and Mucor miehei (Lipozyme) were used as biocatalysts, and yielded the (S)-alcohol (S)-alcohol andin (R)-acetate in high enantioselectivity (>99% ee). After some chemical transformations, and (R)-acetate high enantioselectivity (>99% ee). After some chemical transformations, the (R)-acetate the (R)-acetate isomer was converted into (R)-GABOB and (R)-carnitine [42]. isomer was converted into (R)-GABOB and (R)-carnitine [42].

Scheme 23. Kinetic resolution of the racemic 3-hydroxy-4-tosyloxybutanenitrile to produce (R)-GABOB Scheme 23. Kinetic resolution of the racemic 3-hydroxy-4-tosyloxybutanenitrile to produce and (R)-carnitine [42]. (R)-GABOB and (R)-carnitine [42].

3.13. Quinolone Derivatives 3.13. Quinolone Derivatives The quinolone 2-[2-hydroxy-3-(4-phenylpiperazin-1-yl)propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)-one The quinolone 2-[2-hydroxy-3-(4-phenylpiperazin-1-yl)propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)was chemically synthesized and subsequently subjected to enzymatic kinetic resolution via acylation one was chemically synthesized and subsequently subjected to enzymatic kinetic resolution via reaction (Scheme 24). The best results were obtained using lipase from Candida antarctica B (CAL-B), acylation reaction (Scheme 24). The best results were obtained using lipase from Candida antarctica and vinyl acetate as a both solvent and acyl donor at room temperature. After 48 h of reaction, it was B (CAL-B), and vinyl acetate as a both solvent and acyl donor at room temperature. After 48 h possible to obtain the corresponding (R)-acetate in 36% yield and 96% ee; (S)-quinolone was obtained of reaction, it was possible to obtain the corresponding (R)-acetate in 36% yield and 96% ee; in 41% yield and 98% ee, c 50% and E > 200. The (S)-quinolone as well as the corresponding (R)-acetate (S)-quinolone was obtained in 41% yield and 98% ee, c 50% and E > 200. The (S)-quinolone as well showed significant cytotoxic activity against human neuroblastoma tumor cells (SK-N-SH) and lung cells as the corresponding (R)-acetate showed significant cytotoxic activity against human neuroblastoma (A549) compared to the control doxorubicin [43]. tumor cells (SK-N-SH) and lung cells (A549) compared to the control doxorubicin [43].

29696 Scheme 24. Kinetic enzymatic acylation of the racemic quinolone 2-[2-hydroxy-3-(4-phenylpiperazin-1yl)propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)-one [43].

reaction (Scheme 24). The best results were obtained using lipase from Candida antarctica B (CAL-B), and vinyl acetate as a both solvent and acyl donor at room temperature. After 48 h of reaction, it was possible to obtain the corresponding (R)-acetate in 36% yield and 96% ee; (S)-quinolone was obtained in 41% yield and 98% ee, c 50% and E > 200. The (S)-quinolone as well as the corresponding (R)-acetate showed activity against human neuroblastoma tumor cells (SK-N-SH) and lung cells Int. J. Mol. significant Sci. 2015, 16,cytotoxic 29682–29716 (A549) compared to the control doxorubicin [43].

Scheme 24. Kinetic enzymatic acylation of the racemic quinolone 2-[2-hydroxy-3-(4-phenylpiperazin-1Scheme 24. Kinetic enzymatic acylation of the racemic quinolone 2-[2-hydroxy-3-(4-phenylpiperazinyl)propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)-one [43]. 1-yl)propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)-one [43].

3.14. Key Intermediate of Mevinic Acid Analogues 3.14. Key Intermediate of Mevinic Acid Analogues Analogues of mevinic acid can be obtained from (S)-1-(1-naphthyl)ethanol. A racemic mixture Analogues of mevinic acid can be obtained from (S)-1-(1-naphthyl)ethanol. A racemic mixture of this compound was resolved in the presence of lipases using microwave. Several parameters were of this compound was resolved in the presence of lipases using microwave. Several parameters were studied and the best reaction conditions were Novozym 435 as a biocatalyst, vinyl acetate as an acyl studied and the best reaction conditions were Novozym 435 as a biocatalyst, vinyl acetate as an acyl donor, n-heptane as a solvent, a stirring speed of 400 rpm, temperature at 60 °C and 30 mg of enzyme donor, n-heptane as a solvent, a stirring speed of 400 rpm, temperature at 60 ˝ C and 30 mg of enzyme loading. In such conditions, after 3 h of reaction, the (S)-1-(1-naphthyl)ethanol was obtained with loading. In such conditions, after 3 h of reaction, the (S)-1-(1-naphthyl)ethanol was obtained with c 48%, 90.0% ee and E > 200 (Scheme 25). The study of reuse of the enzyme was performed with a c 48%, 90.0% ee and E > 200 (Scheme 25). The study of reuse of the enzyme was performed with a slight reduction of conversion from 48% to 45% after being used three times. It is noteworthy that the slight reduction of conversion from 48% to 45% after being used three times. It is noteworthy that results from the conventional heating (c 39%, 64% ee and E-value of 164 after 5 h of reaction) were the results from the conventional heating (c 39%, 64% ee and E-value of 164 after 5 h of reaction) were inferior to those obtained under microwave conditions [44]. inferior obtained under microwave conditions [44]. Int. J. Mol.to Sci.those 2015, 16, page–page

14 Scheme 25. enzymatic acylation of rac-1-(1-naphthyl)ethanol to produce the (S)-1-(1Scheme 25. Kinetic Kinetic enzymatic acylation of rac-1-(1-naphthyl)ethanol to produce the naphthyl)ethanol, an intermediate in the synthesis of mevinic acid analogues [44]. (S)-1-(1-naphthyl)ethanol, an intermediate in the synthesis of mevinic acid analogues [44]. 3.15. Key Intermediate of Tetrahydrofuran Derivatives 3.15. Key Intermediate of Tetrahydrofuran Derivatives Compounds (+)- and (−)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine showed Compounds (+)- and (´)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine neuroprotective, antidepressant and antiepileptic activities. Such compounds were prepared by showed neuroprotective, antidepressant and antiepileptic activities. Such compounds were prepared chemoenzymatic synthesis starting from 5,5-diphenyltetrahydrofuran-2(3H)-one. The key step for by chemoenzymatic synthesis starting from 5,5-diphenyltetrahydrofuran-2(3H)-one. The key step introducing chirality in the target molecule was the desymmetrization of the intermediate for introducing chirality in the target molecule was the desymmetrization of the intermediate 3-(hydroxymethyl)-1,1-diphenylbutane-1,4-diol, using vinyl acetate as an acylating agent in the 3-(hydroxymethyl)-1,1-diphenylbutane-1,4-diol, using vinyl acetate as an acylating agent in the presence presence of lipase from Burkholderia cepacia (Amano lipase PS30), to obtain the (+)-4-hydroxy-2of lipase from Burkholderia cepacia (Amano lipase PS30), to obtain the (+)-4-hydroxy-2-(hydroxymethyl)-4, (hydroxymethyl)-4,4-diphenylbutyl acetate (Scheme 26). The latter compound was subjected to two 4-diphenylbutyl acetate (Scheme 26). The latter compound was subjected to two different synthetic different synthetic routes. The first (a) involved a sequence of reactions including tosylation, and routes. The first (a) involved a sequence of reactions including tosylation, and hydrolysis and reaction hydrolysis and reaction with 2,6-lutidine in the presence of trifluoromethane sulfonic anhydride to with 2,6-lutidine in the presence of trifluoromethane sulfonic anhydride to afford the (+) afford the (+) 1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine. In the second 1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine. In the second synthetic route (b), synthetic route (b), the intermediate (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate was the intermediate (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate was subjected to a step of subjected to a step of protecting one of the hydroxyl groups with tert-butyldimethylsilyl chlroride (TBSCl), protecting one of the hydroxyl groups with tert-butyldimethylsilyl chlroride (TBSCl), followed by followed by hydrolysis in the presence of LiOH/H2O, tosylation reaction and deprotection with hydrolysis in the presence of LiOH/H2 O, tosylation reaction and deprotection with tetrabutylammonium tetrabutylammonium fluoride (TBAF). Finally, a reaction with 2,6-lutidine in the presence of trifluoroacetic fluoride (TBAF). Finally, a reaction with 2,6-lutidine in the presence of trifluoroacetic sulfonic sulfonic anydride led to the (−)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine. It should anydride led to the (´)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine. It should be mentioned that the absolute configurations of the products were not reported by the authors [45]. be mentioned that the absolute configurations of the products were not reported by the authors [45].

29697 Scheme 26. Desymmetrization of the intermediate 3-(hydroxymethyl)-1,1-diphenylbutane-1,4-diol to produce (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate, a key intermediate in the

synthetic route (b), the intermediate (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate was subjected to a step of protecting one of the hydroxyl groups with tert-butyldimethylsilyl chlroride (TBSCl), followed by hydrolysis in the presence of LiOH/H2O, tosylation reaction and deprotection with tetrabutylammonium fluoride (TBAF). Finally, a reaction with 2,6-lutidine in the presence of trifluoroacetic sulfonic led29682–29716 to the (−)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine. It should Int. J. Mol.anydride Sci. 2015, 16, be mentioned that the absolute configurations of the products were not reported by the authors [45].

Scheme 26. Desymmetrization of the intermediate 3-(hydroxymethyl)-1,1-diphenylbutane-1,4-diol to Scheme 26. Desymmetrization of the intermediate 3-(hydroxymethyl)-1,1-diphenylbutane-1,4-diol produce (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate, a key intermediate in the to produce (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate, a key intermediate in the synthesis of (+)- and (−)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine [45]. synthesis of (+)- and (´)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine [45].

3.16. Key Intermediates of β-Amino Alcohols 3.16. Key Intermediates of β-Amino Alcohols β-Amino alcohols are part of the structure of numerous active pharmaceuticals such as Vernakalant β-Amino alcohols are part of the structure of numerous active pharmaceuticals such as Vernakalant and Indinavir. The cis- and trans-2-phthalimidocyclopentanol and 2-phthalimidocyclohexanol were and Indinavir. The cis- and trans-2-phthalimidocyclopentanol and 2-phthalimidocyclohexanol were subjected to kinetic resolution in the presence of lipases. The best reaction conditions were obtained subjected to kinetic resolution in the presence of lipases. The best reaction conditions were in the presence of vinyl acetate and TBME. For both trans-2-phthalimidocyclopentanol and trans-2obtained in the presence of vinyl acetate and TBME. For both trans-2-phthalimidocyclopentanol and phthalimidocyclohexanol the most efficient lipase was from Pseudomonas cepacia (PS IM), as seen in trans-2-phthalimidocyclohexanol the most efficient lipase was from Pseudomonas cepacia (PS IM), as Table 3, leading to (1R,2R)-acetates and (1S,2S)-alcohols with >99% ee, c 50% and E > 200. In this case, seen in Table 3, leading to (1R,2R)-acetates and (1S,2S)-alcohols with >99% ee, c 50% and E > 200. the biocatalyst maintained its activity and enantioselectivity after five reaction cycles. For the cis-2In this case, the biocatalyst maintained its activity and enantioselectivity after five reaction cycles. phthalimidocyclopentanol, four lipases were efficient: lipase A from Candida antarctica (CAL-A), For the cis-2-phthalimidocyclopentanol, four lipases were efficient: lipase A from Candida antarctica lipase from Rhizomucor miehei (RM IM), lipase from P. fluorescens (AK) and P. cepacia lipase (PS IM), (CAL-A), lipase from Rhizomucor miehei (RM IM), lipase from P. fluorescens (AK) and P. cepacia as seen in Table 3. In all cases (1S,2R)-cis-alcohol and (1R,2S)-cis-acetate with >99% ee, c 50%, and E > lipase (PS IM), as seen in Table 3. In all cases (1S,2R)-cis-alcohol and (1R,2S)-cis-acetate with >99% ee, 200 were obtained. For the cis-2-phthalimidocyclohexanol the only effective lipase was P. cepacia (PS IM), c 50%, and E > 200 were obtained. For the cis-2-phthalimidocyclohexanol the only effective lipase was Table 3, with a reaction time of 72 h, at 45 °C. In such conditions, (1R,2S)-cis-acetate and (1S,2R)-cisP. cepacia (PS IM), Table 3, with a reaction time of 72 h, at 45 ˝ C. In such conditions, (1R,2S)-cis-acetate alcohol were obtained with >99% ee, c 50% and E > 200 (Scheme 27) [46]. (1S,2R)-cis-alcohol were obtained with >99% ee, c 50% and E > 200 (Scheme 27) [46]. Int. J. and Mol. Sci. 2015, 16, page–page 15

Scheme 27. 27.Kinetic enzymatic acylation trans- and and cis-diastereoisomers cis-diastereoisomers Scheme Kinetic enzymatic acylation ofof racemic racemic transof of 2-phthalimidocyclopentanol produceintermediates intermediates in the 2-phthalimidocyclopentanoland and 2-phthalimidocyclohexanol 2-phthalimidocyclohexanol toto produce in the synthesis of β-amino alcohols [46]. synthesis of β-amino alcohols [46]. Table 3. Efficient lipases the kineticenzymatic enzymatic acylation acylation of transand cis-diastereoisomers Table 3. Efficient lipases forfor the kinetic ofracemic racemic transand cis-diastereoisomers of 2-phthalimidocyclopentanol and 2-phthalimidocyclohexanol [46]. of 2-phthalimidocyclopentanol and 2-phthalimidocyclohexanol [46]. Compound Compound trans-2-phthalimidocyclopentanol trans-2-phthalimidocyclopentanol cis-2-phthalimidocyclopentanol

cis-2-phthalimidocyclopentanol

trans-2-phthalimidocyclohexanol cis-2-phthalimidocyclohexanol trans-2-phthalimidocyclohexanol

cis-2-phthalimidocyclohexanol

Lipase Lipase P. cepacia (PS IM)(PS IM) P. cepacia C. antarctica (CAL-A), R. miehei (RM IM), P. fluorescens (AK) C. antarctica (CAL-A), R. miehei (RM IM), P. fluorescens and P. cepacia (PS IM) (AK) and(PS P. IM) cepacia (PS IM) P. cepacia P. cepacia (PS IM) P. cepacia (PS IM)

P. cepacia (PS IM)

3.17. Key Intermediates of Iminocyclitols Enantiomerically pure 1,3-propanediols 29698 are important intermediates in the synthesis of iminocyclitols (glycosidases inhibitors used as anti-diabetic drugs). The desymmetrization of carboxybenzyl-2-amino-1,3-propanediol (Cbz-serinol) via acetylation reaction in THF mediated by

cis-2-phthalimidocyclopentanol

(AK) and P. cepacia (PS IM) P. cepacia (PS IM) P. cepacia (PS IM)

trans-2-phthalimidocyclohexanol cis-2-phthalimidocyclohexanol Int. J. Mol. Sci. 2015, 16, 29682–29716

3.17. Key Intermediates of Iminocyclitols 3.17. Key Intermediates of Iminocyclitols Enantiomerically pure 1,3-propanediols are important intermediates in the synthesis of iminocyclitols (glycosidasespure inhibitors used as drugs). The desymmetrization of Enantiomerically 1,3-propanediols are anti-diabetic important intermediates in the synthesis of iminocyclitols (glycosidases inhibitors used as anti-diabetic drugs). The desymmetrization of carboxybenzyl-2-amino-1,3-propanediol (Cbz-serinol) via acetylation reaction in THF mediated by carboxybenzyl-2-amino-1,3-propanediol (Cbz-serinol) via acetylation reaction PPL in THF mediated byVI-S) (ii) crude pig pancreatic lipase (PPL, L3126, type II, Steapsin) (i) or purified (L0382, type crude pig pancreatic lipase (PPL, L3126, type II, Steapsin) (i) or purified PPL (L0382, type VI-S) was reported [47]. When the reaction was carried out in vinyl acetate as both a solvent and acyl donor, (ii) was reported [47]. When the reaction was carried out in vinyl acetate as both a solvent and acyl in the presence crude PPLof(i), onlyPPL the(i),(2R)-monoacetate was formed in 98% donor, inofthe presence crude only the (2R)-monoacetate was formed in ee. 98%On ee. the On other the hand, only the (2S)-monoacetate (100% ee) (100% was ee)formed when PPL (ii) catalyzed the other hand, only the (2S)-monoacetate was formed whenpurified purified PPL (ii) catalyzed the desymmetrization (Scheme 28). desymmetrization (Scheme 28).

Scheme Scheme 28. Kinetic enzymatic racemic carboxybenzyl-2-amino-1,3-propanediol 28. Kinetic enzymaticacylation acylation ofof racemic carboxybenzyl-2-amino-1,3-propanediol (Cbz-serinol) (Cbzto produce intermediates in thein synthesis of iminocyclitols [47]. serinol) to produce intermediates the synthesis of iminocyclitols [47]. 3.18. N-Acetyl Phenylalanine and Analogues 3.18. N-Acetyl Phenylalanine and Analogues Phenylalanine and analogues are important building blocks in the preparation of complex

Phenylalanine and analogues are important building blocks in the preparation structures of interest to the medicinal chemistry area. N-Acetyl phenylalanine and a seriesofofcomplex structures of interest to the inmedicinal chemistry area. N-Acetyl phenylalanine a series of analogues were obtained enantiomerically pure form by interesterification reaction in the and presence of lipase from Rhizomucor miehei (RML). Ethyl acetamidocianoacetate was alkylated in the presence analogues were obtained in enantiomerically pure form by interesterification reaction in theofpresence alkyl halides in phase(RML). transfer Ethyl catalysis (PTC), followed by acidicwas hydrolysis, esterification of lipasevarious from Rhizomucor miehei acetamidocianoacetate alkylated in the presence and N-acetylation leading to N-acetyl-phenylalanine methyl and allyl esters derivatives. These of various alkyl halides in phase transfer catalysis (PTC), followed by acidic hydrolysis, esterification derivatives were submitted to an enzymatic kinetic resolution via interesterification reaction. After and N-acetylation leading N-acetyl-phenylalanine and derivatives. screening with various to commercial lipases, it was foundmethyl that only RMLallyl was esters able to promote the These derivatives were submitted to29). an Several enzymatic kinetic resolution reaction. After kinetic resolution (Scheme parameters were studied andvia theinteresterification best reaction conditions were Int. J. Mol. Sci. 2015, 16, page–page obtained in the presence of butyl butyrate interesterification agent,RML with an enzyme:substrate screening with various commercial lipases, asit an was found that only was able to promote the ˝ C, and MeCN as a solvent. Most derivatives (S-configuration) were obtained ratio of 2:1 (w/w), at 30 ratio resolution of 2:1 (w/w), at 30 °C,29). andSeveral MeCNparameters as a solvent.were Moststudied derivatives (S-configuration) were obtained kinetic (Scheme and the best reaction conditions were with >99% ee E and E > 200 [48]. with >99% ee and > 200 [48]. obtained in the presence of butyl butyrate as an interesterification agent, with an enzyme:substrate 16

Scheme 29. Kinetic enzymatic resolution of of racemic racemic N-acetyl and analogues via Scheme 29. Kinetic enzymatic resolution N-acetylphenylalanine phenylalanine and analogues via interesterification reaction [48]. interesterification reaction [48].

3.19. (S)-1-(2-Furyl)ethanol 3.19. (S)-1-(2-Furyl)ethanol The (S)-1-(2-furyl)ethanol is used as an important building block for the synthesis of various

The (S)-1-(2-furyl)ethanol is used as an important building block for the synthesis of various natural products such as flavonoids, polyketide antibiotics and carbohydrate derivatives. This naturalintermediate products was such as flavonoids, polyketide carbohydrate derivatives. obtained by lipase-catalyzed kineticantibiotics resolution ofand racemic 1-(2-furyl)ethanol. After This intermediate was obtained by lipase-catalyzed kinetic resolution of racemic 1-(2-furyl)ethanol. After commercial lipases screening in n-heptane as a solvent and 2 h of reaction time, the lipase from 29699 Candida antarctica B (Novozym 435) was the most efficient enzyme, leading to higher conversion (c 47%) than those observed with the other evaluated enzymes, RM IM Lipozyme (c 1.8%) and Lipozyme TL IM (c 1.5%). Various parameters were studied before establishing the best reaction conditions (vinyl

Scheme 29. Kinetic enzymatic resolution of racemic N-acetyl phenylalanine and analogues via 3.19. (S)-1-(2-Furyl)ethanol interesterification reaction [48].

The (S)-1-(2-furyl)ethanol is used as an important building block for the synthesis of various 3.19. (S)-1-(2-Furyl)ethanol natural products such as flavonoids, polyketide antibiotics and carbohydrate derivatives. This Int. The J. Mol.(S)-1-(2-furyl)ethanol Sci. 2015, 16, 29682–29716 is used as an important building block for the synthesis of various intermediate was obtained by lipase-catalyzed kinetic resolution of racemic 1-(2-furyl)ethanol. After natural products as flavonoids, polyketide antibiotics derivatives. commercial lipases such screening in n-heptane as a solvent and 2and h ofcarbohydrate reaction time, the lipaseThis from intermediate was obtained by lipase-catalyzed kinetic resolution of racemic 1-(2-furyl)ethanol. After Candida antarcticalipases B (Novozym 435) the most to higher conversion (c 47%) commercial screening in was n-heptane as aefficient solventenzyme, and 2 h leading of reaction time, the lipase from commercial lipases Bscreening in 435) n-heptane asmost a solvent and 2 h of leading reactiontotime, theconversion lipase from Candida antarctica (Novozym was the efficient enzyme, higher than those observed with the other evaluated enzymes, RM IM Lipozyme (c 1.8%) and Lipozyme TL Candida antarctica B (Novozym was the most efficient enzyme, higher conversion 47%) 47%) than those observed435) with other evaluated enzymes,leading RM best IMtoLipozyme (c 1.8%) (cand IM (c(c1.5%). Various parameters werethe studied before establishing the reaction conditions (vinyl than those observed with the other evaluated enzymes, RM IM Lipozyme (c 1.8%) and Lipozyme TL Lipozyme TL IM (c 1.5%). Various parameters were studied before establishing the best reaction acetate as the acyl donor, n-heptane as the solvent, a stirring speed of 300 rpm, 5 mg of enzyme loading at IMconditions (c 1.5%). Various parameters studiedn-heptane before establishing the best reaction conditions (vinyl acetate as the were acyl donor, the solvent, speed rpm, 60 °C). In these (vinyl conditions, the (S)-1-(2-furyl)ethanol was as obtained with ac stirring 47% and 89%of ee 300 (Scheme 30). ˝ acetate as the acyl donor, n-heptane as the solvent, a stirring speed of 300 rpm, 5 mg of enzyme loading 5 mg of enzyme loading at 60 C). In these conditions, the (S)-1-(2-furyl)ethanol was obtained with at The study of the reuse of the biocatalyst was also performed, and it was shown that it can be recycled 60c°C). these conditions, the30). (S)-1-(2-furyl)ethanol was obtained with c 47% ee (Scheme 47%Inand 89% ee (Scheme The study of the reuse of the biocatalyst wasand also89% performed, and30). three a reuse small of decrease in conversion 47.0% to 44.5% [49]. Thetimes studywith of the the biocatalyst was alsofrom performed, and it was shown that it can be recycled it was shown that it can be recycled three times with a small decrease in conversion from 47.0% to

three times 44.5% [49].with a small decrease in conversion from 47.0% to 44.5% [49].

Scheme 30. Kinetic enzymatic acylation of the rac-1-(2-furyl)ethanol to produce the (S)-alcohol, an Scheme 30.30.Kinetic enzymatic of the rac-1-(2-furyl)ethanol important building block for the acylation synthesis ofof various natural productsto[49]. Scheme Kinetic enzymatic acylation the rac-1-(2-furyl)ethanol toproduce producethe the(S)-alcohol, (S)-alcohol, an important building block for the synthesis of various natural products an important building block for the synthesis of various natural products[49]. [49].

3.20. Ascorbyl Ester Derivatives 3.20. Ascorbyl Ester 3.20. Ascorbyl EsterDerivatives Derivatives Ascorbic acid (Asc) is a very important natural antioxidant, but with limited industrial Ascorbic acid (Asc) a very important antioxidant, but with limited industrial Ascorbic acid (Asc) is is a hydrophilic very important naturalnatural antioxidant, but with limited industrial application application because of its nature. Alternatively, ascorbyl esters are considered because of its hydrophilic nature. Alternatively, ascorbyl esters are considered antioxidants application because of its hydrophilic nature. Alternatively, ascorbyl esters are considered antioxidants to be used in hydrophobic formulations [50]. A series of lipophilic esters derivedtofrom be used in tohydrophobic formulations formulations [50]. A series of A lipophilic derived from ascorbic antioxidants used inby hydrophobic [50]. series ofesters lipophilic esters from ascorbic acid was be prepared using lipase from Staphylococcus xylosus immobilized onderived silica aerogel. acid was prepared by using lipase from Staphylococcus xylosus immobilized on silica aerogel. The ascorbic acid was prepared by using lipase from Staphylococcus xylosus immobilized on silica aerogel. The regioselective enzymatic esterification of Asc with seven carboxylic acids (acetic acid and six fatty regioselective enzymatic esterification of Asc with seven carboxylic acids (acetic acid andand sixsix fatty The regioselective enzymatic esterification of Asc with seven carboxylic acids (acetic acid fatty acids) was was performed in MeCN and 2-methyl-2-propanol (co-solvent) for 72 h. Yields varied with the acids) performed in MeCN and 2-methyl-2-propanol (co-solvent) for 72 h. Yields varied with the acids) was performed in MeCN and 2-methyl-2-propanol (co-solvent) for 72 h. Yields varied with the length of the fatty acid chain, and obtainedon on thepreparation preparation the of the short chain length thefatty fatty acid chain, andthe thebest bestresult result was was short chain length ofofthe acid chain, and the best result was obtained obtained onthe the preparationofof the short chain derivative ascorbyl acetate (82.6 % yield), as seen in Scheme 31. All Asc derivatives were submitted derivativeascorbyl ascorbylacetate acetate(82.6 (82.6 % % yield), yield), as as seen submitted derivative seen in in Scheme Scheme31. 31.All AllAsc Ascderivatives derivativeswere were submitted to some bioassays (antibacterial, antioxidant, and antileishmanial), and the results suggested them to some bioassays (antibacterial, antioxidant, and antileishmanial), and the results suggested them as asas to some bioassays (antibacterial, antioxidant, and antileishmanial), and the results suggested them candidates to used be used in the cosmetic andpharmaceutical pharmaceutical industries [51]. candidates to to be industries [51]. candidates be usedininthe thecosmetic cosmeticand and pharmaceutical industries [51].

Scheme Kinetic enzymatic acylation theascorbic ascorbic acid produce esters [51]. Scheme 31.31. Kinetic acylation ofofthe acid (Asc) toacid produce ascorbyl estersderivatives derivatives [51]. Scheme 31. enzymatic Kinetic enzymatic acylation of the ascorbic (Asc)ascorbyl to produce ascorbyl esters derivatives [51].

17 17

3.21. Key Intermediates of Stagonolide E, Pyrenophorol and Decarestrictine L Enantiomerically pure hept-6-ene-2,5-diol derivatives were prepared by lipase-catalyzed acetylation of the corresponding racemates, and used as key intermediates in the synthesis of the bioactive compounds Stagonolide E, Pyrenophorol and Decarestrictine L (Scheme 32), all of them isolated from filamentous fungi. Racemic 6-methyl-5-hepten-2-ol was resolved by acetylation with Novozym 435, vinyl acetate in hexane at room temperature, and yielded both the (R)-acetate and (S)-alcohol with 98% ee (E > 195) after 50% conversion. The (R)-alcohol was also produced by treatment of (R)-acetate with LiAlH4 . Then, both the (R)- and (S)-alcohols

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isolated from filamentous fungi. Racemic 6-methyl-5-hepten-2-ol was resolved by acetylation with Novozym 435, vinyl acetate in hexane at room temperature, and yielded both the (R)-acetate and (S)-alcohol with 98% ee (E > 195) after 50% conversion. The (R)-alcohol was also produced by treatment of (R)-acetate with LiAlH . Then, both the (R)- and (S)-alcohols were submitted, individually, to some chemical Int. J. 4Mol. Sci. 2015, 16, 29682–29716 steps that included protection of the hydroxyl group with tert-butyldiphenylsilyl (TBDPS), ozonolysis reaction of the obtained with vinylmagnesium providing the wereand submitted, individually, to somealdehydes chemical steps that included protectionbromide, of the hydroxyl allylic alcohols (3RS,6R) and (3RS,6S).(TBDPS), A similar protocol was usedofinthe theobtained enzyme-catalyzed group with tert-butyldiphenylsilyl ozonolysis and reaction aldehydes withacylation the allylic (3RS,6R) and (3RS,6S). A similar protocol of these vinylmagnesium allylic alcoholsbromide, (Schemeproviding 32). In this case, alcohols both key intermediates (3S,6R)-acetate and (3R,6S)was used in the enzyme-catalyzed acylation of these allylic alcohols (Scheme 32). In this case, both alcohol were obtained with high enantioselectivity (E > 195), c 50%. The enantiomerically pure key intermediates (3S,6R)-acetate and (3R,6S)-alcohol were obtained with high enantioselectivity (3R,6S)-alcohol was used as an intermediate in the synthesis of Stagonolide E. The chemoenzymatic (E > 195), c 50%. The enantiomerically pure (3R,6S)-alcohol was used as an intermediate in the synthesissynthesis of Pyrenophorol and Decarestrictine L involved the enantiomericaly pure allylicL (3S,6R)of Stagonolide E. The chemoenzymatic synthesis of Pyrenophorol and Decarestrictine acetate as key intermediate (Scheme [52]. involved the enantiomericaly pure32) allylic (3S,6R)-acetate as key intermediate (Scheme 32) [52].

Scheme Scheme 32. Kinetic enzymatic acylation derivatives to produce key 32. Kinetic enzymatic acylationofof rac-hept-6-ene-2,5-diol rac-hept-6-ene-2,5-diol derivatives to produce key intermediates the synthesis of the Stagonolide E, andand Decarestrictine L [52]. L [52]. intermediates in theinsynthesis of the Stagonolide E,Pyrenophorol Pyrenophorol Decarestrictine Key Intermediates of Macrolide Antibiotic (´)-A26771B 3.22. Key3.22. Intermediates of Macrolide Antibiotic (−)-A26771B The chemoenzymatic synthesis of the macrolide antibiotic (´)-A26771B involved the lipase-catalyzed

Theacylation chemoenzymatic synthesis of the macrolide involved the lipase-catalyzed for resolving both a methylcarbinol andantibiotic an allylic (−)-A26771B alcohol in order to establish the two acylationstereogenic for resolving methylcarbinol and of ancommercial allylic alcohol order to establish centersboth of theatarget molecule. A series lipases in were investigated on the the two acetylation of the rac-methylcarbinol (tridec-12-en-2-ol) with vinyl acetate in hexane or DIPE at 25 ˝ C. on the stereogenic centers of the target molecule. A series of commercial lipases were investigated The of best was achieved when Novozym 435 (lipase B vinyl from Candida immobilized acetylation theresult rac-methylcarbinol (tridec-12-en-2-ol) with acetateantarctica in hexane or DIPE at 25 °C. on acrylic resin) was used as the biocatalyst and DIPE as the solvent. This condition yielded The best result was achieved when Novozym 435 (lipase B from Candida antarctica immobilized on (S)-methylcarbinol (96% ee) and the corresponding (R)-acetate (92% ee) after 2 h (c 51%), as seen in acrylic resin) wasThe used the biocatalyst DIPE asof the therac-allylic solvent.alcohol This(Scheme condition Scheme 33a. sameas condition was used for and the acetylation 33b), yielded (S)-methylcarbinol (96% ee) and the (98% corresponding (R)-acetate (92% ee) 6after 2 h (c 51%), as seen in which produced (3R,13R)-alcohol ee) and (3S,13R)-acetate (95% ee) after h (c 50%) [53]. Scheme 33a. The same condition was used for the acetylation of the rac-allylic alcohol (Scheme 33b), which produced (3R,13R)-alcohol (98% ee) and (3S,13R)-acetate (95% ee) after 6 h (c 50%) [53].

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Int. J. Mol. Sci. 2015, 16, page–page Int. J. Mol. Sci. 2015, 16, page–page

Scheme 33. Kinetic enzymatic acylation of the (a) racemic tridec-12-en-2-ol and (b) (b) allylic alcohol, alcohol, Scheme Kinetic enzymatic enzymatic acylation acylation of of the the (a) (a) racemic racemic tridec-12-en-2-ol tridec-12-en-2-ol and and Scheme 33. 33. Kinetic (b) allylic allylic alcohol, affording key intermediates in the synthesis of the macrolide antibiotic (−)-A26771B [53]. affording [53]. affording key key intermediates intermediates in in the the synthesis synthesis of of the the macrolide macrolide antibiotic antibiotic(´)-A26771B (−)-A26771B [53].

3.23. Key Intermediate of Vitamin E Acetate 3.23. 3.23. Key Key Intermediate Intermediate of of Vitamin Vitamin E E Acetate Acetate Lipase-catalyzed regioselective transesterification between trimethylhydroquinone diacetate Lipase-catalyzed regioselective transesterification between trimethylhydroquinone diacetate (TMHQ-DA) and n-butanol was applied on the preparation of the trimethylhydroquinone-1(TMHQ-DA) n-butanol waswas applied on theon preparation of the trimethylhydroquinone-1-monoacetate (TMHQ-DA)and and n-butanol applied the preparation of the trimethylhydroquinone-1monoacetate (TMHQ-1-MA) derivative, which is an important aromatic intermediate used in the (TMHQ-1-MA) derivative, which is an important aromatic intermediate used in the synthesis of monoacetate (TMHQ-1-MA) derivative, which is an important aromatic intermediate used in the synthesis of Vitamin E acetate (commercialized form of Vitamin E), as seen in Scheme 34. Seven Vitamin acetate (commercialized form of Vitamin E),ofasVitamin seen in E), Scheme 34. in Seven commercially synthesisE of Vitamin E acetate (commercialized form as seen Scheme 34. Seven commercially available lipases were screened, and the highest yield (99.1%) of the TMHQ-1-MA was available lipases were screened, and the highest yield (99.1%) of the TMHQ-1-MA was obtained commercially available lipases were screened, and the highest yield (99.1%) of the TMHQ-1-MA was obtained using Lipozyme RM IM (lipase from Rhizomucor miehei). No activity was observed when using Lipozyme RM IM (lipase Rhizomucor miehei). miehei). No activity was observed when using obtained using Lipozyme RM IMfrom (lipase from Rhizomucor No activity was observed when using lipase A (from Aspergillus niger) as an enzyme. Except for Lipozyme 435 (CAL-B, lipase B from lipase A (from Aspergillus niger)niger) as anasenzyme. Except forfor Lipozyme using lipase A (from Aspergillus an enzyme. Except Lipozyme435 435(CAL-B, (CAL-B,lipase lipase BB from Candida antarctica immobilized on macroporous polyacrylate resin), all active enzymes showed 100% Candida antarctica immobilized on macroporous polyacrylate resin), all active enzymes showed 100% Candida antarctica resin), regioselectivity on the formation of TMHQ-1-MA. Several reaction parameters were investigated and regioselectivity on the formation of TMHQ-1-MA. Several reaction parameters were investigated and the optimum conditions for the Lipozyme RM IM catalyzed regioselective transesterification were: the optimum optimum conditions conditions for the the Lipozyme Lipozyme RM RM IM IMcatalyzed catalyzedregioselective regioselective transesterification transesterification were: were: TMHQ-DA:n-butanol ratio of 1:1, 200 rpm, 50 °C, TBME:n-hexane (3:7) as a solvent. In such ˝ C, and TMHQ-DA:n-butanol ratio of 1:1, 200 rpm, 50 and TBME:n-hexane (3:7) as aa solvent. TMHQ-DA:n-butanol ratio of 1:1, 200 rpm, 50 °C, and TBME:n-hexane solvent. In such conditions, the enzyme was active even after 20 cycles. A Ping-Pong bi-bi mechanism with n-butanol conditions, the enzyme was active even after 20 cycles. A Ping-Pong bi-bi mechanism with n-butanol inhibition was proposed based on the initial rate data and concentration profiles [54]. inhibition was proposed proposed based based on on the the initial initial rate rate data data and and concentration concentration profiles profiles[54]. [54].

Scheme 34. Lipase-catalyzed regioselective transesterification between TMHQ-DA and n-butanol on Scheme 34. 34. Lipase-catalyzed regioselective transesterification between between TMHQ-DA TMHQ-DA and and n-butanol n-butanol on on Scheme Lipase-catalyzed regioselective transesterification the preparation of TMHQ-1-MA, an intermediate in the synthesis of Vitamin E acetate [54]. the preparation of TMHQ-1-MA, an intermediate in the synthesis of Vitamin E acetate [54]. the preparation of TMHQ-1-MA, an intermediate in the synthesis of Vitamin E acetate [54].

3.24. Rasagiline Mesylate 3.24. Rasagiline Mesylate 3.24. Rasagiline Mesylate A straightforward chemoenzymatic synthesis of Rasagiline mesylate has been developed A straightforward chemoenzymatic synthesis of Rasagiline mesylate has been developed A chemoenzymatic synthesis of Rasagiline mesylate has been (Schemestraightforward 35). This drug is used in monotherapy of Parkinson patients at an early stage,developed and as an (Scheme 35). This drug is used in monotherapy of Parkinson patients at an early stage, and as an (Scheme 35). This drug is used instage monotherapy of Parkinson patientsbegan at anwith earlythestage, and adjunct to moderate the advanced of the disease. The synthesis chemical adjunct to moderate the advanced stage of the disease. The synthesis began with the chemical as an adjunct to moderate advanced stageyield. of the disease. The was synthesis began with the reduction of indanone to givethe rac-indanol in 86% Then, rac-indanol subjected to enzymatic reduction of indanone to give rac-indanol in 86% yield. Then, rac-indanol was subjected to enzymatic chemical reduction of indanone to give rac-indanol in 86% yield. rac-indanol was subjected kinetic resolution using the lipase from Thermomyces lanuginosus (TLL) Then, immobilized on immobead-150 as kinetic resolution using the lipase from Thermomyces lanuginosus (TLL) immobilized on immobead-150 as to enzymatic hexane kinetic resolution using the lipase Thermomyces lanuginosus a biocatalyst, as an organic solvent, vinylfrom acetate as an acyl donor, at 35 (TLL) °C andimmobilized with 15 minon of a biocatalyst, hexane as an organic solvent, vinyl acetate as an acyl donor, at 35 °C and with 15 min˝of immobead-150 a biocatalyst, hexaneacetate as an organic solvent, vinyl acetate aswith an acyl donor, at 35and C reaction time. Inasthis case, (R)-indanyl and (S)-indanol were obtained >99% ee, c 50% reaction time. In this case, (R)-indanyl acetate and (S)-indanol were obtained with >99% ee, c 50% and and minthe of reaction time.was In this case, (R)-indanyl acetate (S)-indanol obtained witha E > with 200. 15 Then, (S)-indanol subjected to a sequence ofand chemical steps,were which included E > 200. Then, the (S)-indanol was subjected to a sequence of chemical steps, which included a Mitsunobu reaction, a Staudinger reaction, the introduction of the propargyl group and, finally, the Mitsunobu reaction, a Staudinger reaction, the introduction of the propargyl group and, finally, the reaction with methanesulfonic acid, which afforded Rasagiline mesylate. Immobilized lipase from reaction with methanesulfonic acid, which afforded 29702 Rasagiline mesylate. Immobilized lipase from 19 19

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>99% ee, c 50% and E > 200. Then, the (S)-indanol was subjected to a sequence of chemical steps, which included a Mitsunobu reaction, a Staudinger reaction, the introduction of the propargyl group and, Int. J. Mol. Sci. 2015, 16, page–page finally, with methanesulfonic acid, which afforded Rasagiline mesylate. Immobilized Int. J. Mol.the Sci. reaction 2015, 16, page–page lipase from T. lanuginosus wastofound to be an efficient biocatalyst to produce(S)-indanol (S)-indanolwith with high T. lanuginosus was found be an efficient biocatalyst to produce high ˝ C andto15 produce T. lanuginosus was found to be an efficient biocatalyst (S)-indanol with high enantioselectivity (>99% ee, E > 200) in hexane, at 35 min. Additionally, this enzyme enantioselectivity (>99% ee, E > 200) in hexane, at 35 °C and 15 min. Additionally, this enzyme was enantioselectivity (>99% ee, E > 200) in hexane, at 35 °C and 15 min. Additionally, this enzyme was was reused 10 times, maintaining both the activity and selectivity unchanged. This preparation reused 10 times, maintaining both the activity and selectivity unchanged. This preparation of of reused 10 mesylate times, maintaining both the and selectivity unchanged. of Rasagiline can considered an activity environmentally sustainable strategy, This since it Rasagiline mesylate can be be considered an environmentally sustainable strategy, since preparation it involved involved the the Rasagiline mesylate can an environmentally sustainable strategy,for since it involved the use that is commercially available, stable, multiple reaction use of of aa biocatalyst biocatalyst thatbe is considered commercially available, low-cost, low-cost, stable, reusable reusable for multiple reaction use of and a biocatalyst that is commercially cycles highly [55,56]. cycles and highly enantioselective enantioselective [55,56]. available, low-cost, stable, reusable for multiple reaction cycles and highly enantioselective [55,56].

Scheme 35. 35. Enzymatic the chemoenzymatic chemoenzymatic synthesis synthesis of of Rasagiline Rasagiline Scheme Enzymatic kinetic kinetic resolution resolution rac-indanol rac-indanol in in the Scheme 35. Enzymatic kinetic resolution rac-indanol in the chemoenzymatic synthesis of Rasagiline mesylate [55,56]. mesylate [55,56]. mesylate [55,56].

Scheme 36. Enzymatic kinetic resolution of rac-N-Boc-HMTHQ to prepare the (R)- and (S)-3-methylScheme 36. Enzymatic kinetic resolution of rac-N-Boc-HMTHQ to prepare the (R)- and (S)-3-methyl1,2,3,4-tetrahydroquinoline, chiral intermediates for the preparation of thethe antithrombotic (21R)- and Scheme 36. Enzymatic kinetic resolution of rac-N-Boc-HMTHQ to prepare (R)- and (S)-3-methyl-1, 1,2,3,4-tetrahydroquinoline, chiral intermediates for the preparation of the antithrombotic (21R)- and (21S)-Argatroban [57]. 2,3,4-tetrahydroquinoline, chiral intermediates for the preparation of the antithrombotic (21R)- and (21S)-Argatroban [57]. (21S)-Argatroban [57].

3.25. Argatroban 3.25. Argatroban 3.25. Argatroban Argatroban is an inhibitor of thrombim, the protease that plays a key role in blood coagulation Argatroban is an inhibitor of thrombim, the protease that plays a key role in blood coagulation and fibrinolysis. The mixture epimersthat ((21R)(21S)-epimers) used as an Argatroban is andiastereoisomeric inhibitor of thrombim, theof protease playsand a key role in bloodiscoagulation and fibrinolysis. The diastereoisomeric mixture of epimers ((21R)and (21S)-epimers) is used an antithrombotic drug, but the (21S)-isomer is twice as potent as 21R. The chiral intermediates (R)-as and fibrinolysis. The diastereoisomeric mixture of epimers ((21R)- and (21S)-epimers) is used asand an antithrombotic drug, but the (21S)-isomerare is twice as potent as 21R. The of chiral intermediates (R)and (S)-3-methyl-1,2,3,4-tetrahydroquinoline for the preparation (21R)antithrombotic drug, but the (21S)-isomer used is twice as potent as 21R. the Theantithrombotic chiral intermediates (S)-3-methyl-1,2,3,4-tetrahydroquinoline are were used prepared for the preparation of the antithrombotic and and (21S)-Argatroban. These intermediates 3-quinoline acid(21R)as a (R)(S)-3-methyl-1,2,3,4-tetrahydroquinoline are used forusing the preparation ofcarboxylic the antithrombotic and (21S)-Argatroban. These intermediates were prepared using 3-quinoline carboxylic acid as a starting material. After some chemical steps, the rac-3-(1’-hydroxymethyl)-1-tert-butyloxycarbonyl(21R)- and (21S)-Argatroban. These intermediates were prepared using 3-quinoline carboxylic acid as a starting material. After some chemical steps, the was rac-3-(1’-hydroxymethyl)-1-tert-butyloxycarbonyl1,2,3,4-tetrahydroquinoline obtained, and subjected to kinetic resolution in starting material. After some(rac-N-Boc-HMTHQ) chemical steps, the rac-3-(1’-hydroxymethyl)-1-tert-butyloxycarbonyl-1, 1,2,3,4-tetrahydroquinoline (rac-N-Boc-HMTHQ) was obtained, and subjected to kinetic resolution in the presence of lipase from Pseudomonas fluorescens (PFL), with and toluene as a solvent and resolution vinyl acetate 2,3,4-tetrahydroquinoline (rac-N-Boc-HMTHQ) was obtained, subjected to kinetic in the presence of lipase from Pseudomonas fluorescens (PFL), with toluene as a solvent and vinyl acetate as an acyl donor. Afterfrom this procedure, thefluorescens (S)-ester (>99% for ctoluene 30%–40%) the (R)-alcohol ee the presence of lipase Pseudomonas (PFL),eewith as aand solvent and vinyl (81% acetate as an acyl donor. After this procedure, the (S)-ester (>99% ee for c 30%–40%) and the (R)-alcohol (81% eea for c 55–65%) were obtained. For obtaining the enantiomerically enriched (R)-alcohol (>99% ee), as an acyl donor. After this procedure, the (S)-ester (>99% ee for c 30%–40%) and the (R)-alcohol for c 55–65%) were obtained. For obtaining thethe enantiomerically enriched (R)-alcohol ee), a second was performed aforementioned conditions using the(>99% (R)-alcohol (81% eekinetic for c resolution 55–65%) were obtained. under For obtaining the enantiomerically enriched (R)-alcohol second kinetic resolution was performed underatthe aforementioned conditions the(Scheme (R)-alcohol (81% ee). case, kinetic the reaction was stopped a conversion from 30%using to 45% 36). (>99% ee),Inathis second resolution was performed under ranging the aforementioned conditions using (81% ee). In this case, the reaction was stopped at a conversion ranging from 30% to 45% (Scheme 36). The (S)-acetate (>99% ee) was converted into the corresponding (S)-alcohol by enzymatic hydrolysis the (R)-alcohol (81% ee). In this case, the reaction was stopped at a conversion ranging from 30% The (S)-acetate ee) Subsequently, was converted both into the (S)-alcohol by enzymatic hydrolysis in the presence(>99% of PFL. (S)-corresponding and (R)-alcohol were subjected to a sequence of in the presence of PFL. Subsequently, both (S)and (R)-alcohol were subjected to a sequence of chemical steps which included tosylation, reaction with lithium aluminum hydride and removal of 29703with lithium aluminum hydride and removal of chemical steps which included tosylation, reaction the Boc protecting group, leading to enantiomerically pure intermediates (S)- and (R)-3-methylthe Boc protecting group, leading to enantiomerically pure intermediates (S)- and (R)-3-methyl1,2,3,4-tetrahydroquinoline [57]. 1,2,3,4-tetrahydroquinoline [57].

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to 45% (Scheme 36). The (S)-acetate (>99% ee) was converted into the corresponding (S)-alcohol by enzymatic hydrolysis in the presence of PFL. Subsequently, both (S)- and (R)-alcohol were subjected to a sequence of chemical steps which included tosylation, reaction with lithium aluminum hydride and removal of the Boc protecting group, leading to enantiomerically pure intermediates (S)- and (R)-3-methyl-1,2,3,4-tetrahydroquinoline [57]. Int. J. Mol. Sci. 2015, 16, page–page 3.26. Intermediate of 3.26. Key Key Intermediate of the the Paclitaxel Paclitaxel Side Side Chain Chain The synthesis synthesis of ofaakey keyintermediate intermediate Paclitaxel chain, (2R,3S)-3-phenylisoserine, forfor thethe Paclitaxel sideside chain, (2R,3S)-3-phenylisoserine, was was performed by kinetic enzymatic resolutionofofracemic racemic N-hydroxymethylated N-hydroxymethylated cis-3-acetoxy-4performed by kinetic enzymatic resolution cis-3-acetoxy-4phenylazetidin-2-one (rac-azetidin-2-one (rac-azetidin-2-one derivative), derivative), via via acylation acylation reaction reaction (Scheme (Scheme 37). 37). Various parameters were evaluated such as lipases from Burkholderia cepacia (PS IM), Candida Burkholderia cepacia Candida antarctica antarctica A (CAL-A), Pseudomonas AY); acyl Pseudomonas fluorescens fluorescens (lipase AK) and C. C. rugosa rugosa (lipase AY); acyl donors such as vinyl ˝ C) and organic acetate, vinyl butyrate and 2,2,2-trifluoroethyl 2,2,2-trifluoroethyl butyrate; butyrate; temperature temperature (4, (4, 25 and 50 °C) solvent (DIPE, (DIPE, toluene, toluene,TBME TBMEand and 2-MeTHF). results obtained the presence of 2-MeTHF). TheThe bestbest results werewere obtained in theinpresence of lipase ˝ lipase IM,asDIPE as avinyl solvent, vinylasbutyrate as an at acyl donor, at 25 C.was The reaction with was PS IM, PS DIPE a solvent, butyrate an acyl donor, 25 °C. The reaction conducted conducted with a higher amount of substrate (100 mg) and, after 20 min, the conversion reached a higher amount of substrate (100 mg) and, after 20 min, the conversion reached 50%, with the 50%, with of thethe formation of the with diester (3R,4S) with ee and the remaining formation diester (3R,4S) >99% ee and the >99% remaining substrate (3S,4R) substrate with 98% (3S,4R) ee, and with 98% ee, traces and Eof > a200, with traces byproductoffrom the migration ofthe the(3R,4S)-diester acyl group. Both E > 200, with byproduct fromof thea migration the acyl group. Both and the (3R,4S)-diester and the remaining (3S,4R)-substrate were subjected to acid hydrolysis leading to remaining (3S,4R)-substrate were subjected to acid hydrolysis leading to (2R,3S)-3(2R,3S)-3-phenylisoserine hydrochloride with (key intermediate the Paclitaxel side chain) phenylisoserine hydrochloride with >99% ee >99% (key ee intermediate for thefor Paclitaxel side chain) and and (2S,3R)-3-phenylisoserine hydrochloride ee [58]. (2S,3R)-3-phenylisoserine hydrochloride withwith 98%98% ee [58].

Scheme 37. Enzymatic kinetic resolution of racemic N-hydroxymethylated cis-3-acetoxy-4Scheme 37. Enzymatic kinetic resolution of racemic N-hydroxymethylated cis-3-acetoxy-4phenylazetidin-2-one (rac-azetidin-2-one derivative), affording the key intermediate for the Paclitaxel phenylazetidin-2-one (rac-azetidin-2-one derivative), affording the key intermediate for the Paclitaxel side chain, (2R,3S)-3-phenylisoserine [58]. side chain, (2R,3S)-3-phenylisoserine [58].

3.27. Piperidine Derivative 3.27. Piperidine Derivative The chiral compound (S)-4-(1-(3,4-dichlorophenyl)-2-methoxyethyl)piperidine is a novel tripleThe chiral compound (S)-4-(1-(3,4-dichlorophenyl)-2-methoxyethyl)piperidine is a novel reuptake inhibitor (inhibiting the reuptake of serotonin, norepinephrine and dopamine), which was triple-reuptake inhibitor (inhibiting the reuptake of serotonin, norepinephrine and dopamine), created as a candidate for a novel antidepressant agent. Among the strategies to synthesize the which was created as a candidate for a novel antidepressant agent. Among the strategies to synthesize aforementioned compound, it is possible to highlight the enzymatic kinetic resolution of racemic tertthe aforementioned compound, it is possible to highlight the enzymatic kinetic resolution of butyl 4-(1-(3,4-dichlorophenyl)-2-hydroxyethyl)piperidine-1-carboxylate (rac-alcohol) via acetylation racemic tert-butyl 4-(1-(3,4-dichlorophenyl)-2-hydroxyethyl)piperidine-1-carboxylate (rac-alcohol) in the presence of lipases [59]. A preliminary enzyme screening was conducted using 38 lipases, via acetylation in the presence of lipases [59]. A preliminary enzyme screening was conducted using wherein the lipase from Pseudomonas sp. immobilized on diatomite (Amano Enzyme Inc.) gave the 38 lipases, wherein the lipase from Pseudomonas sp. immobilized on diatomite (Amano Enzyme Inc.) best E-value (E 243). Several solvents were evaluated such as DIPE, THF, acetone, MeCN, DMF and TBME. In this case, DIPE showed the best performance leading to an E-value of 525. Thus, the kinetic 29704 resolution was performed in the optimized conditions using lipase PS IM (from Pseudomonas sp.), vinyl acetate as an acyl donor and DIPE as a solvent, at 35 °C. After the reaction reached 48% conversion, the product (S)-ester was obtained with 96% ee and (R)-alcohol with >99% ee, and E > 200

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gave the best E-value (E 243). Several solvents were evaluated such as DIPE, THF, acetone, MeCN, DMF and TBME. In this case, DIPE showed the best performance leading to an E-value of 525. Thus, the kinetic resolution was performed in the optimized conditions using lipase PS IM (from Pseudomonas sp.), vinyl acetate as an acyl donor and DIPE as a solvent, at 35 ˝ C. After the reaction reached 48% conversion, the product (S)-ester was obtained with 96% ee and (R)-alcohol with >99% ee, and E > 200 (Scheme 38). Then, after some chemical steps, the (S)-ester was converted into the triple-reuptake inhibitor (S)-4-(1-(3,4-dichlorophenyl)-2-methoxyethyl)piperidine with 96% ee. Int. J. Mol. Sci. 2015, 16, page–page It is worth mentioning that the reaction was performed on a 10 g scale, and after 40 h it was necessary to add additional and vinyl acetate. h of agitation, thekinetic kineticresolution resolution gave gave an additional lipase lipase and vinyl acetate. AfterAfter overover 24 h24of agitation, the an excellent enantioselectivity. excellent enantioselectivity.

Scheme 38. 38. Enzymatic 4-(1-(3,4-dichlorophenyl)-2Scheme Enzymatic kinetic kinetic resolution resolution of of racemic racemic tert-butyl tert-butyl 4-(1-(3,4-dichlorophenyl)-2hydroxyethyl)piperidine-1-carboxylate (rac-alcohol), affording the novel triple reuptake inhibitor (S)hydroxyethyl)piperidine-1-carboxylate (rac-alcohol), affording the novel triple reuptake inhibitor 4-(1-(3,4-dichlorophenyl)-2-methoxyethyl)piperidine [59]. (S)-4-(1-(3,4-dichlorophenyl)-2-methoxyethyl)piperidine [59].

3.28. Tetrahydroquinolinol and Tetrahydrobenzoazepinol Derivatives 3.28. Tetrahydroquinolinol and Tetrahydrobenzoazepinol Derivatives Tetrahydroquinolin-4-ols and tetrahydro-1H-benzo[b]azepin-5-ols, in chiral form, are building Tetrahydroquinolin-4-ols and tetrahydro-1H-benzo[b]azepin-5-ols, in chiral form, are building blocks for drug candidates due to their promising biological activities. A series of racemic blocks for drug candidates due to their promising biological activities. A series of racemic N-protected N-protected tetrahydroquinolin-4-ols were chemically prepared from 1,2,3,4-tetrahydroquinoline. tetrahydroquinolin-4-ols were chemically prepared from 1,2,3,4-tetrahydroquinoline. Subsequently, Subsequently, the rac-N-Boc-tetrahydroquinolin-4-ol (R1 = H; R2 = Boc) was subjected to kinetic the rac-N-Boc-tetrahydroquinolin-4-ol (R1 = H; R2 = Boc) was subjected to kinetic resolution in the resolution in the presence of lipases, using TBME as a solvent and vinyl acetate as an acyl donor, at presence of lipases, using TBME as a solvent and vinyl acetate as an acyl donor, at 30 ˝ C and for 30 °C and for 8 h (n = 1, Scheme 39 and Table 4). Among the evaluated lipases (Amano lipase Type VII, 8 h (n = 1, Scheme 39 and Table 4). Among the evaluated lipases (Amano lipase Type VII, CAL-A, CAL-A, Amano lipase A, Novozym 435, lipase RM IM and lipase TL IM), Novozym 435 lipase Amano lipase A, Novozym 435, lipase RM IM and lipase TL IM), Novozym 435 lipase provided provided the highest enantioselectivity for the (S)-alcohol (98% ee) and the (R)-acetate (97% ee), c 50% the highest enantioselectivity for the (S)-alcohol (98% ee) and the (R)-acetate (97% ee), c 50% and and E > 200. Then, under the same conditions, various acyl donors were tested (vinyl chloroacetate, E > 200. Then, under the same conditions, various acyl donors were tested (vinyl chloroacetate, divinyl heptanedioate, vinyl hexanoate, vinyl undecanoate and vinyl 2,2-dimethylpropionate). In this divinyl heptanedioate, vinyl hexanoate, vinyl undecanoate and vinyl 2,2-dimethylpropionate). In this case, vinyl chloroacetate was the most effective acyl donor, producing both (R)-ester and (S)-alcohol case, vinyl chloroacetate was the most effective acyl donor, producing both (R)-ester and (S)-alcohol with >99% ee, c 50% and E > 200. Besides TBME, other solvents (DIPE, MeCN, toluene and n-hexane) with >99% ee, c 50% and E > 200. Besides TBME, other solvents (DIPE, MeCN, toluene and n-hexane) were evaluated, but TBME proved to be more efficient in the kinetic resolution. After optimizing the were evaluated, but TBME proved to be more efficient in the kinetic resolution. After optimizing the reaction conditions (Novozym 435, TBME as a solvent, vinyl chloroacetate as an acyl donor, 30 °C, 8 h), reaction conditions (Novozym 435, TBME as a solvent, vinyl chloroacetate as an acyl donor, 30 ˝ C, the kinetic resolution was extended to the other racemic N-protected tetrahydroquinolin-4-ols 8 h), the kinetic resolution was extended to the other racemic N-protected tetrahydroquinolin-4-ols containing substituent groups at the aromatic ring (C-6) and different N-protecting groups (Table 4). containing substituent groups at the aromatic ring (C-6) and different N-protecting groups (Table 4). In general, the kinetic resolutions provided the (R)-esters ranging from 37% to 49% yields, 92% to In general, the kinetic resolutions provided the (R)-esters ranging from 37% to 49% yields, 92% to >99% ee, and the (S)-alcohols ranging from 29% to 48% yields, 94% to >99% ee, with E-values ranging >99% ee, and the (S)-alcohols ranging from 29% to 48% yields, 94% to >99% ee, with E-values ranging from 126 to >200. It is worth noting that the formation of any desired products was not observed when the substrate had the benzyl group linked to the nitrogen atom (n = 1; R1 = H; R2 = Bn), as seen 29705 in Scheme 39 and Table 4. In this same study, the authors conducted the kinetic resolution of racemic N-protected tetrahydro-1H-benzo[b]azepin-5-ols (R = Bz, 2-furoyl and Cbz), (n = 2, Scheme 39 and Table 4). As result, the kinetic resolutions produced the corresponding (R)-esters and (S)-alcohols in excellent yields (up to 44%) and enantioselectivity (ee up to 98% and E > 200) [60].

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from 126 to >200. It is worth noting that the formation of any desired products was not observed when the substrate had the benzyl group linked to the nitrogen atom (n = 1; R1 = H; R2 = Bn), as seen in Scheme 39 and Table 4. In this same study, the authors conducted the kinetic resolution of racemic N-protected tetrahydro-1H-benzo[b]azepin-5-ols (R = Bz, 2-furoyl and Cbz), (n = 2, Scheme 39 and Table 4). As result, the kinetic resolutions produced the corresponding (R)-esters and (S)-alcohols in excellent yields to 44%) and enantioselectivity (ee up to 98% and E > 200) [60]. Int. J. Mol. Sci. 2015,(up 16, page–page

Scheme 39. 39. Enzymatic Enzymatickinetic kineticresolution resolutionofof the racemic compounds N-protected tetrahydroquinolinScheme the racemic compounds N-protected tetrahydroquinolin-4-ols 4-ols (n and = 1) N-protected and N-protected tetrahydro-1H-benzo[b]azepin-5-ols to produce building blocks (n = 1) tetrahydro-1H-benzo[b]azepin-5-ols (n = (n 2) =to2)produce building blocks for for drug candidates [60]. drug candidates [60]. Table 4. 4. Substrates tetrahydroquinolinol and and Table Substrates used used for for the the enzymatic enzymatic kinetic kinetic resolution resolution to to produce produce tetrahydroquinolinol tetrahydrobenzoazepinol derivatives derivatives [60]. [60]. tetrahydrobenzoazepinol

Substrates

Substrates

rac-N-protect tetrahydroquinolin-4-ols rac-N-protect tetrahydroquinolin-4-ols

rac-N-protect tetrahydro-1H-benzo[b]azepin-5-ols

rac-N-protect tetrahydro-1H-benzo[b]azepin-5-ols 3.29. Aminohydroxypiperidine Derivatives

n

n

R1

R1

H

1 1

2

2

R2

R2

Boc

H Cl Cl Br Br OMe OMe H H H H H H H H

Boc Boc Boc Boc Boc Ac Ac CO2 Ph CO 2Ph Cbz Bz Cbz 2-furoyl Bz Cbz

H H

2-furoyl Cbz

trans-3-amino-4-hydroxypiperidine moiety is present in the structures of some bioactive 3.29. The Aminohydroxypiperidine Derivatives compounds, such as an inhibitor of non-receptor tyrosine kinase (Scheme 40) that is used for the treatment The trans-3-amino-4-hydroxypiperidine moiety protected is present(3R,4R)-3-amino-4-hydroxypiperidine in the structures of some bioactive of auto-immune disorders. Enantiopure, orthogonally compounds, such as an of non-receptor 40) thatracemate. is used for The the acetate was obtained byinhibitor lipase-mediated kinetic tyrosine acylationkinase of the(Scheme corresponding treatment of auto-immune disorders. Enantiopure, orthogonally protected (3R,4R)-3-amino-4racemic trans-N-benzyl-3-(diallylamino)-4-hydroxypiperidine (trans- hydroxypiperidine derivative) hydroxypiperidine was obtained by lipase-mediated acylation of the corresponding was obtained by theacetate regioselective opening of the epoxide atkinetic the rac-1-benzyl-3,4-epoxypiperidine racemate. The racemic trans-N-benzyl-3-(diallylamino)-4-hydroxypiperidine (trans- hydroxypiperidine (rac-epoxide) with diallylamine. Later, it was subjected to the enzymatic transesterification reaction derivative) was obtained by the regioselective opening of the epoxide at the rac-1-benzyl-3,4with vinyl acetate (5 eq.) as the acyl donor, lipases from Candida antarctica type A (lipase NZL-101) epoxypiperidine with Burkholderia diallylamine.cepacia Later,(PSL it was to fluorescens the enzymatic and C. antarctica (rac-epoxide) B (Novozym 435), IM), subjected Pseudomonas (AK) transesterification reaction with vinyl acetate (5 eq.) as the acyl donor, lipases from Candida antarctica ˝ and Thermomyces lanuginosus (TL IM), in TBME as the solvent, and at 30 C. Although all lipases type A (lipase andthe C. antarctica (Novozym 435), Burkholderia cepacia (PSL IM), Pseudomonas catalyzed the NZL-101) acylation of substrate Bwith the same stereochemical preference, (3R,4R)-product fluorescens (AK) and Thermomyces lanuginosus (TL IM), in(11%–31%). TBME as theThus, solvent, at 30 °C. and (3S,4S)-substrate, the conversions were moderate theand influence ofAlthough different all lipases catalyzed the acylation of the substrate with the same stereochemical preference, (3R,4R)reaction parameters (organic solvent, temperature, and triethylamine as additive) was studied in product and (3S,4S)-substrate, the conversions were moderate (11%–31%). Thus, the influence of order to improve the reaction rate using the lipases CAL-A and CAL-B. The best result was obtained different reaction parameters (organic solvent, temperature, and triethylamine as additive) was ˝ by using CAL-B, TBME/Et3 N (10:1) as the solvent, three days of reaction time, and at 45 C. In this studied order toresolution improve the reaction using the lipases CAL-A and CAL-B. with The best result was case, theinkinetic yielded therate (3R,4R)-product and (3S,4S)-substrate enantiomeric obtained using CAL-B, TBME/Et3N (10:1) the E solvent, days40). of reaction time, and at 45 °C. In excess of by >99% and 81%, respectively, c 45%asand > 200 three (Scheme It should be mentioned that this case, the kinetic resolution yielded the (3R,4R)-product and (3S,4S)-substrate with enantiomeric excess of >99% and 81%, respectively, c 45% and E > 200 (Scheme 40). It should be mentioned that 29706 when the reaction was performed without triethylamine as an additive, a significant decrease in the reaction rate was observed (from three to seven days) [61].

Int. J. Mol. Sci. 2015, 16, 29682–29716

when the reaction was performed without triethylamine as an additive, a significant decrease in the reaction rate2015, was16,observed Int. J. Mol. Sci. page–page(from three to seven days) [61].

Scheme 40. Enzymatic Scheme 40. Enzymatic kinetic kinetic resolution resolution of of the the racemic racemic trans-hydroxypiperidine trans-hydroxypiperidine derivative derivative to to produce the orthogonally protected (3R,4R)-3-amino-4-hydroxypiperidine, which is present produce the orthogonally protected (3R,4R)-3-amino-4-hydroxypiperidine, which is present in in the the structure of an an inhibitor inhibitor of of non-receptor non-receptor tyrosine tyrosine kinase kinase [61]. [61]. structure of

3.30. 3.30. Azole Azole Derivatives Derivatives The azole subunit subunit is is part part of of the the structure structure of The azole of aa number number of of drugs drugs such such as as Econazole, Econazole, Fluconazole, Fluconazole, Ketoconalole, and Ravuconazole. Ravuconazole. It Ketoconalole, Miconazole, Miconazole, Voriconazole Voriconazole and It is is noteworthy noteworthy that that cycloalkyl cycloalkyl azoles azoles have described as as potent potent antileishmanial antileishmanial agents. agents. A have been been described A new new family family of of racemic racemic transtrans- and and cis-azole cis-azole derivatives derivatives was was prepared prepared and and submitted submitted to to aa kinetic kinetic resolution resolution through through transesterification transesterification in in the the presence of lipases (Scheme 41). The kinetic resolution was conducted with the racemic trans-3-(1Hpresence of lipases (Scheme 41). The kinetic resolution was conducted with the racemic trans-3imidazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ol (rac-trans-3-imidazol THN) in theinpresence of vinyl (1H-imidazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ol (rac-trans-3-imidazol THN) the presence of acetate as the acyl donor and THF as the solvent (Scheme 41a and Table 5). Several lipases were vinyl acetate as the acyl donor and THF as the solvent (Scheme 41a and Table 5). Several lipases evaluated, such assuch lipases from Candida antarcticaantarctica B (CAL-B,B immobilized by adsorption in Lewatit E), were evaluated, as lipases from Candida (CAL-B, immobilized by adsorption in from C. antarctica A (CAL-A), from C. rugosa (CRL), lipase AK from Pseudomonas fluorescens, from Lewatit E), from C. antarctica A (CAL-A), from C. rugosa (CRL), lipase AK from Pseudomonas fluorescens, P. cepacia (PSL), fromfrom Rhizomucor miehei (RML), porcine from P. cepacia (PSL), Rhizomucor miehei (RML), porcinepancreatic pancreaticlipase lipase (PPL), (PPL), and and from from Thermomyces lanuginosus (TLL). Among them, only PSL produced an appreciable degree of Thermomyces lanuginosus (TLL). Among them, only PSL produced an appreciable degree of activity activity with commercially available types of PSL werewere evaluated, and with good goodselectivity. selectivity.Subsequently, Subsequently,several several commercially available types of PSL evaluated, PSL-C I (immobilized onto a ceramic carrier) was the most efficient (with a ratio of 1:1 lipase/substrate and PSL-C I (immobilized onto a ceramic carrier) was the most efficient (with a ratio of 1:1 in weight), producing (after 48 h at 30 °C) trans-(2R,3R)-acetate in 95% ee and trans-(2S,3S)-alcohol lipase/substrate in weight), producing (after 48 h at 30 ˝ C) trans-(2R,3R)-acetate in 95% ee and with 83% ee, c 46%, E-value of 102. PSL-C I was also the most efficient in the kinetic resolution of the the trans-(2S,3S)-alcohol with 83% ee, c 46%, E-value of 102. PSL-C I was also the most efficient in cis-isomer. After 24 h at 45 °C, using the ratio 2:1 of lipase:substrate in weight, the cis-(2R,3S)-acetate ˝ kinetic resolution of the cis-isomer. After 24 h at 45 C, using the ratio 2:1 of lipase:substrate in weight, (95% ee) and cis-(2S,3R)-alcohol ee) were obtained with 43%were and obtained an E-value of 83c (Scheme the cis-(2R,3S)-acetate (95% ee) (71% and cis-(2S,3R)-alcohol (71%c ee) with 43% and41a an and Table The strategy used 5). to The prepare theused chiral imidazole derivatives was used to E-value of 835). (Scheme 41a and Table strategy to prepare the chiral imidazole derivatives enantioselectively obtain the transandthe cis-triazol compounds 41b and Table41b 5). Again, PSL-C was used to enantioselectively obtain trans- and cis-triazol(Scheme compounds (Scheme and Table 5). IAgain, was the most efficient enzyme to promote the kinetic resolution of both racemic transand cis-3-(1HPSL-C I was the most efficient enzyme to promote the kinetic resolution of both racemic 1,2,4-triazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ols (rac-trans- and (rac-transrac-cis-3-triazol THN). The trans- and cis-3-(1H-1,2,4-triazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ols and rac-cis-3-triazol experiments were performed using THF as the solvent and vinyl acetate as the acyl donor, 30 °C THN). The experiments were performed using THF as the solvent and vinyl acetate as the acylatdonor, and The 48 reaction the rac-transprovided trans-(2R,3R)-acetate with 99% eewith and at 3048˝ Ch.and h. Thewith reaction with theisomer rac-transisomerthe provided the trans-(2R,3R)-acetate trans-(2S,3S)-alcohol with 93% ee, c 48% and E > 200. In the case of the rac-cis-isomer, it was obtained 99% ee and trans-(2S,3S)-alcohol with 93% ee, c 48% and E > 200. In the case of the rac-cis-isomer, from cis-(2R,3S)-acetate with >99% ee and with cis-(2S,3R)-alcohol 29% ee, c 23%with and E > 200 it wasthe obtained from the cis-(2R,3S)-acetate >99% ee and with cis-(2S,3R)-alcohol 29% ee, (Scheme 41b and Table 5) [62]. Tetrahydronaphthylazoles are known to exhibit antileishmanial properties, which has inspired the authors [62] to resolve the racemate of trans- and cis-1-(1Himidazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ols (rac-trans- and rac-cis-1-imidazol THN) using the 29707 same aforementioned enzymatic methodology. The kinetic resolutions of these isomers were conducted in THF as a solvent and vinyl acetate as an acyl donor, at 30 °C and for 48 h. The lipases CAL-B and PSL-C I were tested and the latter led to the best results. Thus, for the rac-trans-isomer,

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c 23% and E > 200 (Scheme 41b and Table 5) [62]. Tetrahydronaphthylazoles are known to exhibit antileishmanial properties, which has inspired the authors [62] to resolve the racemate of trans- and cis-1-(1H-imidazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ols (rac-trans- and rac-cis-1-imidazol THN) using the same aforementioned enzymatic methodology. The kinetic resolutions of these isomers were conducted in THF as a solvent and vinyl acetate as an acyl donor, at 30 ˝ C and for 48 h. The lipases CAL-B and PSL-C I were tested and the latter led to the best results. Thus, for the Int. J. Mol. Sci. 2015, 16, page–page rac-trans-isomer, both the trans-(1R,2R)-acetate in 96% ee and trans-(1S,2S)-alcohol with 85% ee (c 43% an E-value of 133) In the case of the both theand trans-(1R,2R)-acetate in were 96% eeobtained. and trans-(1S,2S)-alcohol withrac-cis-isomer, 85% ee (c 43%the andcompounds an E-value cis-(1S,2R)-acetate and cis-(1R,2S)-alcohol with 98% ee (c 50% and E > 200) were obtained (Scheme of 133) were obtained. In the case of the rac-cis-isomer, the compounds cis-(1S,2R)-acetate and 41c cisand Table 5). with 98% ee (c 50% and E > 200) were obtained (Scheme 41c and Table 5). (1R,2S)-alcohol

Scheme 41. Enzymatic kinetic resolution of the racemic trans- and cis-3-imidazol THN (a), racemic Scheme 41. Enzymatic kinetic resolution of the racemic trans- and cis-3-imidazol THN (a); racemic trans- and cis-3-triazol THN (b) and racemic trans- and cis-1-imidazol THN (c) [62]. trans- and cis-3-triazol THN (b) and racemic trans- and cis-1-imidazol THN (c) [62]. Table 5. Results from the enzymatic kinetic resolution of the racemic trans- and cis-3-imidazol THN (a), Table 5. Results from the enzymatic kinetic resolution of the racemic trans- and cis-3-imidazol racemic trans- and cis-3-triazol THN (b) and racemic trans- and cis-1-imidazol THN (c) [62]. THN (a), racemic trans- and cis-3-triazol THN (b) and racemic trans- and cis-1-imidazol THN (c) [62].

Scheme 41 Substrate Substrate Scheme 41 a b c

c (%)

trans-3-imidazol THN 46 trans-3-imidazol THN rac-cis-3-imidazol THN 43 a rac-cis-3-imidazol THN rac-trans-3-triazol THN 48 rac-cis-3-triazol THN 23 rac-trans-3-triazol THN 43 brac-trans-1-imidazol THN rac-cis-3-triazol THN rac-cis-1-imidazol THN 50

c 3.31. Benzoin Derivative

eeP (%) cee (%) P (%) Acetate Acetate

eeS (%) E eeS (%) Alcohol Alcohol

48 23

95 95 95 99 95 >99 99 96 98 >99

rac-trans-1-imidazol THN

43

96

85

133

rac-cis-1-imidazol THN

50

98

98

>200

46 43

83 71 93 29

83 71 93 29 85 98

102 83 >200 >200

E 102 83 >200 >200 133 >200

Chiral α-hydroxy ketones are important building blocks in the syntheses of several biologically 3.31. Benzoin Derivative active compounds such as pharmaceuticals, agrochemicals and pheromones. Aiming to obtain the (S)-benzoin butyrate, a study forare theimportant dynamic enzymatic kinetic (DKR) of racemic benzoin Chiral α-hydroxy ketones building blocks inresolution the syntheses of several biologically via acylation was developed [63]; an efficient agrochemicals DKR is neededand for competing with other biocatalytical active compounds such as pharmaceuticals, pheromones. Aiming to obtain the approaches, such as the enantioselective reduction of benzyl which hasofbeen reported as (S)-benzoin butyrate, a study for the dynamic enzymatic kineticketones, resolution (DKR) racemic benzoin avia useful technique for preparing (S)-benzoins [64,65]. The reactions were conducted in batch or acylation was developed [63]; an efficient DKR is needed for competing with other biocatalytical continuous from Pseudomonas stutzeri (lipase TL) immobilized on Accurel carrier approaches,mode such using as the lipase enantioselective reduction of benzyl ketones, which has been reported as a useful technique for preparing (S)-benzoins [64,65]. The reactions were conducted in batch or continuous mode using lipase from Pseudomonas stutzeri (lipase TL) immobilized on Accurel carrier 29708 MP1001 [63]. Various solvents were evaluated in relation to TL lipase activity such as toluene, 2-MeTHF, 1,3-dioxolane, cyclopentyl methyl ether (CPME), as well as some solvents classified as deep eutectic solvents (DESs). At the same time, a study was conducted to verify the influence of the

Int. J. Mol. Sci. 2015, 16, 29682–29716

MP1001 [63]. Various solvents were evaluated in relation to TL lipase activity such as toluene, 2-MeTHF, 1,3-dioxolane, cyclopentyl methyl ether (CPME), as well as some solvents classified as deep eutectic solvents (DESs). At the same time, a study was conducted to verify the influence of the amount of water in the solvents on the enzyme activity. As result, dry CPME was the most Int. J. Mol. Sci. 2015, 16, page–page efficient solvent for the catalytic activity of the lipase TL as well as for catalyst activity of Zr-TUD-1 (a three-dimensional mesoporous silicate containing zirconium), with this latter used for in situ ˝C vinyl butyrate (6 eq.)of as acyl donor, dried as the solvent, catalyst and racemization thethe (R)-benzoin. The DKR of CPME rac-benzoin, in batch mode,Zr-TUD-1 was carriedas outthe at 50 in the TL presence of vinyl butyrate (6 eq.)After as the5acyl donor, dried CPME as the(c solvent, as ee) was immobilized lipase as the enzyme. h, (S)-benzoin butyrate 98.2%Zr-TUD-1 and 99% the catalyst and immobilized TL lipase as the enzyme. After 5 h, (S)-benzoin butyrate (c 98.2% and obtained. Subsequently, the DKR of rac-benzoin was investigated in a continuous flow system in the was obtained. Subsequently, the DKR of rac-benzoin was investigated in a continuous flow presence99% of ee) vinyl butyrate (3 eq.), TL lipase, and Zr-TUD-1 in CPME. After 25 h, the (S)-benzoin system in the presence of vinyl butyrate (3 eq.), TL lipase, and Zr-TUD-1 in CPME. After 25 h, the butyrate(S)-benzoin was obtained a maximum conversion of 40%, decreasing butyratewith was obtained with a maximum conversion of 40%, decreasingtoto 11% 11% atat76 76 h. h. The enantiomeric excess values of(ee) the (S)-benzoin were ≥98% throughout the process The enantiomeric excess (ee) values of the (S)-benzoinbutyrate butyrate were ě98% throughout the process 42) [63]. (Scheme(Scheme 42) [63].

Scheme Scheme 42. Dynamic enzymatic kinetic resolution (DKR)ofofrac-benzoin rac-benzoin in conventional 42. Dynamic enzymatic kinetic resolution (DKR) in conventional batch orbatch or continuous flow [63]. continuous flow [63]. TheinDKR the presence of lipase important approach forfor obtaining enantiomerically pure The DKR the in presence of lipase is isananimportant approach obtaining enantiomerically pure intermediates for the synthesis of biologically active compounds with both ee and conversion up to intermediates for the synthesis of biologically active compounds with both ee and conversion up to 100%. Recently, an elegant review covering this subject was published [66]. 100%. Recently, an elegant review covering this subject was published [66]. 4. Complementary Approaches: Hydrolysis and Esterification

4. Complementary Approaches: Hydrolysis and Esterification 4.1. Acetonide Derivatives

compound ((R)-2,2,4-trimethyl-1,3-dioxolan-4-yl)-methanol ((R)-acetonide alcohol), a chiral 4.1. AcetonideThe Derivatives intermediate used in the synthesis of some bioactive compounds, was prepared in high ee by the

Theenzymatic compound ((R)-2,2,4-trimethyl-1,3-dioxolan-4-yl)-methanol alcohol), a chiral resolution of its racemate form (Scheme 43a). In this case,((R)-acetonide both succinic anhydride and vinyl used as of acylsome donors, and Amano PS-D lipasewas wasprepared the selected intermediate usedlaurate in thewere synthesis bioactive compounds, inenzyme. high ee by the The (S)-acetonide alcohol was selectively acylated (with TBME as the solvent) and yielded the enzymatic resolution of its racemate form (Scheme 43a). In this case, both succinic anhydride and (R)-acetonide semiester (with succinic anhydride as the acyl donor) and (R)-acetonide ester (with vinyl laurate were used as acyl donors, and Amano PS-D lipase was the selected enzyme. The (S)-acetonide vinyl laurate as the acyl donor), besides the remaining (R)-acetonide alcohol (Scheme 43a). The alcohol was selectively acylated (with TBME the solvent) yielded the (R)-acetonide semiester (R)-acetonide alcohol was also obtained byas kinetic resolutionand of the corresponding racemic ester (with succinic anhydride the acyl donor) and ester (with vinylas laurate as the acyl via hydrolysis using as CAL-B (Novozym 435) as (R)-acetonide the lipase, and t-BuOH:H the solvent 2 O (9:1) (Scheme the 43b)remaining [67]. donor), besides (R)-acetonide alcohol (Scheme 43a). The (R)-acetonide alcohol was also obtained by kinetic resolution of the corresponding racemic ester via hydrolysis using CAL-B (Novozym 435) as the lipase, and t-BuOH:H2O (9:1) as the solvent (Scheme 43b) [67].

29709

vinyl laurate were used as acyl donors, and Amano PS-D lipase was the selected enzyme. The (S)-acetonide alcohol was selectively acylated (with TBME as the solvent) and yielded the (R)-acetonide semiester (with succinic anhydride as the acyl donor) and (R)-acetonide ester (with vinyl laurate as the acyl donor), besides the remaining (R)-acetonide alcohol (Scheme 43a). The (R)-acetonide alcohol was also obtained by2015, kinetic resolution of the corresponding racemic ester via hydrolysis using CAL-B Int. J. Mol. Sci. 16, 29682–29716 (Novozym 435) as the lipase, and t-BuOH:H2O (9:1) as the solvent (Scheme 43b) [67].

Scheme 43. Syntheses of (R)-acetonide derivatives by enantiocomplementary kinetic resolution via Scheme 43. Syntheses of (R)-acetonide derivatives by enantiocomplementary kinetic resolution via acylation of the racemic rac-acetonide alcohol (a) and via hydrolysis of (R)-acetonide ester (b) [67]. acylation of the racemic rac-acetonide alcohol (a) and via hydrolysis of (R)-acetonide ester (b) [67]. Int. J. Mol. Sci. 2015, 16, page–page

4.2. Vitamin D Analogues

26 Vitamin levels within thethe normal range. Analogues of Vitamin D Dplays playsaavital vitalrole roleininmaintaining maintainingcalcium calcium levels within normal range. Analogues vitamin D have been been prepared in order obtain with calcemic analogues of vitamin D have prepared in to order to compounds obtain compounds with activity. calcemicSuch activity. Such were obtained separation of a mixture epimers of (atepimers C-24 containing an OH group) using two analogues werebyobtained by separation of of a mixture (at C-24 containing an OH group) approaches, (a) enzymatic resolution esterification (b) solvolysis corresponding using two approaches, (a)kinetic enzymatic kineticviaresolution via or esterification or of (b)the solvolysis of the esters, and the epimer interest (with biological was the one with S was configuration. In the corresponding esters, of and the epimer of interestactivity) (with biological activity) the one with S first approach (route theapproach esterifications carried out usingwere an acylating (especially vinyl configuration. In the a), first (routewere a), the esterifications carried agent out using an acylating acetate), organic solvent hexane orsolvent DIPE) and lipases from Alcalagenes sp. or Pseudomonas sp. agent (especially vinyl(especially acetate), organic (especially hexane or DIPE) and lipases from (in free or immobilized form). sp. The were monitored byThe HPLC to ensure Alcalagenes sp. or Pseudomonas (inreactions free or immobilized form). reactions werediastereomeric monitored by excess the range of 80%–95%. In thisincase, desired epimer is alcohol HPLC amounts to ensureindiastereomeric excess amounts the the range of 80%–95%. Inthe thisremaining case, the desired (S configuration at C-24), as seen in(SScheme 44a. In the secondasapproach (route b),44a. esterified epimer is the remaining alcohol configuration at C-24), seen in Scheme In thevitamin second D analogues have alcoholysis or hydrolysis the presence of the same lipases used in approach (route b),undergone esterified vitamin D analogues havein undergone alcoholysis or hydrolysis in the route a. These gaveused a diastereomeric excess in the gave rangeaof 80%–95% (Scheme and the presence of thereactions same lipases in route a. These reactions diastereomeric excess44b) in the range desired epimer was the non-hydrolyzed (S)-acetate [68].the non-hydrolyzed (S)-acetate [68]. of 80%–95% (Scheme 44b) and the desired epimer was

Scheme 44. Synthesis of vitamin D analogues by enzymatic kinetic resolution via esterification (a) or Scheme 44. Synthesis of vitamin D analogues by enzymatic kinetic resolution via esterification (a) or (b) solvolysis of the corresponding esters [68]. (b) solvolysis of the corresponding esters [68].

4.3. Arylalkylcarbinol Derivatives 4.3. Arylalkylcarbinol Derivatives Candida antarctica lipase B (CAL-B, Novozym 435) was investigated as a biocatalyst in the Candida antarctica lipase B (CAL-B, Novozym 435) was investigated as a biocatalyst in complementary preparation of (S)- and (R)-arylalkylcarbinol acetates, which are important the complementary preparation of (S)- and (R)-arylalkylcarbinol acetates, which are important intermediates for the syntheses of several compounds with industrial application. This enzyme was intermediates for the syntheses of several compounds with industrial application. This enzyme was used in the hydrolysis of racemic arylalkylcarbinyl acetates (route a) and produced both (R)-alcohols used in the hydrolysis of racemic arylalkylcarbinyl acetates (route a) and produced both (R)-alcohols and (S)-acetates in high enantioselectivities. Then, the mixture of these compounds was subjected to and (S)-acetates in high enantioselectivities. Then, the mixture of these compounds was subjected Mitsunobu reaction to yield the (S)-acetates as the only products (94% ee (n = 1) and 99% ee (n = 2)), as seen in Scheme 45a. The complementary process was conducted using CAL-B on the catalyzed 29710 the (S)-alcohols and (R)-acetates in high acylation of the rac-arylalkylcarbinols, producing enantioselectivities (route b). Subsequently, these mixtures were submitted to a Mitsunobu reaction and provided only the (R)-acetates (99% ee (n = 1 or 2)), as seen in Scheme 45b [69].

Candida antarctica lipase B (CAL-B, Novozym 435) was investigated as a biocatalyst in the complementary preparation of (S)- and (R)-arylalkylcarbinol acetates, which are important intermediates for the syntheses of several compounds with industrial application. This enzyme was used in the hydrolysis of racemic arylalkylcarbinyl acetates (route a) and produced both (R)-alcohols Int. J. Mol. Sci. 2015, 16, 29682–29716 and (S)-acetates in high enantioselectivities. Then, the mixture of these compounds was subjected to Mitsunobu reaction to yield the (S)-acetates as the only products (94% ee (n = 1) and 99% ee (n = 2)), to Mitsunobu reaction to yield the (S)-acetates as the only products (94% ee (n = 1) and 99% ee as seen in Scheme 45a. The complementary process was conducted using CAL-B on the catalyzed (n = 2)), as seen in Scheme 45a. The complementary process was conducted using CAL-B on the acylationcatalyzed of theacylation rac-arylalkylcarbinols, producing the the(S)-alcohols and (R)-acetates of the rac-arylalkylcarbinols, producing (S)-alcohols and (R)-acetates in high in high enantioselectivities (route b). Subsequently,these these mixtures submitted to a Mitsunobu reaction reaction enantioselectivities (route b). Subsequently, mixtureswere were submitted to a Mitsunobu and provided only the (R)-acetates (99% ee (n = 1 or 2)), as seen in Scheme 45b [69]. and provided only the (R)-acetates (99% ee (n = 1 or 2)), as seen in Scheme 45b [69].

Scheme Scheme 45. Complementary enzymatic kinetic resolution of rac-arylalkylcarbinyl 45. Complementary enzymatic kinetic resolution of rac-arylalkylcarbinyl acetates acetates or racor rac-arylalkylcarbinols with Mitsunobu reaction preparation of arylalkylcarbinols combined combined with Mitsunobu reaction on on thethepreparation of (S)(S)-andand (R)(R)-arylalkylcarbinyl acetates via hydrolysis (a) or acylation (b), respectively [69]. arylalkylcarbinyl acetates via hydrolysis (a) or acylation (b), respectively [69]. 4.4. Key Intermediate of Levofloxacin

27

The benzoxazine moiety is part of the structures of a series of molecules with biological activities such as antibacterial, anticancer, antifungal and antimicrobial. A number of 1,4-benzoxazine derivatives (with or without substituents at the aromatic ring) were prepared by a combination of chemical steps and lipase-mediated kinetic resolution [70]. The enzymatic step was conducted with rac-1-(2-nitrophenoxy)propan-2-ols (rac-alcohols), via acylation (route a), and with the corresponding rac-acetates, via hydrolysis (route b), as seen in Scheme 46. These two approaches are considered complementary lipase-catalyzed processes. The process of kinetic resolution via acetylation was first evaluated using the rac-1-(2-nitrophenoxy)propan-2-ol as a substrate, in the presence of vinyl acetate as an acyl donor, TBME as a solvent, at 30 ˝ C and with 5 h of reaction time. After screening lipases from Candida antarctica (CAL-A), from Pseudomonascepacia (PSL C-I), from C. antarctica B (CAL-B) and from Rhizomucor miehei in immobilized form (RML IM), it was found that the latter (RM IM) was the most efficient. In the aforementioned conditions, the (R)-acetate (94% ee) and the remaining (S)-alcohol (89% ee) were obtained with c 48%. The study was extended to the rac-alcohols with substituents at the aromatic ring (4-F, 4-OMe and 5-Me), leading to the (R)-acetates (93%–95% ee) and the remaining (S)-alcohols (89%–94% ee) with c 48%–50% and E-values ranging from 103 to >200 (Scheme 46a and Table 6). By a sequence of reactions, the (R)-3-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine derivatives ((R)-3-methyl-1,4-benzoxazines) were synthesized from the (S)-alcohols. The second approach (route b) was related to the resolution of the corresponding rac-acetates via hydrolysis reaction. In this case, the reactions were carried out in the presence of RML IM, at 30 ˝ C, using TBME and water (5 eq.) as solvents. After 52 h, the (R)-alcohols (96% to >99% ee) and the remaining (S)-acetates (91%–97% ee) were obtained with c 48%–50% and E > 200 (Scheme 46b and Table 6). Due to the successful strategies for obtaining the chiral 1,4-benzoxazines, the authors performed the production of the (S)-7,8-difluoro-3-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine, precursor of the potent antibacterial Levofloxacin (Scheme 46c). Then, the kinetic resolution of the rac-1-(2,3-difluoro-6-nitrophenoxy)propan-2-yl acetate was performed, via hydrolysis in the

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IM, at 30 °C, using TBME and water (5 eq.) as solvents. After 52 h, the (R)-alcohols (96% to >99% ee) and the remaining (S)-acetates (91%–97% ee) were obtained with c 48%–50% and E > 200 (Scheme 46b and Table 6). Due to the successful strategies for obtaining the chiral 1,4-benzoxazines, the authors performed the production of the (S)-7,8-difluoro-3-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine, Int. J. Mol. Sci. 2015, 16, 29682–29716 precursor of the potent antibacterial Levofloxacin (Scheme 46c). Then, the kinetic resolution of the rac-1-(2,3-difluoro-6-nitrophenoxy)propan-2-yl acetate was performed, via hydrolysis in the presence presence of lipase RM IM, leading to the (R)-alcohol (>99% ee) and the remaining (S)-acetate of lipase(84% RMee) IM, leading the (R)-alcohol (>99% ee) andsteps, the the remaining (S)-acetate (84% ee) with c with c 46%. to Subsequently, after several chemical (R)-alcohol was converted into 46%. Subsequently, after several chemical steps, the (R)-alcohol into the (S)-7,8the (S)-7,8-difluoro-3-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine (36% was yield,converted >99% ee) Levofloxacin precursor [70]. difluoro-3-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine (36% yield, >99% ee) Levofloxacin precursor [70].

SchemeScheme 46. Enzymatic kinetickinetic resolution of racemic alcohols via 46. Enzymatic resolution of racemic alcohols 1-(2-nitrophenoxy)propan-2-ols 1-(2-nitrophenoxy)propan-2-ols via acetylation or via hydrolysis (b) of the corresponding racemicacetates. acetates. Production Production of acetylation (a) or via (a) hydrolysis (b) of the corresponding racemic of (S)-7,8(S)-7,8-difluoro-3-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine precursor of the antibacterial drug difluoro-3-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine precursor of the antibacterial drug Levofloxacin (c) [70]. Levofloxacin (c) [70]. Table 6. Results from the enzymatic kinetic resolution of racemic alcohols 1-(2-nitrophenoxy) 28 propan-2-ols via acetylation (a) or via hydrolysis (b) of the corresponding racemic acetates [70].

Scheme 46 a b

Acetates ee (%) Yield (%)

Alcohols ee (%) Yield (%)

c (%)

E

93–95 91–97

89–94 96–>99

48–50 48–50

103–>200 >200

45–47 41–48

44–48 44–47

5. Conclusions Recent examples of the use of lipases in the preparation of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates were reviewed, confirming the importance of these enzymes in obtaining compounds with high added value. Most reported biocatalytic processes refer to kinetic resolutions of racemic substrates, which occur under mild conditions with a high degree of regio- or enantioselectivity. Among the examples presented, the action of lipases can be seen in a wide range of substrates with varied structures such as aromatic, heteroaromatic (containing atoms of nitrogen, sulfur, chlorine, etc.), as well as aliphatic with either open or cyclic chain (branched or not). The substances produced by the action of lipases, in the examples herein, are pharmaceutical intermediates or drugs, which makes the biocatalytic process of great interest in

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the pharmaceutical industry. Additionally, it was also possible to confirm the versatility of lipases for acting in either aqueous medium or organic solvents for the hydrolysis of esters, the acylation of alcohols or the esterification of carboxylic acid, allowing the production of the desired stereoisomer. It highlights the operational simplicity, as it is not necessary to add cofactors in the reaction system, and the ease of finding a wide range of commercially available and relatively low-cost lipases. The use of immobilized lipase often allows the reuse of the enzymatic system for several cycles, making the process more effective and economically viable. In summary, it is clear that lipases will continue to be an excellent alternative for obtaining biologically active compounds. Acknowledgments: The authors thank the Brazilian funding agency Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for providing the Special Visiting Researcher fellowship (process 400171/2014-7) under the Brazilian Scientific Program “Ciência sem Fronteira”. Author Contributions: Marcos Carlos de Mattos supervised the manuscript preparation. Marcos Carlos de Mattos, Maria da Conceição Ferreira de Oliveira, Telma Leda Gomes de Lemos, Francesco Molinari, Diego Romano and Immacolata Serra reviewed the literature and co-wrote the manuscript. Ana Caroline Lustosa de Melo Carvalho and Thiago de Sousa Fonseca prepared all illustrations. Conflicts of Interest: The authors declare no conflict of interest.

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