Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D243/00—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms
  • C07D243/06—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms having the nitrogen atoms in positions 1 and 4
  • C07D243/10—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms having the nitrogen atoms in positions 1 and 4 condensed with carbocyclic rings or ring systems
  • C07D243/14—1,4-Benzodiazepines; Hydrogenated 1,4-benzodiazepines
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/10—Nitrogen as only ring hetero atom
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
  • C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
  • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
  • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction

Definitions

• This invention relates to the use of a lipase enzyme to effect the resolution of optical isomers of a chemical compound.
• proteolytic enzymes to hydrolyze esters is well known.
• the proteolytic enzyme reacts much more rapidly with one enantiomer of the racemic mixture than the other enantiomer. With appropriate substrates this chemical selectivity has been used as the basis for resolution of such a mixture.
• the products of such a selective hydro lytic reaction are a carboxylic acid and an alcohol for the reactive enantiomer, while the unreactive enantiomer persists as the ester. The ease of separation of the ester and acid then becomes the basis for the stereochemical purification of the carboxylic acid or alcohol fragment.
• the stereochemical purity of the process is generally dependent upon the relative rates of hydrolysis of the each of the isomers of the racemate, e.g., the greater the difference in relative rates, the higher the purity of the final chiral product.
• the choice of an appropriate enzyme to selectively hydrolyze a given chemical compound is largely empirical, if an appropriate enzyme can be found at all. Thus, in order to be useful, the enzyme must accept the desired compound as a substrate, selectively hydrolyze only the appropriate enantiomer, and produce an acceptable enantiomeric excess (e.e.).
• HPLC methods generally suffer the disadvantage that they are difficult to carry out on a large scale, while chiral syntheses rely upon expensive chiral synthons and may suffer from parital racemisation during the synthetic sequence. Accordingly, new stereoselective methods for preparing such compounds are needed.
• this invention is a process for resolving certain racemic substituted 3-oxo-2,3,4,5-lH-tetrahydro-l ,4-benzodiazepine-2-acetic acid esters using a lipase from Candida Antarctica to selectively hydrolyze one of the chiral esters
• this invention is a substantially enantiomerically pure substituted 3-oxo-2,3,4,5-lH-tetrahydro-l,4-benzodiazepine-2-acetic acid compound, particularly one prepared from a racemic compound by an enzymatic hydrolysis process.
• this invention is a method for improving the stereochemical purity of an ester of a 3-oxo-2,3,4,5-lH-tetrahydro-l,4-benzodiazepine-2-acetic acid.
• this invention comprises specific intermediate compounds which are useful in the synthesis of pharmaceutical products.
• this invention is stabilised immobilised Candida Antarctica lipase B preparation, and a process for producing such a preparation.
• This invention is a process for preparing a compound of the formula (I):
• X is H, halogen, CO2R 3 , OR 4 , COR 5 , or a fibrinogen or vitronectin antagonist side chain;
• R 2 is Ci.galkyl, optionally substituted by Ar, Het or C ⁇ _ 6 cycloalkyl;
• R 3 is C ⁇ _6alkyl or benzyl
• R 4 is C ⁇ -6 alkyl, COR 3 or benzyl
• R 5 is 4,4'-bipiperidin-l-yl, ( -benzyloxycarbonyl)-4,4'-bipiiperidin-l-yl, or (l'-t- butoxycarbonyl)-4,4'-bipiperidin-l-yl; which comprises: treating a compound of the formula (II):
• R 1 is C ⁇ _2 ⁇ alkyl or C3_2 ⁇ alkenyl, optionally substitutedby Ar, NR 2 or NR 3 + , wherein R is C,. 4 alkyl; with Candida Antarctica lipase B; and separating the resulting (S)-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4-benzodiazepine-2- acetic acid carboxylic acid from the corresponding (R)-ester.
• the 3-oxo-2,3,4, 5-tetrahydro-l,4-benzodiazepine acetates of formula (I) are of interest as pharmaceutical products or intermediates in the preparation of pharmaceutical products.
• the (S) stereoisomer of these compounds is responsible for the pharmacological activity.
• either a homochiral synthesis, a physical separation, or chemical resolution is required. While physical resolutions and homochiral syntheses have been reported for such compounds, no chemical resolution has been achieved.
• the Candida Antarctica lipase B has now been found to react with optionally 7- substituted 3-oxo-2,3,4,5-tetrahydro-l,4-benzodiazepine acetates giving the (S)- acid with high stereoselectivity.
• the (R)-enantiomers are effectively not hydrolytic substrates for the enzyme hence the hydrolysis stops spontaneously at about 50% conversion.
• the enzyme is also highly sensitive toward the specific substitution of the (S)-substrate. For instance, the substitution of a methylene group for the 1 -nitrogen of the 1,4 benzodiazepine abolishes activity.
• R 1 is optionally substituted by phenyl. More suitably, R 1 is C ⁇ _ 4 alkyl or benzyl.
• R 2 is H, or C ⁇ alkyl, optionally substituted by phenyl.
• R 1 is methyl and R 2 is methyl.
• X is hydrogen, bromo, iodo, t-butoxycarbonyl, benzyloxycarbonyl, methoxycarbonyl, hydroxy, methoxy, (4,4'-bipiperidin-l-yl)carbonyl, or [ -t-butoxycarbonyl-(4,4'-bipiperidin-l-yl)]carbonyl.
• R 3 is preferably t-butyl.
• the enzyme may be used as a part of a whole cell culture, an enzyme extract, an isolated enzyme, or an isolated enzyme attached to a solid support, such as a macro- porous acrylic resin.
• a solid support such as a macro- porous acrylic resin.
• Supported preparations of the Candida Antarctica lipase B may be purchased commercially from Novo Nordisk, Badsvaerd, Denmark (as Novozym 435) and from Boehringer Mannheim Gmbh, Mannheim, Germany (as L-2 lipase).
• the enzyme is generally thermostable and tolerant of high concentrations of many organic solvents. Supported preparations of the enzyme are preferred for their ease of handling. However, certain problems may occur when a supported preparation of the enzyme is used, particularly in cases where the enzyme is non-covalently bound to resin.
• a cross-linking reagent such as glutaraldehyde.
• Other cross-linking reagents such as dimethyl suberimidate and glutaraldehyde oligomers may also be useful.
• pretreatment consists merely of treating the resin with glutaraldehyde in a mixture of an organic solvent, such as t-butanol, and water for several hours.
• Such treatment stabilises the resin, so that much less enzyme is lost from the resin with each use, so that the number of times the resin may be reused is increased. Additionally, problems with the formation of emulsions on large scale is much reduced.
• the hydrolysis is generally run in water - organic solvent mixtures.
• organic solvents such as acetone, methylethyl ketone, methyl isobutyl ketone, t-butanol, benzene and toluene, and the use of either a solution or two-phase system is suitable. Nevertheless, some experimentation may be necessary in choosing an appropriate solvent to match the solubility characteristics of the substrate.
• An increase in reaction rate may sometimes be achieved by the use of a two phase system, such as water and an aromatic hydrocarbon solvent mixture, such as water and benzene, toluene, xylene or mesitylene.
• a two phase system such as water and an aromatic hydrocarbon solvent mixture, such as water and benzene, toluene, xylene or mesitylene.
• Water/toluene is particularly suitable.
• the reaction mixture can be buffered or run with a constant pH by the addition of base. There is generally little change in reaction rate or stereoselectivity between pH 6.0 and 8.0, however.
• the reaction is run above 20°C.
• room temperature such as about 28 - 45°C the amount of solvent and the reaction time may be reduced.
• the reaction is run at about 36°, 200 volumes of solvent may be reduced to about 40 volumes of solvent, and the reaction time also drops to ⁇ 24 h from 4 days.
• reaction time is not critical. Typically the reaction is run from several hours to several days depending upon the temperature, solvent and substrate used in the reaction. Ten to twenty-four hours is usually suitable.
• the reaction failed when lipases from the following sources were tested: Porcine Pancreatic lipase, Candida lipolytica, Candida Cylindracea, Mucor Javanicus, Pseudomonas Fluorescens, Aspergillus usamii, Geotrichum Candidum, Aspergillus Niger, Humicola Lanuginosa, Ammano SAM2, Rhizopus Arrhizus, Penicillium cycopum, Rhizopus Nivens, Rhizopus Javanunicus, Ammano Lipase A, Rhizopus Delewar, Penicillinium Roqueforti, and Boehringer Lipases- L-1, L-3, L-4, L-5, L-6, and L-8. A second lipase from Candida Antarctica, lipase A, was also unreactive.
• hydrolytic enzymes such as Carlsberg subtilisin and pig liver esterase, will hydrolyse the methyl ester of the 7-unsubstituted benzodiazepine but show little stereoselectivity.
• Conventional techniques for separating an ester from an acid are used to separate the carboxylic acid product from the unreacted ester.
• the reaction mixture is adjusted to a basic pH (e.g., > ⁇ H7) and high salt concentration, and extracted with an appropriate organic solvent. This results in the unreacted ester being dissolved in the organic solvent and the carboxylic acid product as being present in the aqueous layer.
• Drying and evaporation of the organic solvent yields the chiral ester; while acidification of the aqueous layer, extraction, drying and evaporation of the organic solvent yield the desired chiral acid.
• Other techniques such as the use of crystallisation, or chromatography over a silica gel based support, or an ion-exchange or other appropriate resin may be also be used to separate the acid from the ester, and are within the scope of this invention.
• the product prepared by the process of this invention will be substantially enantiomerically pure. Typically it will be greater than 80% (e.e.), preferably greater than 90%), more preferably greater than 95%>, and most preferably greater than 99%>. While this invention is principally a process for preparing a non-racemic 3-oxo-
• the instant invention may also be used as a process for increasing the stereochemical purity of a chiral compound according to formula (I) wherein a significant amount of the (R)-enantiomer is present.
• the instant process may be utilised to increase the e.e. of the product.
• this invention is a process for preparing a compound according to formula (III):
• R 2 is Ci.galkyl, optionally substituted by Ar, Het or Ci.gcycloalkyl.
• R 3 is Ci. ⁇ alkyl or benzyl
• R 4 is Ci- ⁇ alkyl, COR 3 or benzyl; which comprises: (a) treating a compound of the formula (IV):
• R 1 is C ⁇ _2oal yl or C3_2oalkenyl, optionally substitutedby Ar, NR 2 or NR 3 + , wherein R is
• this invention is a process for preparing (S)-7- [(4,4'-bipiperidin-l-yl)carbonyl]-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4- benzodiazepine-2-acetic acid, which comprises treating (R,S)-methyl 7-[(4,4'-bipiperidin- l-yl)carbonyl]-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4-benzodiazepine-2-acetate with a lipase B from Candida Antarctica; and separating the (S)-7-[(4,4'-bipiperidin-l- yl)carbonyl]-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4-benzodiazepine-2-acetic acid from the (R) methyl 7-[(4,4'-bipiperidin-l-yl)carbony
• the substrate is (R,S)-methyl 7-[(l'-t-butoxycarbonyl-(4,4'- bipiperidin-l-yl))carbonyl]-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4-benzodiazepine-2- acetate, or (R,S) methyl 7-[(l'-t-benzyloxycarbonyl-(4,4'-bipiperidin-l-yl))carbonyl]- 2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l ,4-benzodiazepine-2-acetate, further comprising the step of removing the t-butoxycarbonyl or benzyloxycarbonyl group following separation of the enantiomers.
• this invention is a process for preparing (S) 7- iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l ,4-benzodiazepine-2-acetic acid, which comprises treating (R,S) methyl 7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4- benzodiazepine-2-acetate with lipase B from Candida Antartica; and separating the (S) 7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4-benzodiazepine-2- acetic acid from the (R) methyl 7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4- benzodiazepine-2-acetate.
• this invention is a process for preparing (S)- 2,3 ,4,5-tetrahydro-4-methyl-3-oxo- 1 H- 1 ,4-benzodiazepine-2-acetic acid, which comprises treating (R,S)-methyl 2,3,4, 5-tetrahydro-4-methyl-3-oxo-lH-l,4- benzodiazepine-2-acetate with lipase B from Candida Antartica; and separating the (S)-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l ,4-benzodiazepine-2-acetic acid from the (R)-methyl 2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4-benzodiazepine-2- acetate.
• this invention is a process for preparing a nonracemic compound according to formula (I), wherein X is -COR 5 , or a fibrinogen or vitronectin antagonist side chain, which comprises converting a compound of formula (I), wherein X is H, halogen, CO R 3 or OR 4 .
• Compounds of formula (III) are particularly useful intermediates in preparing compounds according to formula (I) wherein X is COR 5 , or a fibrinogen or vitronectin antagonist side chain.
• Preferred moieties for X' are H, Br, I, CO 2 CH 3 , CO 2 -t-Bu and OH.
• Preferred moities for R 1 are C j ⁇ alkyl and benzyl.
• esters would be C ⁇ alkyl, phenyl or benzyl esters, and derivatives thereof, and they may be prepared by conventional esterification reactions from the corresponding carboxylic acids.
• this invention is also a method of preparing compounds of formula (I), wherein X is COR 5 , or a fibrinogen or vitronectin antagonist side chain, which comprises preparing a compound of formula (III) by the above process, and converting the compound of formula (III) into a compound of formula (I), wherein X is COR 5 , or a fibrinogen or vitronectin antagonist side chain.
• Conversion of the intermediate compounds of this invention into compounds of formula (I), wherein X is COR 5 , or a fibrinogen or vitronectin antagonist side chain may be effected by conventional reactions and procedures, such as those described in WO 93/00095 (PCT/US92/05463), WO 94/14776 (PCT/US93/12436), WO 95/18619 (PCT/US95/00248), WO 96/00730 (PCT/US95/08306), WO 96/00574
• this invention is a process for preparing a compound according to formula (I), such as (S)-7-[(4,4'-bipiperidin-l-yl)carbonyl]- 2,3,4, 5-tetrahydro-4-methyl-3-oxo-lH-l,4-benzodiazepine-2-acetic acid, which comprises converting (S) 2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4-benzodiazepine-2- acetic acid, (S) 7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4-benzodiazepine-2- acetic acid; (S) 7-bromo-2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4-benzodiazepine-2- acetic acid; or (S) 7-t-butoxycarbonyl-2,3,4,5-tetrahydro-4-methyl-3-
• a fibrinogen antagonist or vitronectin antagonist side chain may be generally given by the formula: W-(CR'2) q -Z-(CR , R 10 ) U-(CR' 2 )s-V- or W-(CR' 2 )q-U-(CR'2)s-, wherein R' is H, C ⁇ _6alkyl, C3_7cycloalkyl-Co- 4 alkyl or Ar-Co- 4 alkyl;
• R" is R', -C(O)R' or -C(O)OR*;
• R 1 is H, Ci-6alkyl, C3- 7 cycloalkyl-Co- 4 alkyl or Ar-Co- 4 alkyl;
• R 5 is H, Ci- ⁇ alkyl, C3_ 7 cycloalkyl-C ⁇ -4alkyl or Ar-Co- 4 alkyl;
• R 7 is H, halo, -OR 12 , -SR 12 , -CN, -NR'R 12 , -NO 2 , -CF 3 , CF 3 S(O) r , -CO 2 R', -CONR' 2 , R 14 -C 0- 6alkyl-, R 1 -C ⁇ -6 oxoalkyl-, R 14 -C 2 - 6 alkenyl-, R 14 -C 2-6 alkynyl-, R 14 -C 0-6 alkyloxy-, R 14 -C 0 - 6 alkylamino- or R 14 -C 0 - 6 alkyl-S(O) r ;
• R 8 is R', C(O)R', CN, NO 2 , SO 2 R' or C(O)OR 5 ;
• R 9 is R', -CF 3 , -SR 1 , or -OR';
• R i O is H, C ⁇ . 4 alkyl or -NR * R";
• R 12 is R', -C(O)R', -C(O)NR' 2 , -C(O)OR 5 , -S(O)mR' or S(O) 2 NR' 2 ;
• R 14 is H, C3_6cycloalkyl, Het or Ar;
• R 15 is H, Ci-ioalkyl, Cs. ⁇ cycloalkyl-Co-salkyl or Ar-Co-salkyl;
• Q is NR', O or S
• R a is H, Ci- ⁇ alkyl, Ar-C 0 -6alkyl, Het-Cn- ⁇ alkyl, or C 3 .6cycloalkyl-C 0 - 6 alkyl, halogen, OR 1 , SR 1 , COR 1 , OH, NO 2 , N(R 1 ) 2; CO(NR 1 ) 2 , CH 2 N(R 1 ) 2;
• R b and R c are independently selected from H, C ⁇ _6alkyl, Ar-Co- ⁇ alkyl, Het-Co- 6 alkyl, or C 3 . 6 cycloalkyl-Co-6alkyl, halogen, OR 1 , SR 1 , COR 1 , OH, NO 2 , N(R 1 ) 2>
• Y is absent, S or O;
• Z is (CH 2 ) t , Het, Ar or C3- 7 cycloalkyl;
• m is 1 or 2;
• n is 0, 1, 2 or 3;
• q is 0, 1, 2 or 3;
• r is 0, 1 or 2;
• s is O, 1 or 2;
• t is 0, 1 or 2;
• v is 0 or 1 ; and
• w is 0 or 1; q is 0, 1, 2 or 3.
• Such side chains are described, for instance, in WO 93/00095 (PCT/US92/05463), WO 94/14776 (PCT/US93/12436), WO 95/18619 (PCT/US95/00248), WO96/00730 (PCT/US95/08306), WO96/00574 (PCT/US95/08146), and PCT/US96/11108, each of which is incorporated herein by reference as though fully set forth.
• Nitrogen protecting groups are well known to the art and comprise such groups as the acetyl, formyl, trifluoroacetyl, benzoyl, benzyloxycarbonyl and alkyloxcarbonyl groups.
• Typical fibrinogen receptor and vitronectin receptor side chains are:
• C ⁇ _ 4 alkyl as applied herein is meant to include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl.
• Ci- ⁇ alkyl additionally includes pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. likewise includes simple aliphatic hydrocarbons of the indicated number of carbons. Any alkyl group may be optionally substituted by R 7 unless otherwise indicated.
• Co- 4 alkyl and Co-6alkyl additionally indicates that no alkyl group need be present (e.g., that a covalent bond is present).
• C 2- 6alkenyl as applied herein means an alkyl group of 2 to 6 carbons wherein a carbon-carbon single bond is replaced by a carbon-carbon double bond.
• C 2 .galkenyl includes ethylene, 1 -propene, 2-propene, 1 -butene, 2-butene, isobutene and the several isomeric pentenes and hexenes.
• C 3 . 2 oalkenyl likewise includes simple aliphatic hydrocarbons of the indicated number of carbons wherein one or more carbon-carbon single bonds are replaced by a carbon-carbon double bond.. Both cis and trans isomers are included. Any alkenyl group may be optionally substituted by R 7 unless otherwise indicated.
• C 2- 6alkynyl means an alkyl group of 2 to 6 carbons wherein one carbon-carbon single bond is replaced by a carbon-carbon triple bond.
• C 2 .g alkynyl includes acetylene, 1-propyne, 2-propyne, 1-butyne, 2-butyne, 3-butyne and the simple isomers of pentyne and hexyne. Any sp 3 carbon atom in the C 2 - 6 alkynyl group may be optionally substituted by R 7 .
• C ⁇ _ 4 ⁇ xoalkyl refers to an alkyl group of up to four carbons wherein a CH 2 group is replaced by a C(O), or carbonyl, group. Substituted formyl, acetyl, 1-propanal, 2- propanone, 3-propanal, 2-butanone, 3-butanone, 1- and 4-butanal groups are representative.
• C ⁇ _6 ⁇ xoalkyl includes additionally the higher analogues and isomers of five and six carbons substituted by a carbonyl group.
• a substituent on a Cj.galkyl, C -6 alkenyl, C 2 -6 lkynyl or Ci.goxoalkyl group, such as R 7 may be on any carbon atom which results in a stable structure, and is available by conventional synthetic techniques.
• Halogen indicates fluoride, chloride, bromide or iodide.
• Ar, or aryl as applied herein, means phenyl or naphthyl, or phenyl or naphthyl substituted by one to three moieties R 7 .
• R 7 may be C ⁇ . 4 alkoxy, C ⁇ -4 alkthio, trifluoroalkyl, OH, F, Cl, Br or I.
• Het, or heterocycle indicates an optionally substituted five or six membered monocyclic ring, or a nine or ten-membered bicyclic ring containing one to three heteroatoms chosen from the group of nitrogen, oxygen and sulfur, which are stable and available by conventional chemical synthesis.
• heterocycles are benzofuran, benzimidazole, benzopyran, benzothiophene, furan, imidazole, indole, indoline, morpholine, piperidine, piperazine, pyrrole, pyrrolidine, tetrahydropyridine, pyridine, thiazole, thiophene, quinoline, isoquinoline, and tetra- and perhydro- quinoline and isoquinoline.
• a six membered ring heterocycle containing one or two nitrogens, such as piperidine, piperazine, tetrahydropyridine and pyridine, are preferred heterocycles for the moiety Z. Any accessible combination of up to three substituents, such as chosen from R 7 , on the Het ring that is available by chemical synthesis and is stable is within the scope of this invention.
• C3.7cycloalkyl refers to an optionally substituted carbocyclic system of three to seven carbon atoms, which may contain up to two unsaturated carbon-carbon bonds.
• Typical of C3_ 7 cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl and cycloheptyl. Any combination of up to three substituents, such as chosen from R 7 , on the cycloalkyl ring that is available by conventional chemical synthesis and is stable, is within the scope of this invention.
• a nitrogen heterocycle which may be a saturated or unsaturated stable five-, six- or seven-membered monocyclic ring, or a seven- to ten- membered bicyclic ring containing up to three nitrogen atoms or containing one nitrogen atom and a heteroatom chosen from oxygen and sulfur, and which may be substituted on any atom that results in a stable structure.
• the nitrogen atom in such ring may be substituted so as to result in a quaternary nitrogen.
• the nitrogen heterocycle may be substituted in any stable position by R 20 , for instance H, C ⁇ alkoxy, F, Cl, Br, I, NO2, NR' 2 , OH, CO 2 R', CONHR', CF3, R 1 -C 0 - 4 alkyl, R 14 -C ⁇ -4 alkyl-S(O) u (e.g. , where u is 0, 1 or 2) or Ci-4alkyl substituted by any of the aforementioned sustituents.
• R 20 for instance H, C ⁇ alkoxy, F, Cl, Br, I, NO2, NR' 2 , OH, CO 2 R', CONHR', CF3, R 1 -C 0 - 4 alkyl, R 14 -C ⁇ -4 alkyl-S(O) u (e.g. , where u is 0, 1 or 2) or Ci-4alkyl substituted by any of the aforementioned sustituents.
• N of _/ are pyrroline, pyrrolidine, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, piperidine, piperazine, morpholine, pyridine, pyridinium, tetrahydropyridine, tetrahydro- and hexahydro-azepine, quinuclidine, quinuclidinium, quinoline, isoquinoline, and tetra- and perhydro- quinoline and isoquinoline.
• ( G_) may be pyridyl, pyrolidinyl, piperidinyl, piperazinyl, azetidinyl,
• G-— is preferably 4-pyridyl, 4-(2-amino-pyridyl), 4-tetrahydropyridyl, 4-piperidinyl or 4-piperazinyl.
• the ring formed will generally be a five- or six-membered heterocycle selected from those listed above for Het, or will be a phenyl, cyclohexyl or cyclopentyl ring.
• Benzimidazolyl, 4- azabenzimidazolyl, 5-azabenzimidazolyl and substituted derivatives thereof are preferred moieties for W.
• esters of formula (II) and (IV), which are used in this invention are prepared according to procedures describe in WO 93/00095 (PCT/US92/05463), WO 94/14776 (PCT/US93/12436), WO 95/18619 (PCT/US95/00248), WO96/00730 (PCT/US95/08306), WO96/00574 (PCT/US95/08146), WO 97/24336, and PCT/US96/1 1108, each of which is incorporated herein by reference.
• the methyl 3-oxo-2,3,4,5-tetrahydro-l,4-benzodiazepine-2-acetate (0.5 g), or a suitably substituted derivative thereof, was dissolved in acetone (30 mL) or t-butanol and buffer (70 mL, pH 7.0, 0.1 N phosphate).
• Candida Antarctica lipase, supported on macroporous acrylate resin (200 mg, /700 PLU/g, marketed as Novozym 435) was added and the reaction stirred at ambient for 4.0 days.
• the reaction was monitored by HPLC and stopped spontaneously at 47% hydrolysis.
• the pH was adjusted to 8.0 with NaOH solution and EtOAc added (75 mL).
• the filtrate was treated with a solution of benzyl chloroformate (7.8 g, 0.046 mol) in CH 2 C1 2 (130 mL). This mixture was stirred at 30°C for 1 hr keeping the pH at 7.0 by adding 1.5 molar NH 3 solution. The phases were separated and the aqueous phase re- extracted with CH 2 C1 2 (130 mL). The aqueous phase was heated at 55°C under vacuum keeping the pH at 6.8. When the volume of the solution was -150 mL, the solution was cooled to 5°C and left for 12 hr with occasional pH adjustment. The solid was then filtered off, washed with cold water and dried to yield the title compound (16g, 80%); 99.9% (S)-isomer by chiral HPLC analysis.
• Candida Antarctica lipase B supported on macroporous acrylate resin (200 mg, /700 PLU/g, marketed as Novozym 435) was added and the reaction stirred at ambient temperature for 4.0 days. The pH was adjusted to 8.0 with NaOH solution and EtOAc added (75 mL). The mixture was filtered, and the EtOAc layer separated. The aqueous layer was re-extracted with EtOAc, the combined EtOAc extracts were dried (Na2SO 4 ) and the EtOAc evaporated in vacuo to yield the (R)-ester.
• the toluene layer was separated and the aqueous layer was extracted with ethyl acetate (2 x 100 mL) maintaining the pH at 8.0 with ammonia solution (1.5M).
• the combined organic extracts (toluene and ethyl acetate) were stripped to dryness and the residue recrystallised from dichloromethane - hexane to yield the (R)-isomer of the starting ester (8 g, 80%).
• Example 5 Preparation of (S)-7-iodo-2.3.4.5-tetrahydro-4-methyl-3-oxo-lH-1.4-benzodiazepine-2- acetic acid The procedure of Example 3 was repeated to the point of extracting the (R)-ester from the aqueous phase containing (S) 2,3,4,5-tetrahydro-4-methyl-3-oxo-lH-l,4- benzodiazepine-2-acetic acid.
• aqueous solution was then adjusted to pH 7.0 and pyridine IC1 complex (10.2 g, 0.042 mol) was added. The mixture was stirred for one hour maintaining the pH at 6.0 with ammonia solution (1.5 M). The pH was then adjusted to 4.0 with HCl (cone) and stirred for sixteen hours. The product was filtered off and washed with cold pH 4 buffer and dried to yield the title compound (11.2 g, 84%). 99.82%o (S)-isomer by chiral HPLC analysis.
• Novozym 435 Novozym 435 (Novo Nordisk, 3 g) was stirred at room temperature in a mixture of water (4 mL), t-butanol (45 mL) and glutaraldehyde (1 mL, 50% w/w in water) for about 3-4 h without pH adjustment. The resin was filtered and washed with water, and was used without further drying.

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