Title of Invention

PROCESS FOR THE PREPARATION OF SUBSTITUTED 2-AMINO-3-(2-AMINO-PHENYLSULFANYL)-PROPIONIC ACID

Abstract

A process for the preparation of compounds of formula I wherein R<sup>1</sup> is hydrogen or alkyl; R<sup>2</sup> is an amino protecting group; each R<sup>3</sup> is independently halogen, carboxyl, alkoxycarbonyl, alkenyloxycarbonyl or benzyloxycarbonyl; n is 1 or 2, which process comprises reacting compounds of formula II wherein R<sup>1</sup> R <sup>2</sup> R<sup>3</sup> and n are as defined above and R4 is alkyl or benzyl, with a protease in an aqueous system containing an organic co-solvent.

Full Text

The present invention relates to a new enzymatic process for the preparation of . compounds of formula I wherein R1 is hydrogen or alkyl; R is an ammo protecting group; each R is independently halogen, carboxyl, alkoxycarbonyl, alkenyloxycarbonyl or benzyloxycarbonyl; n is 1 or 2. In EP 0407033 A an enzymatic stereoselective hydrolysis of racemic mixtures of esters of 2-substituted acids, other than 2-halo propionic acids, into the corresponding enantiomeric acids is described. The reaction is carried out in the presence of Candida rugosa lipase isoenzymes, an organic solvent (e.g. toluene) and a reducing agent. The process is especially useful for the stereoselective production of S-ketoprofen, S-ibuprofen, S-fenoprofen, S-2-phenyIpropionic acid and S-indoprofen. In EP 0178553 A the preparation of aromatically substituted L-amino acids by selective hydrolysis of the corresponding alkyl esters with chymotrypsin is described. In DE 2804892 the preparation of optically pure N-acyl-L-threonine by hydrolysing N-acyl-DL-threonine ester with serine protease, especially subtilisin Carlsberg, is described. No enzymatic reaction for the preparation of compounds of the formula I has been described in the literature. Attempts to synthesise compounds of formula I by chemical hydrolysis resulted in low yields and decomposition of the starting material. The aim of the present invention therefore is a novel and inventive process for the preparation of substituted 2-amino-3-(2-amino-phenylsulfanyl)-propionic acids. The compounds of formula I may be manufactured by the process of the present invention, which provides a new process for the preparation of substituted 2-amino-3-(2-amino-phenylsulfanyl)-propionic acid of formula I wherein Rl is hydrogen or alkyl; R is an amino protecting group; each R3 is independently halogen, carboxyl, alkoxycarbonyl, alkenyloxycarbonyl or benzyloxycarbonyl; n is 1 or 2, which process comprises reacting compounds of formula II OR4 0 wherein R , R", R and n are as defined above and R4 is alkyl or benzyl, with a protease in an aqueous system containing an organic co-solvent. With the enzymatic approach described herein a selective hydrolysis of the ester under mild reaction conditions is possible. In the structural formula presented herein a wedged bond ( "^ ) denotes that the substituent is above the plane of the paper. The term "alkyl" as used herein denotes an optionally substituted straight or branched chain hydrocarbon residue containing 1 to 12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl including their different isomers. Suitable substituents for the alkyl chain maybe selected from 1-3 halogens such as fluorine or chlorine, or Ci_4-alkoxy such as methoxy or ethoxy. Examples for substituted alkyl are 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl or 2-methoxyethyl. Alkyl in Rl is as defined above and preferably a straight or branched chain hydrocarbon residue containing 1 to 7 carbon atoms. Alkyl in R is more preferred methyl ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert.-butyl. Alkyl in R4 is as defined above and preferably an optionally substituted straight or branched chain hydrocarbon residue containing 1 to 7 carbon atoms. Suitable substituent, for the alkyl chain maybe selected from 1-3 halogen such as fluorine or chlorine, or Q.4-alkoxy such as methoxy or ethoxy. Examples for substituted alkyl are 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl or 2-methoxyethyl. Alkyl in R4 is more preferred a straighl chain hydrocarbon residue containing 1 to 7 or 1 to 4 carbon atoms. Examples are methyl ethyl, propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl. Preferred examples are methyl, ethyl or propyl. Most preferred alkyl in R4 is methyl. The term "amino protecting group" as used herein refers to groups such as those employed in peptide chemistry as described in Green T. Protective Groups in Organic Synthesis, Chapter 5> John Wiley and Sons, Inc. (1981), pp. 218-287, such as an allyloxycarbonyl group (ALLOC), a lower alkoxycarbonyl group (e.g. tert.-butoxycarbonyl (t-BOC))> a substituted lower alkoxycarbonyl group (e.g. trichloroethoxycarbonyl), an optionally substituted aryloxycarbonyl group (e.g. p-nitrobenzyloxycarbonyl, benzyloxycarbonyl (Z) or phenyloxycarbonyl), an alkanoyl group (e.g. formyl, acetyl), an aroyl group (e.g. benzoyl), a halogen-alkanoyl group (e.g. trifluoroacetyl) or a silyl protective group (e.g. tert.-butyldimethylsilyl). Preferred amino protecting groups are benzyloxycarbonyl, tert.-butoxycarbonyl, allyloxycarbonyl or benzoyl, especially preferred amino protecting group is tert.-butoxycarbonyl. The term "alkoxycarbonyl" denotes an Q.yalkoxy residue attached to a carbonyl group (>C=0). Examples for alkoxy groups are methoxy, ethoxy, n-propyloxy, iso-propyloxy, n-butyloxy, 1-sec-butyloxy, iso-butyloxy, tert-butyloxy, pentyloxy, hexyloxy, heptyloxy including their different isomers. Examples for alkoxycarbonyl groups are methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, tert.-butoxycarbonyl and the like. Preferred lower alkoxycarbonyl is tert-butoxycarbonyl. The term "alkenyloxycarbonyl" denotes an alkenyl-oxy residue attached to an carbonyl group (>C=0). The term "alkenyl" as used herein denotes an unsubstituted or substituted hydrocarbon chain radical having from 2 to 8 carbon atoms, preferably from 2 to 4 carbon atoms, and having at least one olefinic double bond, including their different isomers. Examples are vinyl, allyl or isopropenyl. Preferred "alkenyloxycarbonyl" for R3 is allyloxycarbonyl or isopropenyloxycarbonyl, more preferred is allyloxycarbonyl. The term halogen signifies fluorine, chlorine, bromine or iodine. Preferred halogen is bromine. The number n maybe either 1 or 2, preferably the number n is 1. The substituent R may be in any possible position attached to the phenyl ring. If n is 1, R3 may be in the 3,4,5 or 6-position of the phenyl ring. Preferably, the R3 substituent is in the 4-position. If n is 2, both R3 substituents maybe in the independently from each other in the 3,4,5 or 6-position of the phenyl ring. A preferred embodiment of the invention is a process for the preparation of compounds of formula I, characterized in that compound of formula II is wherein R\ R\ R\ R* and n are as defined above. In a further preferred embodiment of the process invention R is hydrogen or alkyl, more preferred R1 is hydrogen; -} R" is an amino protecting group, more preferred R is an amino protecting group; each R3 is independently halogen, carboxyl or alkenyloxycarbonyl; R4 is alkyl or benzyl, more preferred R4 is alkyl; n is 1 or 2, more preferred nis 1. In another preferred embodiment of the invention the process is carried out with a protease in an aqueous system, containing an organic co-solvent, at a pH of 4.0 - 10, preferred 6.0 - 8.5. After the enzymatic hydrolysis of the substrate of formula II the enantiomeric pure product of formula I is separated by acidification and subsequent extraction. Compound of formula II may be used in any possible mixture of the L or D enantiomers. The conversion is accomplished preferably with a racemic mixture or with a mixture which only contains the L-isomer. When employing a mixture of isomers, the unreacted remaining D-ester is removed by an extraction step prior to acidification of the reaction medium and subsequent extraction of the L-acid. Suitable enzymes as catalysts for the reactions are proteases, preferably cheap bulk proteases of microbial origin, more preferred are Bacillus proteases (like Savinase from Novo Nordisk) or subtilisins e.g. subtilisin Carlsberg from Novo Nordisk (Alkalase) or from Solvay (Protease-L) or Aspergillus proteases (like Prozyme 6 from Amano) or Tritachium proteases (Proteinase K from Fluka). Most preferred enzyme as catalyst for the reactions is subtilisin Carlsberg (e.g. Alcalase from Novo Nordisk). As an alternative the enzymes may be used in immobilized form. The reaction is carried out in an aqueous system with an organic co-solvent, such as a water-immiscible solvent or a water-miscible organic co-solvent. The water-immiscible solvent may be used in any ratio with the aqueous phase, preferred ratio is 25-75% (v/v). The water-miscible organic co-solvent may be used in an amount as high as tolerated by the enzyme, typically 5-25% (v/v) but which might exceed even 50% (v/v), e.g. in case of subtilisin Carlsberg. The reaction is carried out at a reaction temperature from 0°C to 50°C, preferred at a reaction temperature between 15°C and 40°C, and most preferred at a reaction temperature between 15°C and 25°C. As to the aqueous phase, common buffer solutions known in the art for biochemical conversions may be used, such as sodium or potassium phosphate in a concentration of up to 1M, preferably between about 5mM and about 50mM. Such a buffer solution may additionally contain one of the usual salts like e.g. sodium or potassium chloride, and also LiSCN, Na2S04 or a polyhydric alcohol e.g. a sugar; in a concentration up to 1M. Suitable organic co-solvents are technically common solvents. Examples are ethers (e.g. tetrahydrofuran (THF), dioxan or tert.-butyl methyl ether (TBME)), lower alcohols, esters (e.g. ethyl acetate), polar aprotic solvents (e.g. dimethylsulfoxide (DMSO), dimethylacetamide, N,N-dimethylformamide (DMF) or acetone). Preferred organic co-solvents are tetrahydrofuran (THF), tert-butyl methyl ether (TBME) and ethyl acetate. The term "lower alcohol" as used herein denotes straight chain or branched alkyl residues containing 1 to 8 carbon atoms with one hydroxy group, such as methanol, etnanoi, propanol, isopropanol, butanol, isobutanol, tert.-butanol, pentanol, hexanol, heptanol or octanol, preferably methanol, ethanol, propanol, isopropanol, butanol, isobutanol, tert.-butanol and more preferred alcohol are methanol or ethanol. The substrate is suitably applied as a solution in a 0.1 - 25% overall concentration (w/w). A more preferred overall concentration is 1 - 10%. After addition of the enzyme, the pH of the reaction mixture is maintained under vigorous stirring at the selected pH-value by the controlled addition of a base. Preferred bases are aqueous NaOH or KOH solutions. After termination of the reaction, the enantiomerically pure product of formula I is worked up conventionally after phase separation, by acidification of the aqueous phase with a suitable acid and subsequent extraction with a suitable organic solvent. Compounds of formula I are synthesized, according to the present invention, in high enantiomeric purity, which means that enantiomeric excesses of 90% or higher, preferred an enantiomeric excess of 95% or higher maybe achieved. Compounds of formula II are prepared according the following reaction scheme or in a conventional manner known to the skilled in the art. ■ * wherein R1, R2> R3 and R4 are as described for compounds of formula I and II. In reaction scheme 1, substituted 2-nitro fluoro-aromatic of formula a) is reacted with protected cysteine (e.g. BOC-Cysteine-methylester) of formula b) to obtain the corresponding nitro-phenyl substituted protected cysteine of formula c). The reaction is conveniently carried out under basic conditions (with a diisopropylamine, e.g. ethyldiisopropylamine) in an appropriate organic solvent, such as hexane, diisopropylether, ethyl acetate, methanol, ethanol, propanol, dichloromethane, DMF or DMSO. The reaction temperature is preferably between -30°C to +150°C. Most preferred, the reaction is carried out with BOC-Cysteine-methylester in ethanol in the presence of ethyldiisopropylamine at a temperature of 110°C. Compounds of formula a) and the protected cysteine (e.g. BOC-Cysteine-methylester) of formula b) are commercially available or synthesized according to methods known from textbooks about organic chemistry e.g. from J. March (1992), "Advanced Organic Chemistry: Reactions, Mechanisms, and Structure" 4th ed. John Wiley & Sons). The second step of the reaction is carried out in that the nitro group of the nitro-phenyl substituted protected cysteine of formula c) is reduced to the corresponding amino-phenyl substituted protected cysteine of formula II. The reduction reaction is carried out according to methods known in the art for example known in textbooks on organic chemistry e.g. J. March (1992), "Advanced Organic Chemistry: Reactions, Mechanisms, and Structure", 4* ed. John Wiley & Sons). The reaction is conveniently carried out with a suitable reducing agent (e.g. Zinc and optional NH4CI) in acidic media with organic solvents such as hexane, diisopropylether, ethyl acetate, methanol, ethanol, propanol, dichloromethane, DMF, DMSO, preferably methanol. The reaction temperature is preferably between -30°C to +150°C. The NH2-group of compound of amino-phenyl substituted protected cysteine of formula II may be alkylated with RiHal, wherein Ri is as defined above and Hal is chlorine or bromine. The compounds of formula Il-a and Il-b (see below) are novel intermediates and therefore also subject of the present invention Compounds of the formula I are versatile building blocks for the synthesis of 1,5-benzothiazepines. Such benzothiazepine scaffolds have been used as a constrained dipeptide mimics in various enzyme inhibitors (protease, interleukin-l|3-converting enzyme, elastase or angiotensin-converting enzyme, but also as GPCR antagonists (cholecystokinin, angiotensin II receptor). The possible different uses is described in G.C. Morton et al., Tet. Lett, 41 (2000) 3029-3033. The benzothiazepines are prepared according reaction scheme 2: wherein R\ R\ R3, R* and n are as described for compounds of formula I and II. The cyclisation reaction of compound of formula I to obtain benzothiazepine of formula III is carried out thermally or in the presence of an appropriate reagent, for example as described in G.C. Morton et al., Tet. Lett, 41 (2000) 3029-3033). The reaction is conveniently carried out with a carbodiimide in an appropriate solvent such as hexane, xylene, diisopropylether, ethyl acetate, methanol, ethanol, propanol, dichloromethane, DMF or DMSO. The reaction temperature is preferably between -30°C to +150°C. The reaction is preferably carried out at room temperature in DMF in the presence of EDAC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride). In the following examples the abbreviations used have the following significations. ISP-MS ion spray positive mass spectroscopy ISN-MS ion spray negative mass spectroscopy EI-MS electron impact mass spectroscopy SFC super critical fluid chromatography NMR nuclear magnetic resonance spectroscopy IR infra red spectroscopy HV high vacuum min minute(s) Example 1 (preparation of the starting material) fS)-3-(4-Bromo-2-nitro-phenylsulfanyD-2-tert.-butoxycarbonylamino-propionicacid methyl ester The reaction is carried out as described in M. K. Schwarz et al., J. Org. Chem. 1999, 64,2219-2231. To a solution of commercially available BOC-Cysteine-Methylester (27.38g, 116.35mmol, 1 eq), N-Ethyldiisopropylamine (DIPEA) (4.4mL, 25.75mmoI, 2.5 eq) in EtOH (310 mL) was added 5-bromo-2-fluoronitrobenzene (25.62g ; 116,45mmol; 1 eq) and heated at 110°C for 3 h. The progress of the reaction was monitored by HPLC. The solvent was evaporated to a red-brown oil and this was partitioned between water (600mL) and ethyl acetate (3 X 300mL). Drying (Na2S04), and evaporation gave crude product which was recrystallised with cyclohexane to give yellow crystals (38.75g, 77%). Analytical data: *H-NMR (CDC13,400MHz): 1.430 (s, 9H , 0(CH3)); 3.350 (dd, 1H, H(4')> h-4»=5.2Hz, J4*.5=14.4Hz); 3.500 (dd, 1H, H(4"), J4«-4'=5.2Hz, J4».5=14.4Hz);. 3.755 (s ,3H, OCH3); 4.621 (m, 1H,H(5)); 5.301 (d, 1H, H(6), J6-5=6.4Hz ); 7.449 (d, 1H,H(1), j!.2=8.8Hz); 7.670 (dd, 1H, H(2), J1.2=8.8Hz, J2-3=2.0Hz); 8.278 (d, 1H, H(3), J2.3=2Hz). IR: 3353 cm*1 (NH); 1765 cm"1 (Ester -C=0); 1686 cm"1 (Carbamate -C=0); 1546 cm_1(Amide -OO); 1460 and 1329 cm_1(N02); 1220 cm"1 (Ester). MS: m/z=452.3 [M+NH4+] with 79Br; m/z=454.3 [M+ NH4+] with 81Br; m/z=457.3 [M+Na+] with 79Br; m/z=459.3 [M+Na+] with 81Br. Rf=0.38 in Ethyl acetate / Hexane 1:2 on Si02. Example 2 (S)-3-(2-Amino-4-bromO"phenylsuIfanyl)-2-tert.-butoxycarbonyIamino--prQpionicacid methyl ester The reaction is carried out as described in J. Slade et.al., J. Med. Chem. 1985,28, 1517-1521). A mixture of the nitro compound from above (40.34g, 92.6mmol, 1 eq), NH4C1 (19.81g, 370.6mmol, 4 eq) is added and Zn (79.37g, 1.204mmol, 13 eq) in MeOH (950 mL) was heated at reflux for 16h and the resultant mixture filtered through Celite and washed with boiling MeOH. After concentration, the crude product was partitioned between ethyl acetate and a NaHC03 (aq.). The resultant oil was chromatographed using ethyl acetate / hexane / 3% triethylamine) to give the product (21.11g, 56%). Analytical data: *H-NMR (CDCI3,400MHz): 1.395 (s, 9H, 0(CH3)); 3.185 (d, 2H, H(4), J4-5=4Hz); 3.617 (s, 3H, OCH3); 4.446 (s, 2H, H(7)); 4.528 (m, 1H, H(5)); 5.502 (d, 1H, H(6), J6-5=8Hz); 6.793 (dd, 1H, H(3), J3-2=2Hz, J3-i=8.4Hz) ;6.868 ( d, 1H, H(3), J3-2=2Hz), 7.225 (d, 1H, H(l), Ji.2=8.4Hz). IR: 3356cm"1 (-NH2, -NH); 1744cm-1 (Ester -C=0); 1707cm"1 (Carbamate -C=0); 1504cm'1 (Amide); 1250cm'1 (Ester). MS: m/z=405.3 [M+H+] with 79Br; m/z=407.3 [M+H+] with 81Br. Rfc0.47 in ethyl acetate / hexane 1:2 on Si02. Example 3.1 (large scale enzymatic hydrolysis) (S)-3-(2-AminQ-4-brQmo-phenylsulfanyl)"2-tert.-butoxycarbonylaminO"propionic acid 19.9g(48.02mmol)ofN4ert."butoxycarbonyl-3-(2-amino-4-bromophenylthio)-L-alanine methyl ester (97.8%) was dissolved in 750ml TBME and emulsified in 31 buffer solution (0.1M sodium chloride, 20mM sodium phosphate pH 7.5) under vigorous stirring. 12.0ml Alcalase 2.4 L and 30mg Substilisin A [both enzyme preparations are subtilisin Carlsberg from Novo Nordisk] were added and the pH was maintained at 7.5 under vigorous stirring by the controlled addition (pH-static) of 1.0N sodium hydroxide solution. After 7d 44.75ml of 1.0N sodium hydroxide solution was consumed and the conversion degree was >99% (HPLC analysis). The biphasic reaction mixture was separated. The aqueous phase was washed briefly with 11 TBME for the separation of small amounts of lipophilic impurities and traces of the remaining substrate. The combined organic phases were extracted with 1 x 250ml 0.1 M potassium phosphate buffer pH 7.6. The combined aqueous phases were acidified to pH 2 with 32% hydrochloric acid and extracted with 1.51 ethyl acetate. The resulting emulsion was separated by the addition of 10% Dicalite under stirring and a subsequent filtration. The aqueous phase was extracted with 2 x 11 ethyl acetate. The combined ethyl acetate phases were dried on anhydrous sodium sulfate and evaporated. The residue was dissolved in dichloromethane, evaporated and dried atHV to give 18.79g N-tert.-butoxycarbonyl-3-(2-amino-4-bromophenylthio)-L-alanine as a pale yellow solid (yield: 81.8%). Analytical data: *H-NMR (CDC13,400MHz) 1.399 (s, 9H, OC(CH3)); 3.184 (dd, 1H, SCH2> J=14.2Hz, J=6.6Hz); 3.235 (dd, 1H, SCH2, J=14.2Hz, J=6.6Hz); 4.515 (m, 1H, COCHNH); 5.517 (d, 1H, CONHCH, J=6.8Hz); 6.796 (dd, 1H, arom., J=2Hz, J=8.4Hz); 6.876 (d, 1H, arom., J=2Hz); 7.250 (d, 1H, arom., J=8.4Hz). IR (ATR-IR) 3445 and 3355cm"1 (-NH, -NH2); 2978 and 2929cm"1 broad (-COOH); 1693cm"1 (COOH -OO, carbamate -C=0); 1508cm"1 (amide -CO-NH); 1247 and 1157cm"1 (COOH). MS (ESI-positive ionization) m/z=391.1 [M+H+] with79Br; m/z=393.1 [M+H+] with81Br;m/z=413.2 [M+Na+] with79Br;m/z=415.2 [M+Na+] with 8lBr. OR [a]D= +53.3 ° (CHC13; c=1.0). HPLC analysis: column: ABZ+plus; mobile phase: A: 0.1% TFA in H20; B: MeCN; gradient B: 30 - 80% 0-15 min, 80 - 3Qo/0 15 - 16 min, 30% 16 - 19.5 min; flow:l ml/min; pressure: 50 - 80 bar; detection: UV, 300nm; retention times: 12.1 min (product-acid) 13.5 min (substrate-ester). Example 3.2 (small scale enzymatic hydrolysis) fS)-3"(2-Amino-4-bromo-phenvlsulfanyl)-2-tert.-butoxycarbonylamino-propionicacid 1.65g(3.99mmol) ofN-tert.-butoxycarbonyl-3-(2-amino-4-bromophenylthio)-L-alanine methyl ester (98%) was dissolved in 65ml TBME and emulsified in 240ml buffer solution (0.1M sodium chloride; 20mM sodium phosphate pH 7.5) under vigorous stirring. 0.75ml Alcalase 2.4 L [a subtilisin Carlsberg from Novo Nordisk] was added and the pH maintained at 7.5 under vigorous stirring by the controlled addition (pH-static) of 1.0N sodium hydroxide solution. After 33h 4.23ml of 1.0N sodium hydroxide solution was consumed and the conversion degree was >99% (HPLC analysis). The reaction mixture was acidified to pH 2 with 32% hydrochloric acid, filtered over Decalite. After phase separation the aqueous phase was extracted with 3 x 275ml ethyl acetate. The combined organic phases were dried on anhydrous sodium sulfate and evaporated. The residue was dissolved in dichloromethane, evaporated and dried at HV to give L56g N-tert.-butoxycarbonyl-3-(2-amino-4-bromophenylthio)-L-alanine as a pale yellow solid (yield: 99.8%). Example 3.3 (enzymatic hydrolysis with different solvents) (SV3-(2-Amino-4-bromo-phenylsulfanvl)-2-tert.-butoxvcarbonylamino-propionicacid lOOmg (0.242mmol) of N-tert.-butoxycarbonyl-3-(2-amino-4-bromophenylthio)-L-alanine methyl ester (98%) was added to a system consisting of 20% organic co-solvent (see following table) and 80% buffer solution (0.1M sodium chloride; 3mM sodium phosphate pH 7.5) under vigorous stirring. lOOul Alcalase 2.4 L [a subtilisin Carlsberg from Novo Nordisk] was added and the pH maintained at 7.5 under vigorous stirring by the controlled addition (pH-static) of 0.1N sodium hydroxide solution. At the end of the reaction the conversion degree was determined by HPLC. Example 3.4 (enzymatic hydrolysis with other enzymes) (S)-3-f2-Amino-4"bromo-phenylsulfanyn-2-tert.-butoxycarbonyIamino-propionicacid 200mg (0.493mmol) of N-tert.-butoxycarbonyl-3-(4-bromo-2-aminophenylthio)-L-alanine methyl ester (93.8%) was added to a system consisting of 5ml TBME and 20ml buffer solution (0.1M sodium chloride; 3mM sodium phosphate pH 7.0) under vigorous stirring. Enzyme [see following table] was added and the pH maintained at 7.0 under vigorous stirring by the controlled addition (pH-static) of 0.1N sodium hydroxide solution. After termination of the reaction the reaction mixture was washed twice with 25ml TBME, acidified to pH 2.5 with 25% HCl and extracted with 3 x 25ml ethyl acetate. The combined organic phases were dried over sodium sulfate, the solvent evaporated and the residue dried on a high vacuum. Example 4 (S)-(7-Bromo-4-oxo-23,4,5"tetrahydro-benzo[biri,4lthiazepin-3-yl)-carbamicacidtert.-butyl ester The reaction is carried out as described in G.C. Morton et al., Tet. Lett., 41 (2000) 3029-3033.). The product from the enzyme hydrolysis (15.2g, 38.8mmol, leq) and EDAC (l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (7.44g>38.8mmol> 1 eq) in DMF (500 mL) was stirred at room temperature for 16h, concentrated and the residue dissolved in ethyl acetate (500 ml) and extracted with aq. NaHCOs (1M) (500mL) and water (500mL). Drying (NaSO^ and concentrated to give the product (14.5g, quantitative). Analytical data: ^-NMR (CDC13,400MHz): 1.409 (s, 9H, 0(CH3)); 2.939 (t, 1H, H(4')> J=11.6Hz); 3.794 (dd, 1H, H(4")> J4-.5=llHz, J4»-4'=6.4Hz); 4.439 (m, 1H, H(5)); 5.567 (d, 1H, H(6), J6-5=8Hz); 7.306 (d, 1H, H(2), J2-i=8Hz, J2.3=2Hz); 7.337 (d, 1H> H(3), J3-2=2Hz); 7.748 (d, 1H, H(l), Ji-2=8Hz); 8.042 (s, 1H, H(7)). IR: 3284 and 3184 cm'1 (-NH); 1729cm"1 (Carbamate -C=0); 1678cm"1 (Amide -C=0); 1574cm'1 (Amide); 1251cm"1 (Ester). MS: m/z=373.3 [M+H+] with 79Br; m/z=375.3 [M+H+] with 81Br; m/z=395 [M+Na+] with 79Br; m/z=397 [M+Na+] with 81Br. Rf=0.23 in Ethyl acetate / Hexane 1 :2 on Si02. Example 5 (S)-4-(2-tert.-Butoxvcarbonvlamino-2-methoxycarbonvl-ethylsulfanvD-3-nitro-benzoic acid allyl ester 4-Fiuoro-3-nitrobenzoic acid (49.95g, 269.8mmol, leq) was dissolved in methanol (350mL) and Cs2C03 (44.06g, 135.2mmol, 0.5eq) added and the solvent evaporated. Then the Cs-salt was dissolved in DMF, heated to 50°C and treated with allylbromide (22.8mL, 269.8mmol, leq). After 5 mins. the solvent was evaporated and the resultant solid triturated in Et20. Filtration and evaporation gave the crude allylester quantitatively. The next step is carried according to M. K. Schwarz et al., J. Org. Chem. 1999,64,2219-2231. The allyl ester (30g, 133.2mmol, leq), BOC-Cysteine-Methylester (31.35g, 133.2mmol, 1 eq) and DIPEA (68.3mL, 399.6mmol, 3eq) is added slowly (exothermic reaction is observed) in EtOH was stirred at rt for 3 hours, and concentrated to give a solid which was recrystallised from diisopropylether (46.16g, 79%). Analaytical data: jH-NMR (CDC13, 400MHz): 1.443 (s, 9H, O(CH3));3.401 (dd, 1H, H(7J), Jy-r=2.4Hz, J7'-8=6Hz); 3.506 (dd, 1H, H(7")> J7--r=2.4Hz, J7»-8=6Hz); 3.777 (s ,3H, OCH3); 4.608 (m, 1H, H(8)); 4.860 (dt, 2H, H(3',3")> J3-2=5.6Hz; Js-^l^Hz); 5.322 (q, 1H, H(l')> J=0.8Hz); 5.347 (t, 1H> H(l"), J=0.8Hz); 5.406 (d, 1H, H(9), J9-8=1.6Hz); 6.032 (m, 1H, H(2)) ; 7.625 (d, 1H, H(5), J5-4-8.4Hz); 8.201 (dd, 1H, H(4), J4-5=8.4Hz, J4-6=L6Hz); 8.830 (d, 1H, H(6), J6^=1.6Hz). IR: SSSOcm^C-NH); 1742cm"1 (Ester-C=0); H^cm^Conj. Ester -C=0); 1686cm" 1 (Carbamate -0=0); 1523 and 1334cm~l(-N02); 1245cm"1(Ester). MS: m/z=441.3 [M+H+]; m/z=458.4 [M+ NH4+]; m/z=463.2 [M+Na+].Rf=0.40 in Ethyl acetate / Hexane 1:2 on Si02- Example 6 fS)-3-Amino-4-(2-tert-butoxycarbonylamino-2-methoxvcarbonvl-ethylsulfanvD-benzoic acid allyl ester The reaction is carried out as described in J. Slade et.al., J. Med. Chem. 1985,28, 1517-1521. The above product (506mg, 1.15mmol, leq), aq. NH4C1 (20mL) and Zn (976mg, 14.9mmol, 13eq) in DME (15mL) was stirred and heated at 80°C for 16h. The solvent was evaporated and the residue dissolved in ethyl acetate (250mL) and extracted with (aq.) sodium NaHC03 (1M) (3 X 50mL). The organic phase was dried with NaS04 and evaporated to give a yellow oil which crystallized after few days (276mg> 60%). Analytical data: *H-NMR (CDC13,400MHz): 1.380 (s, 1H, 0(CH3)); 3.283 (d, 2H, H(7', 7"), J7-8=4.4Hz); 3.579 (s ,3H, OCH3); 4.445 (s> 2H, H(10?, 10")); 4.554 (m, 1H, H(8)); 4.787 (ddd,2H>H(2,J2")>J=1.2Hz); 5.283 (dd, 1H,H(1'), Jr-2=10.6HzJiM»=L2Hz); 5.390 (dd, 1H> H(l")> Jr.2=17.8Hz, Ji».r=1.2Hz); 5.506 (d, 1H, H(9)> J9-s=7.6Hz ); 6.015 (m, 1H, H(8)); 7.360 (dd, 1H, H(2), J4-5=SHz, J4_6=1.6Hz); 7.398 (d, 1H, H(6), J6-4=1.6Hz); 7.425 (d, 1H, H(5), J5.4=8Hz). IR: 3378cm_1(-NH, -NH2); 1751cm_1(Ester-C=0); 1713cml(Conj. Ester -C=0); 1685cm'1 (Carbamate -C=0); 1511cm"1 (-Amide) ; 1220cm"'(Ester). MS: m/z=411.3 [M+H+]; m/z=433.3 [M+Na+].Rf=0.27 in Ethyl acetate / Hexane 1:2 on Si02- Example 7.1 (large scale enzymatic hydrolysis) (S)"3"Amino-4-(2-tert.-butoxycarbonylamino-2-carboxy-ethylsulfanyl')-benzoicacid allyl ester 6.4g (15.658mmol) of N-tert.-butoxycarbonyl-3-(4-alloxycarbonyl-2-aminophenylthio)-L-alanine methyl ester (99%) was dissolved in 480ml TBME and emulsified in 1.51 buffer solution (0.1M sodium chloride; 160mM sodium phosphate pH 7.5) under vigorous stirring. 12.0ml Alcalase 2.4 L [a subtilisin Carlsberg from Novo Nordisk] was added and the pH maintained at 7.5 under vigorous stirring by the controlled addition (pH-static) of 1.0N sodium hydroxide solution. After 48.5h 16.6ml of 1.0N sodium hydroxide solution was consumed and the conversion degree was >97% (HPLC analysis). After phase separation the aqueous phase was extracted once with 11 TBME. The combined TBME phases were extracted with 2 x 0.410.1 M potassium phosphate buffer pH 7.6. The combined aqueous phases were acidified to pH 2 with 32% hydrochloric acid and extracted with 3 x 0.51 ethyl acetate. In case a stabile emulsion was formed the phase separation was achieved by filtration on Dicalite. The combined ethyl acetate phases were dried on anhydrous sodium sulfate and evaporated. The residue was dissolved in dichloromethane, evaporated and dried at HV to give 5.85g N-tert.-butoxycarbonyl-3-(4-alloxycarbonyl-2-aminophenylthio)-L-alanine as a yellow highly viscous oil (yield: 94.6%). Analyticat data: !H-NMR (DMSO, 400MHz) 1.338 (s, 9H> OC(CH3)); 3.077 (dd,1H, SCH2, J=12.8Hz, J=6.8Hz); 3.238 (dd, 1H, SCH2, J=13.8Hz, J=4.6Hz); 3.867 (m, 1H, COCHNH); 4.753 (d, 2H, COOCH2, J=5.2Hz); 5.526 (d, 1H, CH=CH2, J=10Hz); 5.538 (d, 1H, CH-CH2, J=17.6Hz); 5.595 (m, 2H, NH2); 6.018(ddtr5 1H, CH2CH=CH2, J=16.6Hz, J=10.6Hz, J=5.6Hz); 6.519 (m, 1H, CONHCH); 6.876 (d, 1H, arom. CSCH, J=8.4Hz); 7.330 (d, 1H, arom. CSCHCH, J=8.4Hz); 7.338 (s, 1H, arom. CHCNH2). IR (ATR-IR) 3420 and 3355cm*1 (-NH, -NH2); 2980 and 2935 cm'1 broad (-COOH); 1705cm-1 (COOH-OO, carbamate -C=0); 1510cm"1 (amide-CO-NH); 1486cm"1 aromate; 1229 and 1160cm'1 (COOH); 982 and 932 cm"1 (vinyl, -C=CH2). MS (ESI-positive ionisation) m/z=397.1 [M+ET]; m/z=419.4 [M+Na+]. OR [a]D= +16.16 ° (CHC13; c=1.0 ). HPLC analysis: column: ABZ+plus; mobile phase: A: 0.1% TFA in H20; B: MeCN; gradient B: 30 - 80% 0-15 min, 80 - 30% 15 - 16 min, 30% 16 - 19.5 min; flow: 1 ml/min; pressure:50 - 80 bar; detection: UV, 300nm; retention times: 11.2 min (product-acid); 12.7 min (substrate-ester). Example 7.2 fsmall scale enzymatic hydrolysis) (S)-3-Amino-4-(2"tert.-butoxvcarbonyIamino-2"Carboxy-ethylsuIfanyD--benzoicacid allyl ester 0.769 g (L658mmol) of N-tert.-butoxycarbonyl-3-(4-alloxycarbonyl-2-aminophenyl-thio)-L-alanine methyl ester was dissolved in 40ml TBME and emulsified in 220ml 0.1M sodium chloride solution; 3mM sodium phosphate buffer pH 7.5 under vigorous stirring. 1.0ml Alcalase 2.4 L [a subtilisin Carlsberg from Novo Nordisk] was added and the pH maintained at 7.5 under vigorous stirring by the controlled addition (pH-static) of 1.0N sodium hydroxide solution. After 45.4h 23.6ml of 1.0N sodium hydroxide solution was consumed and the conversion degree was > 97% (HPLC analysis). The reaction mixture was extracted with 100ml TBME. After phase separation the aqueous phase was acidified to pH 2.3 with 32% hydrochloric acid and extracted with 3 x 250ml ethyl acetate. In case a stabile emulsion was formed the phase separation was achieved by filtration on Dicalite. The combined ethyl acetate extracts were dried on anhydrous sodium sulfate and evaporated. The residue was dissolved in dichloromethane, evaporated and dried at HV to give 604mg N- tert.-butoxycarbonyl-3-(4-alloxycarbonyl-2-aminophenylthio)-L-alanine as a pale yellow solid (yield: 93.9%, purity: 95%). Example 7.3 (enzymatic hydrolysis with different solvents) fS)-3-Amino-4-(2-tert.-butoxvcarbonylamino-2-carboxy-ethvIsulfanyl)-benzoicacidallyl ester 200mg (0.487mmol) of N-tert.-butoxycarbonyl-3-(4-alloxycarbonyl-2-aminophenylthio)-L-alanine methyl ester (96.7%) was added to a system consisting of 2 to 5ml of an organic co-solvent (see following table) and 20ml buffer solution (0.1M sodium chloride; 3mM sodium phosphate pH 7.0) under vigorous stirring. Alcalase 2.4 L [a subtilisin Carlsberg from Novo Nordisk, amount see following table] was added and the pH maintained at 7.0 under vigorous stirring by the controlled addition (pH-static) of 0. IN sodium hydroxide solution. The reaction was monitored by the consumption of titrating agent, and the formation of the product confirmed by HPLC. Example 7.4 (enzymatic hydrolysis with other enzymes) (S)-3-Amino-4-(2-tert.-butoxvcarbonvlamino-2-carboxv-ethylsulfanyI)"benzoicacidallyl ester 200mg (0.487mmol) of N-tert.-butoxycarbonyl-3-(4-alloxycarbonyl-2-aminophenylthio)-L-alanine methyl ester (96.7%) was added to a system consisting of 5ml TBME and 20ml buffer solution (0.1M sodium chloride; 3mM sodium phosphate pH 7.0) under vigorous stirring. Enzyme [see following table] was added and the pH maintained at 7.0 under vigorous stirring by the controlled addition (pH-static) of Q.1N sodium hydroxide solution. The reaction was monitored by the consumption of titrating agent, and the formation of the product confirmed by HPLC. Example 7,5 (enzymatic hydrolysis at higher temperature) (S)-3-AminO"4-(2-tert.-butoxycarbonylamino-2-carboxy"ethylsulfanyl)-benzoicacidallyl ester 200mg (0.487mmol) of N-tert.-butoxycarbonyl-3-(4-alloxycarbonyl-2-aminophenylthio)-L-alanine methyl ester (96.7%) was added to a system consisting of 5ml TBME and 20ml buffer solution (0.1M sodium chloride; 3mM sodium phosphate pH 7.0) under vigorous stirring at 40°C. IOOJJI of Alcalase 2.4 L [a subtilisin Carlsberg from Novo Nordisk] was added and the pH maintained at 7.0 under vigorous stirring at 40°C by the controlled addition (pH-static) of 0.1N sodium hydroxide solution. After a consumption 5.44ml of titrating agent (112% of theory; after 17.4h) the reaction mixture was washed with 25ml TBME, acidified to pH 2.5 with IN HCl and extracted with 3 x 25ml ethyl acetate. The combined organic phases were dried over sodium sulfate, the solvent evaporated and the residue dried on a high vacuum to give 190mg (0.479mmol; 98%) of the product acid in 94.5% HPLC-purity (area-%). Example 8 fS)-3-tert.-ButoxycarbonyIamino-4-oxo-23,4,5-tetrahydro-benzofb1[l,4lthiazepine-7-carboxylic acid allvl ester The reaction is carried out as described in G.C. Morton et al., Tet. Lett., 41 (2000) 3029-3033. The product from the enzyme hydrolysis (5.6g, 14.1mmol, leq) was dissolved in DMF (75mL) and treated with EDAC (2.70g, 14.1mmol, leq) and stirred at room temperature for 16h. After concentration the residue was dissolved in ethyl acetate (500mL) and extracted with (aq.) NaHC03 (1M) (3 X 150mL) and water (3 X 150mL). The organic phase was dried (Na2S04) and evaporated to give yellow oil. After chromatography with ethyl acetate / hexane 1:2. yellow crystals were obtained (4.96g, 93%). Analytical data: !H-NMR (CDC13,400MHz): 1.400 (s, 1H, 0(CH3)); 3.014 (t, 1H, H(7'or 7"), J=12Hz); 3.815 (dd, 1H, H(7'or 7"), J7-8=12Hz, Jr-7»=6.4Hz); 4.466 (m, 1H, H(8)); 4.835 (d, 2H, H(3*, 3"), J3-2= 5.7Hz); 5.316 (dd, 1H, H(l'), Ji..2=10.5Hz, Jr. i«=1.2Hz); 5.413 (dd, 1H, H(l"), Jr.2=17.2Hz, Jr.r=1.2Hz); 5.581 (d, 1H, H(9), J9. 8=8Hz ); 6.013 (m, 1H, H(8)); 7.338 (d, 1H, H(5), J5-4=H.lHz); 7.704 (s, 1H, H(10)); 7.751 Example 9 (S)-3-Amino-4-(2-tert.-butoxycarbonylamino-2-carboxv-ethylsulfanyl')-benzoicacid 200mg (0.540mrnol) of N-tert-butoxycarbonyl-3-(4-carboxy-2-aminophenylthio)-L-alanine methyl ester was added to a system consisting of 5ml TBME and 20ml buffer solution (0.1M sodium chloride; 3mM sodium phosphate pH 7.0) under vigorous stirring. 100^1 of Alcalase 2.5 L [a subtilisin Carlsberg from Novo Nordiskjwas added and the pH maintained at 7.0 under vigorous stirring by the controlled addition (pH-static) of 0.1N sodium hydroxide solution. After a consumption 5.847ml of titrating agent (108% of theory; after 1.5h) the reaction mixture was washed twice with 25ml TBME, acidified to pH 2.5 with 25% HC1 and extracted with 3 x 25ml ethyl acetate. The combined organic phases were dried over sodium sulfate, the solvent evaporated and the residue dried on a HV to give 187mg (0.479mmol) N-tert.-butoxycarbonyl-3-(2-amino-4-carboxyphenylthio)-L-alanine as yellow crystals (yield: 93.4%). Analytical data: HPLC-purity: 96.4%% (area). lH-NMR (DMSO, 400MHz) 1.378 (s, 9H, OC(CH3)); 2.982 (dd, 1H, SCH2(J=13.2Hz, J=9.6Hz); 3.150 (dd, 1H, SCH2, J=13.2Hz, J=4.4Hz); 3.950 (m, 1H, COCHNH); 5.542 (m, 2H, -NH2); 7.067 (dd, 1H, CONHCH, J=8.0Hz, J=1.6Hz); 7.201 (d, 1H, arom. SCCH, J=8.0Hz); 7.298 (d, 1H, arom. SCCHCH, J=8.0Hz); 7.323 (d, 1H, arom. CHCNH2, J=1.6Hz); 12.73 (bs, 2H, 2 -COOH). IR: 3445 and 3347cm-1 (-NH, -NH2); 2977 and 2930cm"1 broad (-COOH); 1720 and 1692cm'1 (COOH -0=0, carbamate-C=0); 1608cm"1 (-COO"); 1509cm"1 (amide -CO-NH); 1486 cm"1 (aromate); 1247 and 1163cm"1 (COOH). ISN-MS: m/z=355.1 [M-H*]. OR [a]D= +13.00° (EtOH; c=1.0). WE CLAIM: 1. A process for the preparation of compounds of formula I wherein Rl is hydrogen or alkyl; R2 is an amino protecting group; each R3 is independently halogen, carboxyl> alkoxycarbonyl, alkenyloxycarbonyl or benzyloxycarbonyl; n is 1 or 2, which process comprises reacting compounds of formula II wherein R1'R2 , R and n are as defined above and R4 is alkyl or benzyl, with a protease in an aqueous system containing an organic co-solvent. 2- The process as claimed in claim 1, wherein the compound of formula II is 1 O \ A. wherein R', R\ R3, R* and n are as defined in claim 1. 3# The process as claimed in claims 1 and 2, wherein Rl is hydrogen or alkyl; R2 is an amino protecting group; each R is independently halogen, carboxyl or alkenyloxycarbonyl; R4 is alkyl or benzyl; n is 1 or 2. 4. The process as claimed in any one of claims 1 to 3, wherein R1 is hydrogen; R is an amino protecting group; R3 is halogen, carboxyl or alkenyloxycarbonyl; R4 is alkyl; nis 1. 5. The process as claimed in any one of claims 1 to 4, wherein the reaction is carried out at pH of 6.0-8.5. 6. . The process as claimed in any one of claims 1 to 5, wherein the reaction is carried out with a Bacillus protease, subtilisin, an Aspergillus protease or a Tritachium protease. 7. The process as claimed in any one of claims 1 to 6, wherein the organic co-solvent is tetrahydrofuran, dioxan, tert-butyl methyl ether, a C1-8-alkohol, ethyl acetate, dimethylsulfoxide, dimethylacetamide or acetone.

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