CZ229399A3 - Process of enzymatic splitting of benzodiazepine acetic acid esters by lipase - Google Patents

Abstract

Process for the preparation of benzodiazepine acetic acid of the general formula (1) from a compound of formula (II) wherein the substituents have of particular importance lies in the processing of a racemic mixture of esters acid by stereoselective hydrolysis of lipase B from CANDIDA ANTRACTICA

Description

Method of enzymatic cleavage of benzodiazepinacetic acid esters by lipase

Technical field

The invention relates to the use of a lipase for the enzymatic resolution of the optical isomers of a chemical compound.

BACKGROUND OF THE INVENTION

The use of proteolytic enzymes for ester hydrolysis is known in the art. In some cases, where the ester substrate has one or more chiral carbon atoms, the proteolytic enzyme reacts with one of the enantiomers of the racemic mixture much more rapidly than with the other. In the case of suitable substrates, said chemical selectivity has been used as the main principle for the cleavage of said mixtures. The products of such a selective hydrogenation reaction of a reactive enantiomer are a carboxylic acid and an alcohol, while the non-reactive enantiomer remains in the form of an ester. The ease of separation of ester and acid is then the basis for the stereochemical purification of the residue with a carboxylic acid or alcohol.

In general, if the chiral and complex arrangements are on the remainder of the carboxylic acid molecule, they are used to prepare the corresponding esterase or protease acids, and when the complex and chiral arrangements are on the alcohol moiety, lipases are used to prepare the chiral alcohols. However, there are known cases of some chiral acid cleavage lipases.

The stereochemical purity achieved in this process usually depends on the relative rate of hydrolysis of each of the isomers.

· • 9 • 9 • ¹ · racemate for example, so that the greater the difference in these relative speeds, the higher the purity of the final chiral product. The choice of a suitable enzyme for the selective hydrolysis of a given chemical compound, if any such enzyme can be found, is mainly empirical. Thus, if the method is to be applicable, the desired compound must be accepted by the enzyme as a substrate, the enzyme must only hydrolyze the respective enantiomer and produce an acceptable enantiomeric excess (ee).

WO 93/00095 (PCT / US / 92/05463), WO 94/14776 (PCT / US93 / 12436), WO 95/18619 (PCT / US95 / 00248) and PCT / US96 / 11108 disclose certain benzodiazepine compounds with pharmaceutical use. As is often the case with such compounds, one of the enantiomers in the racemates exhibits pharmacological activity. The methods disclosed in these patents include separation of enantiomers by chiral high performance liquid chromatography (HPLC) and homochiral synthesis. However, HPLC methods have the general disadvantage that they are difficult to carry out on a commercial scale, while chiral syntheses are dependent on expensive chiral synthons and partial racemization may occur during the synthesis steps. Thus, the need for new, stereoselective methods for the preparation of these compounds is still relevant.

The present inventors have now found that certain compounds disclosed in WO 93/00095 (PCT / US / 92/05463), WO 94/14776 (PCT / US93 / 12436), WO 95/18619 (PCT / US95 / 00248) can be prepared in non-racemic form by a method comprising cleaving the racemic mixture by stereoselective hydrolysis using a lipase isolated from the yeast Candida antarctica. This cleavage is easy to perform and highly selective and represents a significant step in the field of chiral chemistry of 3-oxo-1,4-benzodiazepine-3-acetic acid.

SUMMARY OF THE INVENTION

One aspect of the invention is a method of resolving certain esters of substituted racemic 3-oxo-2,3,4,5-1H-tetrahydro-1,4-benzodiazepine-2-acetic acid using a Candida antarctica lipase to selectively hydrolyze one of said esters.

In another aspect, the invention provides a substantially enantiomerically pure compound, substituted 3-oxo-2,3,4,5-1H-tetrahydro-1,4-benzodiazepine-2-acetic acid, in particular the above compound obtained from a racemic compound by an enzymatic hydrolytic method .

Another aspect of the invention is a method of improving the stereochemical purity of a 3-oxo-2,3,4,5-1H-tetrahydro-1,4-benzodiazepineacetic acid ester.

In another aspect, the invention includes specific intermediate compounds that are useful in the synthesis of pharmaceutical products.

Finally, the invention encompasses a stabilized immobilized composition comprising Candida antarctica lipase B and a process for preparing the composition.

Detailed description

The invention relates to a process for the preparation of a compound of formula (I):

• · • ·

• · · · · · · · · ♦ ··· ···

where

X is H, halogen, CO ₂ R ³ , OR ⁴ , COR ^S , or a fibrinogen or vitronectin antagonist side chain;

R ² is C _x _ _s alkyl group optionally substituted with Ar, Het, or C cycloalkyl;

R ³ represents C _x alkyl or benzyl group;

R ⁴ represents a group consisting of C _alkyl, COR ^3, and benzyl;

^R represents a group consisting of 4,4'-bipiperidine-l-yl, (1'-benzyloxycarbonyl) -4,4'-bipiperidin-l-yl, and (1'-tert-butoxycarbonyl) -4,4'-bipiperidine -1-yl;

which includes:

treating a compound of formula (II):

(Π) • · where

R ¹ represents a radical comprising _{a C} _ C _2o alkyl or alkenyl optionally substituted with Ar, _NR2 or _NR3 ', wherein R is C _i _ ₄ alkyl;

with lipase B from Candida antarctica; and separating the obtained carboxylic acid, (S) -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid from the corresponding (R) -ester.

Said 3-oxo-2,3,4,5-tetrahydro-1,4-benzodiazepine acetates of formula (I) are important as pharmaceutical products or as intermediates in the preparation of pharmaceutical products. As disclosed in WO 95/18619 (PCT / US95 / 00248), the (S) stereoisomer is pharmacologically active in these compounds. Thus, homochiral synthesis, physical separation, or chemical cleavage is required for preparation. While physical separation and homochiral synthesis are already known in the art, chemical cleavage has not yet been disclosed.

The present inventors have now found that Candida antarctica lipase reacts with optionally 7-substituted 3-oxo-2,3,4,5-1,4-benzodiazepine acetates to form a (S) -acid with high stereoselectivity. The (R) -enantiomers are not suitable hydrolytic substrates for this enzyme since hydrolysis stops spontaneously at about 50% conversion. Said enzyme is also highly selective for the specific substitution of the (S) -substrate. For example, replacement of 1-nitrogen in 1,4-benzodiazepine with a methylene group in loss of activity. Similarly, when the substituent X is CO ₂ R ³ , displacement of this substituent from the 7- to 8- position results in a loss of hydrolytic activity. However, at the 7-position of the benzodiazepine ring there is considerable tolerance to various types of substituents. Suitably, R ^x in formula (II) is C 1 -C 10 alkyl or C 3 alkenyl optionally substituted by phenyl. More preferably, R ^{1 is} C 1-6 alkyl or benzyl. R ² is suitably H or C _i _ ₄ alkyl group optionally substituted by a phenyl group. Preferably, in formula (II), R ¹ is methyl and R ² is methyl. X suitably represents hydrogen, bromo, iodo, t-butoxycarbonyl, benzyloxycarbonyl, methoxycarbonyl, hydroxy, methoxy, (4,4'-bipiperidin-l-yl) carbonyl, and [1 ¹ -Boc-Pipecolyl- (4.4 1'-bipiperidin-1-yl)] carbonyl. R ³ preferably represents tert-butyl.

Said enzyme may be used in the form of a proportion of the total cell culture, as an enzymatic extract, as an isolated enzyme or as an isolated enzyme on a solid support such as a macroporous acrylic resin. Lipid B-containing preparations of Candida antarctica on carriers can be obtained commercially from Novo Nordisk, Badsvaerd, Denmark (as Novozym 435) and from Boehringer Mannheim Gmbh, Mannheim, Germany (as L-2 lipase). In general, this enzyme is thermostable and tolerates high concentrations of many organic solvents. Of these, enzyme-based formulations on carriers are preferred for ease of handling.

However, there may be some problems with the use of carrier formulations, especially when the enzyme is not bound to the carrier by a covalent bond. For example, it has been found that due to protein leaching, the number of additional uses of the enzyme on the resin support is somewhat reduced. In addition, it has been found that when the reaction is carried out in a process.

Ί scale, so that leaching of protein can lead to the formation of emulsions.

The present inventors have now found that these problems can be overcome by treating the resin prior to use with a crosslinking agent such as glutaraldehyde. It is also possible to use other crosslinking agents such as dimethylsuberimidate and oligomeric glutaraldehyde. Typically, prior to use, this resin treatment only involves treating the resin with glutaraldehyde in a mixture of an organic solvent such as tert-butanol with water for several hours. This treatment stabilizes the resin, thereby causing a much smaller loss of enzyme from the resin in each assay, thus allowing the number of reuses of the resin to be increased. In addition, problems with emulsion formation in process preparation are greatly reduced.

In general, the hydrolysis is carried out in water-organic solvent mixtures. Various solvents may be suitable, such as solvents including acetone, methyl ethyl ketone, methyl isobutyl ketone, tert-butanol, benzene and toluene, and are suitable either in solution or in a two-phase system. However, preliminary experiments may be necessary to select a suitable solvent to achieve appropriate solubility parameters for the substrate. For example, when selecting an acetone-water mixture, the substrate is (R, S) -methyl-7 - [(1'-tert-butoxy-carbonyl- (4,4'-bipiperidin-1-yl)) carbonyl] -2,3 4,5-1-tetrahydro-4-methyl-3-iso-1H-1,4-benzodiazepine-2-acetate low reactivity. However, if a tert-butanol-water mixture is used, the reaction proceeds continuously. Particularly suitable organic solvents for the single-phase system are ketones and secondary and tertiary alcohols. In some cases, it is possible to increase the reaction rate by using a two-phase system, for example using a mixture of water and an aromatic hydrocarbon such as water and benzene, toluene, xylene or mesitylene. Water / toluene is particularly suitable.

The reaction mixture may be buffered, or the reaction may be carried out at a constant pH with the addition of a base. However, reaction rates or stereoselectivity generally vary little in the pH range of 6.0 to 8.0.

A suitable temperature for the reaction is above 20 ° C. In general, however, if the reaction is carried out at temperatures above room temperature, such as about 28-45 ° C, it is possible to reduce the amount of solvent and to reduce the reaction time. For example, if the reaction temperature is about 36 ° C, the solvent volume can be reduced from 200 volumes to 40 volumes and the reaction time is reduced from 4 days to about 24 hours.

Typically, the reaction is allowed to proceed until 50% of the starting ester is consumed. This can be monitored by HPLC, preferably chiral HPLC. Since the (R) -ester is a poorly acceptable substrate for said enzyme, the reaction time is not critical. Typically, the reaction is allowed to proceed for several hours to several days, depending on the temperature, solvent and substrate used in the reaction. Typically, a reaction time of ten to twenty-four hours is appropriate.

The selective effect on the 3-oxo-2,3,4,5-tetrahydro-1,4-benzodiazepine compounds of the invention has been tested in several other lipases, but only for Candida antarctica lipase B this ring system has been found to be acceptable, selective hydrolytic substrate. This reaction failed to test lipases from the following sources: 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 javanicus, Ammano lipase A, Rhizopus Delewar, Penicillium roqueforti, and Boehringer L1, L-3, L-4, L-4, L-4 , and L-8. The second Candida antarctica lipase, lipase A, is also unreactive.

Other hydrolytic enzymes, such as subtilisin Carlsberg and porcine liver esterase, hydrolyze the 7-unsubstituted benzodiazepine methyl ester, but have little stereoselectivity.

Conventional methods of separating esters from acids can be used to separate the carboxylic acid product from the unreacted ester. Typically, the reaction mixture is brought to a basic pH (e.g., > pH 7) with a high salt content, and the mixture is extracted with a suitable organic solvent. This procedure results in the unreacted ester being dissolved in an organic solvent and the carboxylic acid present in the aqueous layer. Drying and evaporation of the organic solvent yields the chiral ester, while acidifying the aqueous layer, extracting, drying the layer and evaporating the organic solvent yields the desired chiral acid. Other methods may be used to separate the acid from the ester, such as crystallization, silica gel or ion exchange chromatography, or other suitable resin, the use of which is within the scope of the invention.

Other related methods can be used to separate specific ester substrates from the carboxylic acid product obtained. For example, methyl- (R) -7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine Acetic acid is very soluble in water, making it difficult to extract from the aqueous layer even at basic pH. In this case, the acid and ester, as reaction products, may be treated with a reactive reagent that reacts preferentially with one substrate component to facilitate separation. For example, it has been found that processing of (S) -7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H- 1,4-benzodiazepine acetic acid and its corresponding (R) methyl ester isomers at pH around 7.0 with carbobenzyl oxy chloride, tert-butyloxycarbonyl chloride or the corresponding anhydrides results in preferential acylation of the piperidinyl ring ester. Once the piperidine ring is acylated, the ester preferably dissolves in the organic solvent and can be separated by extraction.

Preferably, the product prepared by the process of the invention is substantially enantiomerically pure. Typically, this purity is 80% (e.e.) preferably greater than 90%, even more preferably greater than 95%, and most preferably greater than 99%.

While the present invention is mainly formulated as a process for preparing the non-racemic 3-oxo-2,3,4,5-tetrahydro-1,4-benzodiazepine product of Formula (I), it should be appreciated that the invention may also be applied as a method for enhancing the stereochemical purity of the chiral compound of formula (I) in which a significant proportion of the (R) -enantiomers is present. For example, when a chemical synthesis or physical method such as chromatography yields a product of unsatisfactory enantiomeric purity, the present method can be used to increase the e.e. of this product.

In a specific embodiment, the present invention provides a process for preparing a compound of formula (III):

where

X 'is H, halogen, CO ₂ R ³ , OR ⁴ ;

R ² is C _ig alkyl group optionally substituted with Ar, Het, or C _ _cycloalkyl;

R ³ is C _is alkyl or benzyl group;

R ⁴ represents a group consisting of C alkyl, COR ³ and benzyl;

which includes:

(a) treating a compound of formula (IV):

where

R ¹ represents a radical comprising _{a C 2o} alkyl or C _32o alkenyl optionally substituted with Ar, • • · ·

NR _a or NR ₃ *, wherein R represents a C _1-4 alkyl group; and

R ² and X 'are as defined for formula (III);

with lipase B from Candida antarctica; and (b) separating the obtained carboxylic acid, (S) -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid from the corresponding (R) -ester .

According to one preferred embodiment, the invention includes a process for the preparation of (S) -7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H- 1,4-benzodiazepine-2-acetic acid, which comprises treatment of (R, S) -methyl-7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro- 4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetate with Candida Antarctica lipase B; and separating (S) -7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine- 2-acetic acid from (R) -methyl-7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1 4-benzodiazepine-2-acetate. Other variations of this preferred embodiment include those wherein the substrate is (R, S) -methyl-7 - [( ^11- tert-butoxycarbonyl- (4,4'-bipiperidin-1-yl) carbonyl] -2,3, 4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetate or (R, S) -methyl-7 - [(1'-benzyloxycarbonyl- (4,4'-bipiperidine) -1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetate, and wherein these variations include the step of removing tert-butoxycarbonyl or benzyl These protecting groups are usually removed by conventional methods such as acid treatment or catalytic hydrogenation.

According to another preferred embodiment, the invention comprises a process for the preparation of (S) -7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid, which comprises treating (R, S) -methyl 7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetate with Candida antarctica lipase B; and separating (S) -7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid from (R) -methyl-7-iodo -2,3,4,5t etahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetate.

According to yet another preferred embodiment, the invention comprises a process for the preparation of (S) -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid, which comprises treating (R, S) -methyl-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetate with Candida antarctica lipase B; and separating (S) -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid from (R) -methyl-2,3,4,5tet rahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetate.

In yet another aspect, the invention includes a process for preparing a non-racemic compound of formula (I) wherein X is -COR ⁵ , or a fibrinogen- or vitronectinantagonist side chain, the process comprising converting a compound of formula (I) wherein X is H, halogen, CO ₂ R ³ or OR ⁴ .

Compounds of formula (III) are particularly useful intermediates for the preparation of compounds of formula (I) wherein X is COR ^S or a fibrinogen- or vitronectinantagonist side chain. Preferred X 'groups are H, Br, I, CO ₂ CH ₃ , CO ₂ CH ₃ , CO ₂ -t-Bu and OH. Preferred R ¹ groups are C 1-6 alkyl and benzyl.

Typical suitable compounds prepared by the methods set forth in the methods of this application are: (S) -2, 3,4,5-tetrahydro-4-methyl-3-oxo-1 H -1,4-benzodiazepine-2-acetic acid; (S) -7-Iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1 H -1,4-benzodiazepine-2-acetic acid;

(S) -7-bromo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1 H -1,4-benzodiazepine-acetic acid; or (S) -7-tert-butoxycarbonyl-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid and their single esters. Typical esters may be C alkylphenyl or benzyl esters, and derivatives thereof, and may be prepared by conventional esterification reactions from the corresponding carboxylic acids.

In another aspect, the invention provides a process for the preparation of compounds of formula (I) wherein X is COR ^S , or a fibrinogen- or vitronectin-antagonist side chain, the process comprising preparing a compound of formula (III) by the above method and converting a compound of formula (III) to a compound of formula (I) wherein X is COR ^S or a fibrinogen- or vitronectinantagonist side chain. Conversion of intermediate compounds of the invention to compounds of formula (I) wherein X is COR ^S or a fibrinogen- or vitronectinantagonist chain can be accomplished by conventional reactions and methods such as those disclosed 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 (PCT / US95 / 08146), PCT / US97 / 18001 and WO 97/24336 which are incorporated herein by reference.

In a more specific embodiment, the invention includes a process for preparing a compound of formula (I) such as (S) -7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl -3-oxo-1H-1,4-benzodiazepine-2-acetic acid, which comprises the conversion of (S) -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-15

1,4-benzodiazepine-2-acetic acid; (S) -7-Iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid;

(S) -7-bromo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-acetic acid; or (S) -7-tert-butoxycarbonyl-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid to (S) -7 - [( 4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid.

An example of a method that can be used to convert the above intermediates to a compound of formula (I) is shown in Scheme I.

Scheme I

a) pyridin.IC1 or CBr ₄ / Ph ₃ P; (b) CO, Pd (OAc) ₄ , Ph ₃ P, and (4,4 ^1- bipiperidine or 1'-Boc-4,4'-bipiperidine; or

1'-Cbz-4,4'-bipiperidine).

However, they are also prerequisites for using several other conventional methods for application to individual chiral intermediates of formula (III) to prepare compounds of formula (I).

Common abbreviations and symbols used in the chemical arts are used herein to describe the compounds. In addition, certain terms have the following meanings:

The term fibrinogen- or vitronectin-antagonist side chain may generally have the meaning given by the general formula: W- (CR ') -Z- (CR'R ¹⁰ ) -U- (CR') -V- or W '- (CR') - U- (CR ') <3 X · 2 s 2 <3 2 s where

R 'is H, _C _ _g alkyl, C ₃ _ _{in cycloalkyl-C} _ ₄ alkyl, and _Ar-Q _ C ₄ alkyl;

R '' represents R ', -C (O) R' or -C (O) OR ';

R ¹ represents a group comprising H, C alkyl,

C ₃ _ _{7 cycloalkyl-C} _ ₄ alkyl and Ar-C _Q4 alkyl;

R ⁵ is H, C _alkyl,

Cycloalkyl-C alkyl and Ar-C alkyl

R ⁷ is H, halogen, -OR ¹² , -SR ¹² , -CN, -NR'R ¹² , -NO, -CF, CF S (O) -, -CO R ', -CONR', R ¹⁴ -Cosalkyl-, -Cigoxoalkyl- ^{R14, R14} -C2_salkenyl-, -C26alkinyl- ^{R14, R14} -Co_salkyloxy-, ^R14 -CQ_ _with amino and ^R14 _of -C _ _alkyl-s (O) _R ~;

R ⁸ is R ', C (O) R', CN, NO ₂ , SO-R 'or C (O) OR ^S ;

R ⁹ is R ', -CF, -SR', or -OR ';

R ¹⁰ is H, C _i4 alkyl and -NR'R '';

R ¹² is R ', - C (O) R', -C (O) NR ' ₂ , -C (O) OR ^S , -S (O) m R' or S (O) ₂ NR '₂;

• · · · · · · · · · ·

R ^{X 4} is H, C _ cycloalkyl, Het and

Ar;

R ^x is H, C alkyl, C ₃ _ _{7 cycloalkyl-C} _ _alkyl and _Ar-C _ _alkyl;

U and V are absent or are CO, CR ' ₂ , C (= CR ^XS 2) S (O), O, NR ^XS , CR ^XS ' OR ^XS , CR '(OR'')CR', CR 'CR' (OR 1 '), n 2 2

C (O) CR ' ₂ , CR ^1s 2C (O), CONR ^xs , NR ^{X 5} CO, OC (0), C (O) 0, C (S) O,

OC (S), C (S) NR ^XS , NR ^{X 5} C (S), SOsNR ^xs , NR ^xs SO 2, N = N, NR ^XS NR ^XS , γώ ¹⁵

NR CR ^XS , CR ^XS 0, OCR '2' 2 ¹⁵ C = C, CR ^XS = CR ^XS , Het, or Ar, provided that U and V are absent simultaneously;

W represents R'R''N-, R'R''NR'N-, R'R''NR'NCO-, R ' ₂ NR'NC (= NR') R'ONR'C (= NR ') ) -,

NR "

NR *

AND.

or ^{2 of} l | 11

Ά = ' ₂ n ^ n- R ^ nr · - ^nr ', 5 ³

Θ;

W 'is a_y

(O) w

R =

R '

V-Q

N

R ^a

Q is NR 1, O or S;

^And R is H, _C _ _{alkyl, Ar-C} _ _alkyl, Het-C alkyl, cycloalkyl or C -C alkyl, halogen, OR ³ ·,

O - β 3 - β O ^- o

SR ¹ , COR ¹ , OH, NO 2, N (R ¹ ) 2, CO (NR ¹ ) 2, CH 2 N (R ¹ ) 2;

and R ^c is independently selected from the group consisting of H, C alkyl, Ar-C _axis alkyl, _Het-C _ _alkyl, or C ₃ _ _{cycloalkyl-C axis} alkyl, halo, OR ^1, SR ^1, COR ¹ , OH, NO 2, N (R ¹ ) 2, CO (NR ¹ ) 2, CH 2 N (R ¹ ) 2, or, and R ^c combine to form a 5- or 6-membered aromatic or non-aromatic ring optionally substituted by a group selected from the group consisting of halogen, C _i _ ₄ alkyl, oR ^1, SR ^1, COR ^1, OH, N02, N (R ¹⁾ 2, CO (NR ^x) 2, CH 2 N (R ¹⁾ 2, CN, or R b R NC (= NR ') -;

X is N = CR ', C (O) or O;

Y has no meaning, or is S or O;

Z represents a group comprising _{(CH2) t,} Het, Ar and _C3 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;

··? 4 Ρ Ρ Ρ * * s s s s s s s s s s s s s znamená s s znamená znamená znamená znamená , 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 example, 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 (PCT / US95 / 08146) and PCT / US96 / 11108, all of which are fully incorporated herein by reference. It will be appreciated that the basic nitrogen of group W or W 'may optionally be protected when applying the process of the invention. Nitrogen protecting groups are well known in the art and include groups such as acetyl, formyl, trifluoroacetyl, benzoyl, benzyloxycarbonyl and alkoxycarbonyl.

Typical fibrinogen and vitronectin receptor side chains include:

t ·

(I) r-ch ₂ nr'CO

NR'COΗ

CHR'NR'CO

2- (CH 2) 3 N

The term C _i _ ₄ alkyl as used herein means a group comprising methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. The term C _i6 alkyl additionally includes pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. Similarly, the term C _I - _2o alkyl includes simple aliphatic hydrocarbons of the indicated number of carbon atoms. Each alkyl group may be optionally substituted with R ⁷ unless otherwise specified. The term C _o _ ₄ alkyl and C _oe alkyl additionally indicate that may not be any alkyl group (e.g., is only a covalent bond is present).

The term C ₂ _ _g alkenyl as used herein represents an alkyl group having 2-6 carbon atoms in a single bond is replaced by a carbon-carbon double carbon-carbon bond. Among the C ₂ _ _g alkenyl groups include ethylene, 1-propene, 2-propene, 1-butene, 2-butene, isobutene and the several isomeric pentenes and hexenes. Similarly, C ₃ _ _2Q alkenyl includes simple aliphatic hydrocarbons of the indicated number of carbon atoms in which one or more single carbon-carbon bonds is replaced by a carbon-carbon bond. The invention includes both cis and trans isomers. Each alkenyl group may be substituted with R ⁷ unless otherwise specified.

• · · ·

The term C ₂ _ _of alkynyl as used herein, represents an alkyl group having 2-6 carbon atoms in a single bond is replaced by a carbon-carbon triple carbon-carbon bond. Among the C ₂ _ _alkynyl groups include acetylene, 1-propyne, 2-propyne, 1-butyne, 2-butyne, 3-butyne and the simple isomers of pentyne and hexyne. Any ^sp3 carbon atom in the C ₂ _ _g alkynyl group may be substituted with a substituent R ^7th

The term ₄ oxoalkyl as used herein refers to an alkyl group of up to four carbon atoms wherein the CH ₂ group is replaced by a C (O) or carbonyl group. Typical examples of these groups include substituted formyl, acetyl, 1-propanal, 2-propanone, 3-propanal, 2-butanone, 3-butanone, and 1a 4-butanal. The term C _is oxoalkyl includes additionally the higher analogues and isomers of five and six carbons substituted by a carbonyl group.

The substituent on the group consisting of C alkyl, C alkenyl, C _{2 6} alkynyl and C _ie oxoalkyl moieties such as ^R7, may be on any carbon atoms of the substitution results in a stable structure and the method of preparation is available by conventional synthetic techniques.

Halogen means fluoride, chloride, bromide or iodide.

The term Ar, or aryl, as used herein, means a phenyl or naphthyl group substituted with one to three R ⁷ substituents. Particularly R ⁷ may represent a group consisting of C _i _ ₄ alkyl, C _i4 alkoxy, C _i4 alkylthio, trifluoroalkyl, OH, F, Cl, Br or I.

Het or heterocycle means an optionally substituted five- or six-membered monocyclic ring, or a nine- or ten-membered bicyclic ring containing one to three heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, which are stable and available by conventional chemical syntheses. Examples of these heterocycles include benzofuran, benzimidazole, benzopyran, benzothiophene, furan, imidazole, indole, indoline, morpholine, piperidine, piperazine, pyrrole, pyrrolidine, tetrahydropyridine, pyridine, thiazole, thiophene, quinoline, isoquinoline, and tetra- and perhydroquinoline and isoquinoline. Preferred heterocycles forming part of the Z group are six-membered ring heterocycles containing one or two nitrogen atoms, and include heterocycles such as piperidine, piperazine, tetrahydropyridine and pyridine. Within the scope of the invention, each available combination with up to three substituents on the Het ring, such as substituents selected from R ⁷ , which is available by chemical syntheses and which is stable.

The term C cycloalkyl means an optionally substituted carbocyclic system of three to seven carbon atoms, which may contain up to two unsaturated carbon-carbon bonds. Typical C ₃₇ cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl and cycloheptyl. Within the scope of the invention is any combination with up to three substituents such as selected from R ⁷ which is available by chemical syntheses and which is stable.

The symbol used herein means a nitrogen heterocycle which may be a stable saturated or unsaturated 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 selected from oxygen and sulfur atoms , which may be substituted on any atom to form a stable structure. The nitrogen atom in this ring may be substituted to form a quaternary nitrogen.

The nitrogen heterocycle may be substituted in any stable position by R ² ° for example selected from the group consisting of H, Ci_4alkoxy, F, Cl, Br, I, N02, NR'2, OH, CO2R, CONHR ', CF 3, R ¹⁴ -Co_ ₄ alkyl-S (O) _U (for example, is 0, 1 or 2), or C alkyl substituted with any of the foregoing substituents. Typical groups are pyrroline, pyrrolidine, imidazole, imidazoline, pyrazole, pyrazoline, pyrazolidine, piperidine, piperazine, morpholine, pyridine, pyridinium, tetrahydropyridine, tetrahydro and hexahydroazepine, quinuclidine, quinuclidinium, quinoline, isoquinoline, and tetra- and isoquinoline, and tetra- and perhydroquinoline, and tetra- and perhydroquinoline. Θ may in particular be pyridyl, pyrrolidinyl, piperidinyl, piperazinyl, azetidinyl, quinuclidinyl or tetrahydropyridinyl. preferably represents 4-pyridyl,

4- (2-Amino-pyridyl), 4-tetrahydropyridyl,

4-piperidinyl, or 4-piperazinyl.

When R ^t and R ^c combine to form a five or six membered aromatic or non-aromatic ring fused to the ring to which R ^t and R ^{c are} attached, the ring thus formed is generally a five or six membered heterocycle belonging to those listed for Het, or it is a phenyl, cyclohexyl or cyclopentyl ring. Preferred W 'groups include benzimidazolyl, 4-azabenzimidazolyl, 5-azabenzimidazolyl and substituted derivatives thereof.

Esters of formula (II) and (IV) used in the process of the invention are prepared by the methods disclosed 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 (PCT / US95 / 08146), WO 97/24336 and PCT / US96 / 11108, all of which are incorporated herein by reference.

··· ···· · · Λ · · · ♦ · · · «« «« · · · · · · · · · · · · ·

It will be apparent to those skilled in the art that the process of the invention may also be applied to the preparation of compounds of formula (V):

wherein R ¹ , R ² and X are as defined for formula (II), and that these compounds can be hydrolyzed by conventional means to prepare the non-racemic (R) isomer of the carboxylic acid of formula (II) wherein R ¹ is H.

The examples which follow are given to illustrate the preparation and use of the compounds of the invention and are not intended to limit the invention in any way. Many other embodiments will be apparent and available to those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

General method of hydrolysis by Candida antartica lipase B

Methyl 3-oxo-2,3,4,5-tetrahydro-1,4-benzodiazepine-2-acetate (0.5 g) or a suitably substituted derivative thereof is dissolved in acetone (30 ml) or tert-butanol and buffer (70 mL, pH 7.0, 0.1 M phosphate). A Candida antartica lipase on a macroporous acrylate carrier (200 mg / 700 PLU / g sold as Novozym 435) was added and the mixture was stirred at room temperature for 4.0 days. The progress of the reaction was monitored by HPLC and ended spontaneously at 47% hydrolysis. The pH was adjusted to 8.0 with NaOH solution and EtOAc (75 mL) was added. The mixture was

filtered to remove the enzyme catalyst and the EtOAc layer was separated. The aqueous layer was re-extracted with EtOAc (2 x 75 mL). The combined EtOAc extracts were dried (Na ₂ SO ₄ ) and the EtOAc evaporated in vacuo to give a residue containing (R) -ester (0.28 g). Stereochemistry was determined by comparison with the original (R) -ester (chiral HPLC); ee > 99%.

The aqueous phase was adjusted to pH 5 with concentrated HCl and extracted with EtOAc (5 x 75 mL). The combined EtOAc extracts were dried (Na ₂ SO ₄ ) and evaporated to give (S) acid (0.23 g). The ee value is 99%) (determined by chiral HPLC).

The following 3-oxo-2,3,4,5-tetrahydro-1,4-benzodiazepine-2-acetic acid was cleaved as described above.

X R ¹ e.e H CH (WITH) 99% (R) 99% Br CH 3 (WITH) 99.4% (R) 99.4% AND CH 3 (WITH) 98% (R) 95% C0 ₂ -terc .Bu CH 3 (WITH) 99.8% (R) 99.1% [1'-Cbz- (4,4'-bi- CH 3 (WITH) 98.3

-piperidin-1-yl)] carbonyl

Example 2

Preparation of (S) -7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2 (R, S) -methyl-7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1-acetic acid 4-Benzodiazepine-2-acetate (34 g, 0.077 mol) was slurried with water (580 mL) and 1.0 N HCl (34 mL). The mixture was stirred at 30 ° C for 0.5 hour while maintaining the pH at about 6.2 by the addition of 1.0N HCl. Immobilized on resin (Boehringer L-2 lipase, 8.2 g) was added and the reaction mixture was stirred at 30 ° C while maintaining the pH by adding 1.5 M NH ₃ solution. If it is no longer necessary to add the titrant, the enzyme on the resin is filtered off and washed with water.

The filtrate was treated with a solution of benzyl chloroformate (7.8 g, 0.046 mol) in CH ₂ C1 ₂ (130 mL). The mixture was stirred at 30 ° C for 1 hour while maintaining the pH at 7 by adding 1.5 molar NH 3 solution. The phases were separated and the aqueous phase was re-extracted with CH ₂ Cl ₂ (130 mL).

The aqueous phase is heated at 55 ° C under vacuum while maintaining the pH at 6.8. When the volume of the solution reaches ~ 150 ml, the solution is cooled to 5 ° C and left for 12 hours with occasional pH adjustment. The solid was filtered off, washed with cold water and dried to give the title compound (16 g, 80%); 99.9% of the (S) -isomers, determined by chiral HPLC.

Evaporation of the dichloromethane extracts gave the title compound, R-methyl-7- [1'-benzyloxycarbonyl- (4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3 -oxo-1H-1,4-benzodiazepine-2-acetic acid as a pale yellow crystalline solid (23 g, 100%); > 98% of the (R) -isomers as determined by HPLC.

Example 3

Preparation of methyl- (S) -7 - [[N'-benzyloxycarbonyl) -4,4'-bipiperidinyl] carbonyl] -3-oxo-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2 -acetic acids

a) methyl 7 - [[N '-benzyloxycarbonyl-4,4'-bipiridinyl] carbonyl] -3-oxo-2,3,4,5-tetrahydro-1,4-benzodiazepine-2-acetate

A mixture of methyl 7-iodo-3-oxo-2,3,4,5-tetrahydro-1,4-benzodiazepine-2-acetate (50 g, 134 mmol),

1'-benzyloxycarbonyl-4,4'-bipiperidine hydrochloride (52.12 g, 154 mmol), Hunings base (67.9 mL, 402 mmol), NMP (200 mL), water (5.2 mL, 289 mmol) ) and bistriphenylphosphine-palladium (II) chloride (1.88 g, 2.68 mmol) were shaken in a Paar shaker at 95 ° C and under a pressure of 8-14 psi of carbon monoxide. After 4 hours carbon monoxide uptake is stopped, the reaction mixture is allowed to cool to room temperature. Dichloromethane (400 mL) was then added, the solution was filtered and washed with water. The organic layer was concentrated to dryness and the residue slurried with methanol (770 mL). The product was filtered off, washed with methanol and dried by suction to give the title compound (54 g, 70%).

b) (S) -7- (4,4'-bipiperidinyl) carbonyl-3-oxo-2,3,4,5-tetrahydro-1 H -1,4-benzodiazepine-2-acetic acid

The compound of Example 3 (a) is dissolved in tert-butanol and in a buffer (70 ml, pH 7.0, 0.1N phosphate). Add Candida antarctica lipase B onto a macroporous acrylate resin (200 mg, / 700 PLU / g, sold as:).

Novozyme 435) and the reaction mixture was stirred at room temperature for 4.0 days. The pH was adjusted to 8.0 with NaOH solution and EtOAc (75 mL) was added. The mixture was filtered and the EtOAc layer was separated. The aqueous layer was re-extracted with EtOAc, the combined EtOAc extracts were dried (Na ₂ SO ₄ ) and evaporated in vacuo to give the (R) -ester.

The aqueous phase was adjusted to pH 5 with HCl and extracted with EtOAc. The combined EtOAc extracts were dried (Na ₂ SO ₄ ) and evaporated to give the (S) -acid (99% ee)

A solution of this (S) acid (0.15 mmol) in methanol (40 mL) in acetic acid (8 drops) was shaken for 30 minutes under a hydrogen atmosphere (45 psi) with 10% Pd / C (20 mg). Filter the mixture through CELITE ^R and concentrate the filtrate in vacuo to give the title compound.

Example 4

Preparation of (S) -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid (R, S) -methyl-2,3,4,5 - Tetrahydro-4-methyl-3-oxo-1,4-benzodiazepino-2-acetic acid (20 g, 0.081 mol) was slurried with toluene (200 mL) and heated at reflux for 5 min. The solution was then cooled to 40 ° C and water (200 mL) and Novozym 435 (20 g) were added. The reaction mixture was stirred at 40 ° C and the pH was maintained at 7.0 with ammonia solution (1.5 mol). After 16 hours, HPLC analysis indicated complete reaction. The enzyme on the resin was filtered off and washed with hot water (45 mL) and hot toluene (75 mL).

The toluene layer was separated and the aqueous layer was extracted with ethyl acetate (2 x 100 mL) while maintaining the pH at 8.0 with ammonia solution (1.5M). Combined organic extracts

(toluene and ethyl acetate) was evaporated to dryness and recrystallization of the residue from dichloromethane-hexane gave the (R) -isomer of the starting ester (8 g, 80%).

The aqueous layer was partitioned with ethyl acetate, acidified to pH 4.0 with hydrochloric acid, evaporated to dryness and recrystallized to give the title compound.

Example 5

Preparation of (S) -7-Iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid

The procedure of Example 3 was repeated until the (R) -ester was extracted from the aqueous phase containing (S) -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2. -acetic acid. The pH of the aqueous solution was adjusted to 7.0 and pyridine-IC1 complex (10.2 g, 0.042 mol) was added. The mixture was stirred for one hour while maintaining the pH at 6.0 (1.5M) with ammonia solution. The pH was adjusted to 4.0 with HCl (concentrated) and the mixture was stirred for 16 hours. The product was filtered, washed with cold pH 4 buffer and dried to give the title compound (11.2 g, 84%). Chiral HPLC analysis revealed 99.82% of the (S) -isomers.

Example 6

Preparation of stabilized enzyme on a support

Novozym 435 (Novo Nordisk, 3g) was stirred at room temperature in a mixture of water (4ml), tert-butanol (45ml) and glutaraldehyde (1ml, 50% w / w in water) at room temperature for 3- 4 hours without pH adjustment. The resin is then filtered off, washed with water and dried.

Claims (14)

Ε’ΑΤΕΓΤΤΟΛΖΕ ISTÁROKY A process for the preparation of a compound of formula (I) (D wherein X is H, halogen, CO ₂ R ³ , OR ⁴ , COR ^S , or fibrinogen or vitronectin antagonist side chain; R ² is C _1-6 alkyl, optionally substituted with Ar, Het or C _6-6 cycloalkyl; R ³ is C alkyl or benzyl group; R ⁴ represents a group consisting of _Cis alkyl, COR ^3, and benzyl; ^R represents a group consisting of 4,4'-bipiperidine-l-yl, (1'-benzyloxycarbonyl) -4,4 bipiperidin-l-yl, and (1 'tertbutoxycarbonyl) -4,4'-bipiperidine -1-yl; characterized by comprising: (a) treating a compound of formula (II): Where R ¹ represents a radical comprising _{a C} _ _2o alkyl or C ₃ _ _2o alkenyl, optionally substituted by Ar, NR ₂ or NR ₃ *, wherein R represents a C _1-4 alkyl group; Candida antarctica lipase B; and (b) separating the obtained carboxylic acid, 2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid from the corresponding ester. 2. The method of claim 1, wherein the enzyme, Candida antarctica lipase B, is fixed to a solid support. 3. The method of claim 2, wherein the enzyme, Candida antarctica lipase B, is stabilized by treatment with a crosslinker. 4. The process of claim 1 wherein the (S) isomer is separated from the (R) isomer by extraction of the aqueous solution with an immiscible organic solvent. 5. The method of claim lvyznačující in that R ¹ represents a methyl group. • · · · · · · · · · · · · · · · · · · · · 6. The method of claim Ivyznačující in that R ² represents a methyl group. 7. The process of claim 1 wherein the reaction temperature is above room temperature. A process for preparing (S) -7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4 benzodiazepine-2-acetic acids, characterized in that they comprise: a) treatment of (R, S) -methyl-7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1 4-benzodiazepine-2-acetate with Candida antarctica lipase B; and b) separating (S) -7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine -2-acetic acid from (R) -methyl-7 - [(4,4'-bipiperidin-1-yl) carbonyl] -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H- 1,4-benzodiazepine-2-acetic acid. A process for the preparation of (S) -7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid, comprising: a) treatment of (R, S) -methyl-7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-ΙΗ-1,4-benzodiazepine-2-acetate with Candida antarctica lipase B ; and b) separating (S) -7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid from (R) -methyl-7- iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetate. • · · · · · · · · · A process for the preparation of (S) -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid, comprising: (a) treating (R, S) -methyl-2,3,4,5-tetrahydro-4-methyl-3-iso-ΙΗ-1,4-benzodiazepine-2-acetic acid with Candida antarctica lipase B; and b) separating (S) -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid from (R) -methyl-2,3,4, 5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid. 11. A compound which is: (S) -2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid, (S) -7-iodo-2,3,4,5 -tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid, (S) -7-bromo-2,3,4,5-tetrahydro-4-methyl-3-oxo- 1H-1,4-benzodiazepine-2-acetic acid or (S) -7-tert-butoxycarbonyl-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2 -acetic acid. A process for the preparation of a compound of formula (I) according to claim 1, wherein X is -COR ⁵ or a fibrinogen- or vitronectin-antagonist side chain, comprising converting a compound of claim 11 into said compound of formula (I). A process for preparing a compound of formula (I) wherein X is COR ^S , or a fibrinogen- or vitronectinantagonist side chain; R ² is C _is alkyl, optionally substituted by Ar, Het or 0 _χ _ ₆ cycloalkyl; R ³ represents a _{C t} _alkylovou or benzyl; R ⁴ represents a group comprising _{a C} _ _alkyl, COR ^3, and benzyl; ^R represents a group consisting of 4.4 ¹ bipiperidin-l-yl, (1'-benzyloxycarbonyl) -4,4'-bipiperidin-l-yl, and (1'-tert-butoxycarbonyl) -4,4'-bipiperidine -1-yl; characterized by comprising: (a) preparing a compound of formula (III): (DD or OR ⁴ where X 'is H, halogen, CO ₂ R ³ R ² is C _1-6 alkyl optionally substituted with Ar, Het or C _1-6 cycloalkyl; R ³ represents C _x _ _s alkyl or benzyl group; R ⁴ represents a group consisting of C alkyl, COR ³ or benzyl; by treating the compound of formula (IV): where R ¹ represents a radical comprising alkyl or C _{I2O 32o} C alkenyl optionally substituted with Ar, NR _2, or -NR *, where R is C _i4 alkyl group; with lipase B from Candida antarctica; and (b) separating the obtained carboxylic acid, (S) acid. 2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid from the corresponding (R) -ester of formula (III); and (c) converting a compound of formula (III) to a compound of formula (I) wherein X is COR ⁵ or a fibrinogen- or vitronectin-antagonist side chain. 14. The method according to claim 14 characterized in that R ¹ is C _i4 alkyl or benzyl group; and X 'is H, Br, I, CO CH, CO-t-Bu or OH.