NZ270913A

NZ270913A - Phenylalanyl-prolylaminobutyl boronic acid derivatives and anti-thrombotic compositions

Info

Publication number: NZ270913A
Application number: NZ270913A
Authority: NZ
Inventors: Sergio Mallart; Gilbert Lassalle; Thomas Andrew Purcell; Jean Claude Muller
Original assignee: Synthelabo
Priority date: 1994-04-12
Filing date: 1995-04-11
Publication date: 1996-05-28
Also published as: EP0677531A1; FI951726A0; CA2146833A1; IL113332A0; PL308074A1; JPH07285990A; CN1113493A; FR2718451A1; KR950032225A; FR2718451B1; AU1638095A; HU9501041D0; NO951418D0; NO951418L; SK47695A3; ZA952983B; HUT71617A; CZ92295A3; FI951726A; RU95105419A

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £70913 27091 Priority Date(s): Complete Specification Filed: Class: I Publication Date: .2L.&HA3LI99S. ' P.O. Journal No: No.: Date: NEW ZEALAND PATENTS ACT, 1953 COMPLETE SPECIFICATION BORONIC PEPTIDE DERIVATIVES, PREPARATION AND APPLICATION THEREOF We, SYNTHELABO, a French body corporate, of 22 Avenue Galilee, 92350 Le Plessis-Robinson, Cedex, France hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: - -1 - (followed by page la) 2709H - lcx- The subject of the present invention is boronic peptide 5 derivatives# the preparation and the therapeutic application thereof. The present invention provides a compound of formula (I) 0 10 (I) 15 c in which R represents a hydrogen atom, a straight or branched (C1-C4)alkyl group, a straight or branched -CO (Cr-C4) alkyl group 20 or a straight or branched -C02 (C,-) alkyl group, R, represents a phenyl group or a cyclohexyl group, Rj represents a group or a group \—r/ where S, is a hydrogen atom or a (C^-C^) alkyl group «5 X and Rj and R4 either each represent a hydrogen atom or together- 27091 - 2 - represent the residue of a dihydroxy compound or an addition salt thereof with a pharmaceutically acceptable acid or base. Preferred dihydroxy residues are of 2,3-butanediol, 2,3-dixnathyl-2,3-butanediol or (la,3a,5a) -2,6,6-5 trimethylbicyclo-[3.1.1]heptane-2,3-diol [(+)-o-pinanediol]. Depending on the definition of the substituents £3 and R4, the compounds of the invention preferably possess 3 or 7 asymmetric centres. The compounds of the invention may exist in the form 10 of optical or geometrical isomers, which are pure or in the form of mixtures which also form part of the invention. According to the invention, the preferred compounds having 3 asymmetric centres are the D-alanyl-L-prolinamide derivatives of [2 (R) ] configuration and the preferred compounds 15 having 7 asymmetric centres are the D-alanyl-N- [ (4,6 -methano-l,3,2-benzodioxaborol-2-yl)methyl] -L-prolinamide derivatives of [3aS,[2 (R) , 3aa,46,6fi,7aa]] configuration. The compounds of the invention may exist in the form of free bases or in the form of addition salts with 20 pharmaceutically acceptable acids and bases, which also form part of the invention. Preferred are salts of hydrogen chloride. Among the compounds of the invention, the preferred compounds are those for which Rj represents a group *5 H*. 25 where Rg is a hydrogen atom or a (C,-^)alkyl group. 27D91 3 Among these, the preferred compounds are those in which R represents a hydrogen atom or a straight or branched (cn—CA) alkyl group, 5 R1 represents a phenyl group and Rj and R4 each represent a hydrogen atom. Finally# the most advantageous compound is (R) - [1-[ (D-phenylalanyl-L-prolyl) amino] -4- (lH-imidazol-4-yl) butyl] boronic acid or an addition salt thereof with a 10 pharmaceutically acceptable acid or base. as defined above, Rj and R4 together represent a residue of a 15 dihydroxy compound as defined above, R6 represents either a hydrogen atom when Rj represents a straight or branched -CO (Cj-C^) alkyl group, or a straight or branched -COz (C1 -C4) alkyl group when R7 represents a hydrogen atom or a straight or branched (C,-C4) alkyl group) is reacted with a compound of 20 formula (III) (in which Rj is a hydrogen atom or a (C,-C4) alkyl group) , in a solvent such as dioxane, for example, to give a compound of formula (la) . Then, if it is desired to obtain a compound of formula (lb) (in which R, R, and a group _ Vl__/ may be synthesized according to Scheme 1. "5 A boronic tripeptide of formula (II) (in which R, is Scheme 1 <^*vO i OB^ C 00 <00 H £J C o 270913 OH r \ <*> = OH c 5 91. Sj are as defined above), the compound of formula (la) may be reacted with, hydrochloric acid or with boron trichloride. The compounds of the invention in which Rg represents 5 group may be synthesized according to Scheme 2. [3aS- (3aa,4£, 6ft,7aa] -2- (3-Bromopropyl) -3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborole of formula (IV) is reacted with sodium iodide in a solvent such as acetone, for example, to give [3aS- (3aa,4S,6&,7aa) ] -2- (3-iodopropyl) -3a,5,5-10 trimethylhexahydro-4, 6-methano-l, 3,2-benzodioxaborole of formula (V) which is coupled with a compound of formula (VT) (in which Kg is as defined above) to give the compound of formula (VII); the reaction is performed in a solvent such as tetrahydrofuran, at a temperature between -78 and +20°C. Then, 15 according to a process similar to that described by Mattheson in Organometallies, (1984), £., 614, the compound of formula (VII) is reacted with dichloromethyllithium, in the presence of zinc chloride, in a solvent such as tetrahydrofuran, at a temperature between -100 and +20°C, to give the compound of 20 formula (VIII) which is reacted with lithium bis (trimethylsilyl)amide; the reaction is ic^NH a group Scheme 2 2709 ,ft*n V* Jtal (no W (CH3J3P (cHaVl ££ avKCHaJa (VD (CKJJjHOJS <ca,M <C"3^ (vn) 1) UMtSKCHg^lj <) Ha 270 7 Scheme 2 (continued) Sji^. «' [ Nrl <o° -/ (X) (XD (xn) (xm) - 8 - Scheme 2 (continued) *91$ performed in a solvent such as tetrahydrofuran, at a temperature between -78 and +20°C. A compound is obtained which 20 is treated with hydrochloric acid in a solvent such as dioxane and the hydrochloride of formula (XX) is obtained, which is hydrolysed to give (R) -a-aminobutaneboronic acid dihydrochloride of formula (X), which is reacted with a diol of formula (XX) (in which Rj and R4 together represent the residue 25 of a. dihydroxy compound as defined above) to give the hydrochloride of formula (XII); then, the compound of formula (XIX) is coupled with an activated form of a dipeptide of formula (XIII) (in which R1, as defined above, R6 represents either a hydrogen atom when R7 represents a straight or - 9 - 2709! 3 branched -CO (C^-C^) alkyl group, or a straight or branched -C02(CrC4) alkyl group when Ry represents a hydrogen atom or a straight or branched (C.,-C4) alkyl group and X represents either 5 the pyrrolidin-l-yl-2,5-dione group or the 2- methylpropyloxycarbonyl group) to give a compound which is treated with hydrochloric acid, and the compound of formula (Ic) is obtained. Then, if it is desired to obtain a compound of formula (Id) (in which R, R1 and 1^ are as defined above) , 10 the compound of formula (Ic) may be reacted with hydrochloric acid or with boron trichloride. The starting compounds are commercially available or are described in the literature or may be prepared according to methods which are described therein or which are known to those 15 skilled in the art. Thus, tripep tides of formula (II) are described in European Patent Application Publication No. 293,881. N-Acetyl-D-phenylalanyl-L-proline, [3aS- [2 (R) , 3ao, 4&,6£,7aa]] -N-acetyl-D-phenylalanyl-N- [4-bromo-l- (3a,5,5-20 trlmethylhexahydro-4,6-methano-l,3,2-benzodioxaborol-2-yl) butyl] -L-prolinamide and 1- (N- [(1,1-dimethylethoxy] -D-phenylalanyl-L-proline are described by Kettner et al. In J. Biol. Chem. (1990), 265. 18289. [3aS-(3aa,4£,6S,7aa)]-2-(3-Bromopropyl)-3a,5,5-25 trimethylhexahydro-4,6-methano-l, 3,2-benzodioxaborole la described In European Patent Publication No. 293,881. 2- t(l# 1-Dimethylethyl)dimethylsilyl] -N, N-dimethyl-1H-imidazole-l-aulphonamide is described by Ngochindo in J. Chem. Soc. Perkin Trans., (1990), 1, 1645. 270913 - 10 - The preparation of the compounds of formula (XXI) is described in European Patent Application No. 0677525. The preparation, of the compounds of formula (XXXX) (in which R6 represents a straight or branched -C02 (C,-C4) alkyl 5 group and Rj. represents a straight or branched (C,-CA) alkyl group) is performed using the corresponding compounds of formula (XIII) (in which Ry represents a hydrogen atom) according to a method similar to that described by Bajusz in J. Med. Chem., (1990), 33. # 1729-1735. 10 The present invention also provides a process for the production of a compound of formula (I) in which Rj represents a group as hereinbefore defined, which process comprises reacting a boronic tripeptide of formula (II) as hereinbefore defined with a compound of formula (III) as 15 hereinbefore defined to give a compound of formula (la) as hereinbefore defined followed, if desired, by reacting the compound of formula (la) with hydrochloric acid or with boron trichloride to obtain a compound of formula (lb) as hereinbefore defined and optionally converting the resulting 20 compound into the free base or an addition salt thereof. The present invention also provides a process for the production of a compound of formula (X) in which Rg represents a group as • hereinbefore defined, which process X 270 Q w - 11 - comprises coupling the free base of a compound of formula (XIX) or an addition salt thereof as hereinbefore defined with an activated form of a dipeptide of formula (XXII) as hereinbefore defined, to give the free base of a compound of formula Ic or 5 an addition salt thereof as hereinbefore defined and, where appropriate, optionally converting the compound of formula (Xc) into the free base or an addition salt thereof and/or optionally reacting with hydrochloric acid or boron trichloride to give a compound of formula (Id) as hereinbefore defined and, 10 if desired, converting the resulting compound into a free base or other addition salt thereof. The Examples which follow illustrate the preparation of certain compounds in accordance with the invention. The elemental microanalyses and the IR and NMR 15 spectra confirm the structure of the compounds obtained. The numbers of the example compounds refer to those in the table given later. The ratio in parentheses is the acid/base molar r ratio. 20 gifwmpl i i (compound 1) [3aS- [2 (R) , 3aot,4£,6S,7aa]] -N-Acetyl-D-phenylalanyl-N- [4- (1H-imidarol-l-yl) -1- (3a,5,5-trimethylhexahydro-4, € -me thano-1,3,2 -benzodioxaborol-2-yl) butyl] -L-prolinamide hydrochloride (1:1) 25 1.1. [3aS-[2(R), 3aar,4fi,6S,7aa]] -N-acetyl-D-phenylalanyl-N- [4-(lH-imidazol-l-yl) -1- (3a, 5, 5-trimethyl-hexahydro-4,6-methano-1,3,2 -benzodioxaborol-2-yl)butyl] -L-prolinamide 3g (4.8 mmol) of [3aS- [2 (R), 3aa, 4&, 6£, 7aa) ] -N-acetyl-D-phenylalanyl-H- [4-bromo-l- (3a, 5,5-trimethylhexahydro- 270 12 4,6 - me thano-1, 3 , 2 -benxodioxaborol-2 -yl) butyl] -L-prolinamide and 0.902 g (9.6 mmol) of 1H-Imidazole are dissolved in 10 ml of dioxane. The solution is heated at 80 *C for 6 hours and the solvent is then evaporated off under vacuum. The residue is 5 taken up in 50 il of dichloromethane and the organic phase Is then washed successively with aqueous 5 % sodium hydrogen carbonate solution and with saturated sodium chloride solution. The solution is dried over sodium sulphate, filtered and evaporated. The product is purified by chromatography on a 10 column of silica gel, eluting with a methanol/dichloromethane mixture (5/95). Yield - 41 % [a]£° « -77.5° (c ■ 1.5; chloroform) 15 1.2. [3aS-[2(R)( 3aa,4£, 6£, 7aa] ]-N-Acetyl-D-phenylalanyl-N-[4- 20 acetyl-D-phenylalanyl-IT- [4- (lH-ixnidazol-l-yl) -1- (3a, 5,5-t r ime thyl-hexahydro - 4,6-me thano-1,3,2 -benzodioxaborol-2 -yl) butyl]-L-prolinamide are dissolved in 3 ml of chloroform and the solution is cooled to 0°C. 20 ml of a 0.1 N hydrochloric acid solution in isoprqpanol are -added, and the mixture is 25 stirred for 15 minutes at O'C and concentrated under vacuum. The residue is dried over phosphorus pentoxide and triturated with ether. 1.2 g of product are obtained in the form of an oil (lH-imidasol-l-yl) -1- (3a, 5, 5-trimethylhexahydro-4,6-methano-1,3,2 -benzodioxaborol-2 -yl) butyl] -L-prolinamide hydrochloride (111) 1.2 g (1.97 amol) of [3aS-[2(K), 3aa,4fi,6&,7ao] J -N- 1 g of product is obtained in the form of an amorphous solid. - 13 - Melting point ■ 99WC Yield ■ 78 % If 270 [a]n° » -90.4° (c ■ 1.1; chloroform) utifurpi ^ 2 (compound 2) 5 (R) -N-acetyl-E-phenylalanyl-N- [l-borono-4- (lH-imidazol-1-yl) butyl]-L-prolinamide hydrochloride (1:1) 700 mg (1.2 mmol) of [3aS-[2(R), 3aa,4&, 6S, 7aa] ] -N-acetyl-D-phenylalanyl-N- [4-lH-±midazol-l-yl) -1- (3a, 5,5-trime thylhexahydro-4,6 -me thano-1,3,2 -benzodioxaborol-2-10 yl) butyl]-L-prolinamide hydrochloride are dissolved in 6 ml of anhydrous dichloromethane. The solution is cooled to -78°C and 5 ml (5 mmol) of a 1 M solution of boron trichloride in dichloromethane are added dropwise over 15 minutes. The mixture is stirred for 15 minutes at -7B°C and for 45 minutes at 0°C, 15 followed by slow addition of 15 ml of water. The solution is stirred for 30 minutes at 0°C, followed by addition of 10 % acetic acid solution. The phases are separated, and the aqueous phase is extracted with 3 times 10 ml of ether and evaporated to dryness. The residue is purified by chromatography on a 20 Biogel P2® column, eluting with 10 % acetic acid. The fractions containing the product are concentrated and the residue is triturated in ether. A product is obtained in the form of a white powder, which is diluted with 2 ml of water. A solution is obtained, which is purified by chromatography on a Biorad AG 25 1W8® column (OH* form), eluting with water and with IN hydrochloric acid solution. The fractions containing the product are concentrated and the residue is triturated in ether. 50 mg of product are obtained. - 14 - Melting point « 168-175°C (d) [a]^° ■ -118.5* (c - 0.6; water) 270 Yield - 10 % i Example 3 (compound 3) 5 [3aS- [2 (R) , 3aa,4S, €&, 7aa] ] -N-Acetyl-D-phenylalanyl-K- [4- (1H-imidazol-4 (5) -yl) -1- (3a, 5,5-trimethylhexahydro-4,6-methano-l,3,2-benzodioxaborol-2-yl)butyl] -L-prolinamide hydrochloride (1:1) 3.1. [3aS- [2 (R) , 3aa,4&, 6fi,7aa] ] -a- (3a,5,5-trimethylhexahydro-10 4,6-methano-l,3,2-benzodioxaborol-2-yl) -1H-Imidazole-4(5) - butanamine hydrochloride (2:1) 3.1.1. [3aS- (3aac,4£,6S,7aa) ] -2- (3-iodopropyl) -3a,5,5- trimethylhexahydro-4,6-methano-l, 3,2-benzodioxaborole A solution of 37 g (122 mmol) of [3aS-(3aa, 4fi, 6&, 15 7aa) ] -2- (3 -bromopropyl) -3a,5,5-trimethylhexahydro-4, 6-m©thano-1,3,2-benzodioxaborole and 72.7 g (488 mmol) of sodium iodide in 500 ml of acetone is maintained at reflux temperature for 24 hours. The solvent is evaporated off and the residue is taken up in a mixture of 500 ml of ether and 100 ml of water 20 containing 1 g of sodium sulphite. The organic phase is dried over magnesium sulphate, filtered and evaporated. 40 g of product are obtained, which are used directly in the following step. Yield - 95 % 25 3.1.2. [3aS- (3aa,4&,6S,7aor)] -2- [(1,1- dimethylethyl) dimethylsilyl] -N,N-dimethyl-4 (5) - [3-(3a, 5,5-trimethylhexahydro-4,6-methano-l, 3,2-benz odioxaborol-2-yl)propyl]-1H-imidazole-1-sulphonamide • • • 270§|5 70.5 g (244 mmol) of 2-1(1,1-dimethylethyl) dimathylsilyl] -N,N-dim«thyl-lH-imidazole-1-sulphonamide are dissolved in 250 ml of tetrahydrofuran. The reaction medium is cooled to -78*C and 152 ml (244 mmol) of a 5 1.6 H solution of n-butyllithium in hexane are added. The mixture is left stirring for 1 hour at -78°C, followed by addition of 40 g (115 mmol) of [3aS- (3aat,4£, 6£,7aa) ] -2-(3-iodopropyl) -3a,5,5-trimethylhexahydro-4,6-methano-l,3,2-benzodioxaborole dissolved in 100 ml of tetrahydrofuran. The 10 reaction "T" is stirred between -78°C and +20°C over 1 hour and then at 20°C for 2 hours. The reaction medium is poured onto 350 ml of an ice-water mixture containing 14.5 g (121 mmol) of sodium hydrogen sulphate. The aqueous phase is extracted with 3 times 100 ml of ether and the ether phases are 15 combined, dried over magnesium sulphate, filtered and evaporated. The residue is purified by chromatography on a column of silica gel eluting with a 20 % ethyl acetate solution in hexane. 45 g of product are obtained. 20 yield - 73 % [«]J° - +12.5° (c ■ 1.9; chloroform) 3.1.3. [3aS- [2(S) , 3aa,4£,6£,7aa]l-4(5) - [4-chloro-4-(3a,5,5-tri methylhexehydro-4 , 6 -me thano-1,3,2 -bengodioxaborol-2-yl)butyl] -2- [1,1-dimethylethyl)dimethylsilyl] -N,N-25 dimethyl - 1H- imidazole-1-sulphonewl de X solution of 8.3 g (98 mmol) of dichloromethane in 100 ml of tetrahydrofuran is cooled to -100*C. 39 .1 ml (98 mmol) of a 2.5 H solution of n-butyllithium in hexane are added. The mixture is left for 15 minutes at this temperature, 0;O * ^ -IS - followed by addition of 45 g (89 mmol) of [3aS-(3aa,4fi, 6£, 7aa) ] -2- [ (1,1-dimethylethyl)dimathylsilyl] -N,N-dimethyl-4 (5) -[3- (3 a, 5,5-trimethylhexahydro-4,6-methano-l,3,2-benzodloxaborol - 2 -yl) propyl] -1H-imidazole-1 - sulphonamide 5 dissolved In 50 ml of tetrahydrofuran. The reaction mixture is left for 15 minutes at -100°C, and 9.8 g (70 mmol) of zinc chloride solution In 50 ml of tetrahydrofuran are added. The mixture is allowed to warm to +20°C over 16 hours. It is evaporated under vacuum and the residue is taken up in a 10 mixture of 200 ml of dichloromethane and 50 ml of water. The phases are separated and the aqueous phase is extracted with 100 ml of dichloromethane. The organic phases are combined, dried over magnesium sulphate, filtered and evaporated. The coloured residue obtained is purified by chromatography on a 15 column of silica gel, eluting with an ethyl acetate/hexane mixture (20/80). 40 g of product are obtained in the form of a colourless oil. Yield - 80 % 20 [a]^° m +15.9° (c ■ 2.65; chloroform) 3.1.4. [3aS- [2 (R) , 3aa, 4ft, 6£, 7acr]] -4(5) - [4-amino-4-(3a,5,5-trlmethylhexahydro-4,6-methano-l, 3,2 -benzodioxaborol-2 -yl) butyl] -N, N-dime thyl - 1H- imidazole -1 - sulphonamide hydrochloride (ltl) ■ . 25 12.6 g (78 mmol) of 1,1,1,3,3,3-hexamethyl-disilazane are dissolved in 80 ml of tetrahydrofuran and 31 ml (78 mmol) of 2 .5 N solution of n-butyllithium in hexane are added. The mixture is left for 1 hour at -78°C and 40 g (71 mmol) of [3aS-[2 (S), 3aa,4S,6S,7aa] ] -4(5) - [4-chloro-4- (3a,5,5-trimethylhexa- J 0 f 1 - 17 - hydro-4,6-methano-l,3,2-benzodioxaborol-2-yl)butyl] -2- [ (1,1-dimethylethyl) dimethylsilyl] -N, N-dimethyl-lH-imidazole-1-sulphonamide, dissolved in 80 nl of tetrahydrofuran, are added. The mixture is left stirring for 1 hour at -78°C and for 5 16 hours at +20°C. The reaction medium is cooled to -78"C, 78 ml (312 mmol) of a 4 N solution of hydrochloric acid in dioxane are added and the mixture is left stirring for 1 hour at -78°C and for 2 hours at +20°C. The mixture is evaporated under vacuum and the residue is taken up in 200 ml of 10 chloroform. This solution is filtered and evaporated. 32 g of product are obtained in the form of an oil, which is triturated in ether. The product is obtained in the form of a solid. Yield - 89 % 15 Melting point « 90-92°C [a]£° - +11° (c ■ 1; methanol) 3.1.5. [3aS-[2(R), 3aa, 4ft, 6ft, 7aa] ] -a-(3a,5,5- trimethylhexahydro-4,6-methano-l, 3,2-henzo-dioxaborol-2-yl)-lH-imidazole-4(5) -butanamine 20 hydrochloride (2:1) 32 g (70 mmol) of [3aS-[2(R), 3aa,4fi, 6ft, 7 aa] ] -4 (5) -[4-amino-4- (3a, 5,5-trimethylhexahydro-4,6-methano-l, 3,2-benzodioxaborol - 2 -yl) butyl] -N, N-dimethyl - IB- imidazole -1 -sulphonamide hydrochloride dissolved in 200 ml of 4 K 25 hydrochloric acid are maintained at reflux for 3 hours. The solution is extracted with 4 times 100 ml of ether and evaporated to dryness . The residue is taken up in 100 ml of methanol, and 11.9 g (70 mmol) of [1R-(la, 2a, 3a, 5a)]-2,6,6-trimethylbi-cyclo-[3.1.1]heptane-2,3-diol are added. The 270Q1J - 18 - mixture is left stirring for 16 hours at 20°C and is evaporated to dryness. 27 g of product are obtained in the form of an oil, which is triturated in ether. 5 The product is obtained in the form of a solid. Melting point » 75-80°C 3.2. Ester of N-acetyl-D-phenylalanyl-L-proline with l-hydroxypyrrolidine-2, 5-dione 6 g (20 mmol) of N-acetyl-D-phenylalanyl-L-proline 10 are suspended in a mixture of 100 ml of ethyl acetate asid 5 ml of dimethylformamide. 2.53 g (22 mmol) of 1 -hydr oxypyr r o 1 idine-2,5-dione are added and the mixture is cooled to 0°C. 4.53 g (22 mmol) of 1,3-dicyclohexylcarbodiimide in solid form are then added portionwise. The mixture is stirred for 20 hours at 15 20°C and the suspension is filtered. The filtrate is washed successively with 20 ml of 5 % sodium hydrogen carbonate solution and then with saturated sodium chloride solution and is dried over magnesium sulphate. The residue obtained is triturated in ether. 20 8 g of vitreous product are obtained, which product is used as it is in the following step. 3.3 [3aS- [2 (R) , 3aa,4S, 6S,7aa] ] -N-acetyl-D-phenylalanyl-N- [4-(lH-imidazol-4 (5) -yl) -1- (3a,5,5-trimethylhexahydro-4,6-methano-l,3,2-benzodioxa-boxol-2-yl)butyl] -L-prolinamide 25 hydrochloride (lxl) 2.4 g (6.4 mmol) of [3aS-[2(R), 3aa,4£,6S,7aa]]-a-(3a, 5,5-trimethylhexahydro-4, 6-methano-l, 3,2-benzodioxaborol-2 -yl) -lH-imidazole-4 (5) -butanamine hydrochloride are dissolved in 20 ml of dichloro-methane, 2.5 g (6.4 mmol) o£ the ester of - 19 - ^ N-acetyl-D-phenylalanyl-L-proline with 1 - hydr oxypyrrol i dine -2,5-dione are added and the mixture is cooled to -30°C. 3.6 ml (25.6 mmol) o£ triethylamine are added dropwise and the mixture is stirred for 2 hours between -30°C and +20°C and then for 2 5 hours at +20°C. 20 ml of aqueous 5 % sodium hydrogen carbonate solution are then added, followed by extraction of the aqueous phase with twice 20 ml of dichloromethane. The organic phases are combined, dried over sodium sulphate, filtered and evaporated. 10 The residue is taken up in 10 ml of isopro- panol and is treated, at 0°C, with 64 ml of a 0.1 N solution of hydrochloric acid in isopropanol. After evaporation, the residue is decolorized with animal black in ethyl acetate and is purified on a Sephadex® LH-20 column, eluting with methanol. 15 The solvent is evaporated off and the residue is triturated in ether. 2 g of product are obtained in the form of a colourless solid. Melting point ■ 130-135°C Yield - 51 % 20 [a]jj° - -112.1° (c ■ 1; chloroform) Example 4 (compound 4) (R) -N-acetyl-D-phenylalanyl-lt- [l-borono-4- (lH-imidazol-4 (5) -yl)butyl]-L-prolinamide hydrochloride (1:1) 25 2 g (3 mmol) of [3aS- [2(R), 3aa, 4&,6&, 7aa] ]-N-acetyl- D-phenylalanyl-N-[4-(lH-imidasol-4 (5)-yl)-1-(3a, 5, 5-trlmethylhexahydro-4,6 - me thano-1,3,2 -benzodioxaborol -2 -yl)butyl]-L-prolinamide hydrochloride are dissolved in 30 ml of dichloromethane. The solution is cooled to -78°C and 12 ml of a 20 2 7 0 Q t x 1 H solution of boron trichloride in dichloromethane are added dropwise. The mixture is left for 15 minutes at -78°C and is then placed in an ice-bath and stirred for 45 minutes at 0°C. 30 ml of water are added to the reaction medium and the phases 5 are separated. The organic phase is extracted with twice 25 ml of 10 % acetic aoid solution and the aqueous phase with 3 times 25 ml of ether. The aqueous phases are combined and concentrated, and the residue is diluted in 10 ml of methanol and evaporated. The residue is purified by chromatography on a 10 Bio-gel0 P2 column eluting with 10 % acetic acid solution, and then by chromatography on a Bio-gel® P2 column eluting with 1 zoM hydrochloric acid solution. 15 [a]g° - -98.5° (c -0.65; water) •g-ympi e 5 (compound 5) 1,1-dimethylethy 1 [3aS- [2 [R[S(R)] ] , 3aa,4£,6&,7aa]]-[2- [2-[[[4-(lH-imidasol-4-yl) -1- (3a,5,5-trimethylhexahydro-4,6-methano-20 1,3,2, -benzodioxaborol-2-yl)butyl] amino] carbonyl]pyrrolidin-1 -yl] -2-oxo-l- (phenylmethyl) ethyl] -carbamate hydrochloride (1:1) ethoxy) carbonyl] -D-phenylalanyl-L-proline dissolved in 30 ml of tetrahydrofuran are treated, at iroom temperature, with 2.3 ml to -20°C and 2.72 ml (21 mmol) of isobutyl chloroformate are added. The mixture Is left stirring for 15 minutes at -20°C, 7.5 g (20 mmol) of [3aS-[2(R), 3aa,4£,6£,7aa] ] -a- (3a,5,5-trimethylhexahydro-4, 6-methano-l,3,2-benzodi-oxaborol-2-yl) -1H- 800 mg of product are obtained. Melting point « 155-160°C Yield - 52 % 7.2 g (7.2 mmol) of N-[(1,1-dimethyl- 25 (21 mmol) of N-methylmor-pholine. The reaction medium is cooled 27 Qg f 3 21 imidazole-* (5) -butanamine hydro- chloride dissolved in 10 ml of chloroform and 5.6 ml (40 mmol) o£ triethylamine are added. The mixture is left stirring for 30 minutes at -20°C and then for 24 hours at room temperature. It is diluted with 300 ml of 5 ethyl acetate and washed successively with 200 ml of water and 200 xnl of saturated sodium chloride solution. The resulting solution is dried over sodium sulphate, the solvent is evaporated to dryness and the residue is taken up in 50 ml of dichloromethane. The hydrochloride is prepared by adding 20 ml 10 of a 0.1 N solution of hydrochloric acid in isopropanol and is purified on a Lichroprep® RP 18 column, eluting with a gradient of acetonitrile in 0.02 N hydrochloric acid (20 % to 100 %) . [a]£° « - 106° (c - 1; chloroform) Example 6 (compound 6) (R) - [1- [(D-phenylalanyl-L-prolyl) amino] -4- (lH-imidazol-4 20 yl)butyl] boronic acid hydrochloride (3:1) [3aS- [2 [R[S (R) ] ] , 3aa,4fi, 68,7ao] ] - [2- [2- I [ [4- (lH-imidazol-4-yl) -1- (3a,5,5-trimethylhexa-hydro-4,6-methano-l,3 ,2, -benzodioxaborol-2-yl)butyl-amino].carbonyl]pyrrolidin-l-yl] -2-25 oxo-1- (phenyl-methyl) ethyl] carbamate hydrochloride, according to the procedure described in Example 4 and after purification on a Lichroprep® RP 18 column, eluting with a gradient of acetonitrile and 0.02 N hydrochloric acid, 300 mg of the expected compound are obtained. 5 g of product are obtained in the form of the hydrochloride. 15 Melting point - 115-120°C Yield « 45 % Starting with 698 mg (1 mmol) of 1,1-dimethylethyl - 22 - Melting point « 194°C I? c y if A [a] g0 . - 131® (c - 1.3; water) Example 7 (compound 7) 5 1,1-dimethyl ethyl [3aS- [2 [R[S (R) ] ], 3aa,4S,6S,7aa] ] - [2- [2- [ [ [4-(5-methyl-lH-imidagol-4-yl) -1- (3a, 5,5-trimethylhexahydro-4, 6-methano-1, 3,2, -benzodioxaborol-2 -yl)butyl] amino] carbonyl]pyrrolidin-l-yl] -2-oxo-l-(phenylmethyl) ethyl] carbamate hydrochloride (lxl) 10 Thia compound is prepared according to the method described in Example 5, starting with [3aS-[2(R), 3aar, 4fi, 6£, 7aa] ] -5-methyl-a- (3a,5,5-trimethylhexahydro-4,6-methano-l,3,2-benzodioxaborol-2-yl) -lH-imidazole-4-butwnamine hydrochloride. 600 mg o£ product art obtained. 15 Melting point « 85-90°C [a]p° ■ - 53.2 (c - 0.86; methanol) The table which follows illustrates the chemical structure and the chemical properties of some compounds according to the invention. Xn the "31," column, -C6Hg represents 20 the phenyl group and -C^H^ represents the cyclohexyl group. Xn the "Salt" column, "HC1" represents a hydrochloride and the ratio in parentheses represents the acid/base molar ratio. Xn the "sa.p. (#C) ■ column, "(d)" means "melting with 25 decomposition". TABLE I (I) c No. R R, *a K Salt t«3? (•) (c; solvent) m.p. (*c) 1 -COCHj -Q.H, W HCl (1»1) -90,4 (1.1; ehlorofora) 99 2 -COCHj -CA w -H -H HCl (1*1) -118,5 (o.c; water) 168-175 (d) M W IS5 Si No. R R| R» *♦ Salt C«l£ C) (°f .solvent) m.p. (*C) 3 -COCH, -<!A c^ BCl (1*1) • i -112,1 (1; ohlorofom) 130-135 4 -COCH, -PA C(™ -H -H HCl (111) -98.5 (0« 65; water 3 155-160 S -COOCfCH,), -CA HCl (1*1) -106 (l; ohlorofom) 115-120 6 -H -CA -M -M HCl (3*1) -131 (1.3; water) 194 7 -COOC(CH,), -CA NS^NIH cO HCl (1*1) -53.2 (0#86 t Bothanol) 85-90 10 tlfc r /*> No. R Rl *1 RJ R4 Salt t*J? C) (of solvent) m.p. (*c) 8 -H -CA & -H -H HCl (2:1) » -129 (o.4 /water) 195-197 9 -COOCfCtt,), Cr 3 CHg HCl (ltl) -31.2 (0*8 ; methanol) 105-110 10 -H QH -H -M HCl (2tl) -84.5 (0*46 / water) 199-201 11 -CM, -<v»3 -H -H HCl (2:1) -131 (1.3 /water) 212-215 12 -CH, -CA o- -H -H HCl (211) -104 (0.55 ;water) 190-195 (0 U1 f\3 CaJ - 26 - 270 The compounds of the Invention are tested with respect to thrombin and trypsin in-vitro in the following tests. 5 1. fibrinogen bv bovine thrombin. The compound to be tested or the vehicle thereof (10 /tl) is incubated, for 2 minutes at 37 °C, in a solution of human fibrinogen (200 pi, 2mg/ml in physiological saline) . 10 200 /tl of bovine thrombin dissolved in distilled water are then added. The final concentration of the thrombin is 0.5 NXH units/ml. The mixture is stirred and the time, expressed in seconds, for formation of a visible fibrin network is noted. The inhibition of fibrin formation is quantified by 15 calculating the concentration of compound which increases the precipitation time by 100 % (CA^g) . 2. Precipitation of fibrinogen bv human thrombin. » The compound to be tested or the vehicle thereof 20 (10 /tl) is incubated, for 2 minutes at 37°C, in a solution of humsn fibrinogen (200 /tl, 2 mg/ml in physiological saline) . 200 /tl of human thrombin dissolved in distilled water * are then added. The final concentration of the thrombin is 2 NXH units/oil. The mixture is stirred and the time, expressed in 25 seconds, for formation of a visible fibrin network is noted. The inhibition of fibrin formation is quantified by calculating the concent:?ation of compound which Increases the precipitation time by 100 % (CA100) . 3. Coagulation of rat plasma bv bovine thrombin. Male CD rats, weighing 150 to 200 g, are anaesthetized with nembutal (60 ing/kg, 0.1 tnl/kg) . Blood is withdrawn over 3.8 % trisodium citrate (1 vol/9 vol of blood) 5 from the retro-orbital sinus. The plasma is prepared by centrifugation at 3600 g for 15 minutes, at room temperature. The compound to be tested or the vehicle thereof (10 fil) is incubated with 200 /xl of plasma, at 37°C fox 2 minutes, before the addition of 200 pi of a solution of bovine thrombin. The 10 final concentration of thrombin is 0.75 NIH units/ml. The coagulation time, expressed in seconds, is noted. The inhibition of thrombin is quantified by calculating the concentration which increases the coagulation time by 100 % (CA^). 15 4. Aggregation of rabbit platelets induced bv human thrombin. Blood is withdrawn, by cardiac puncture, over 3.8 % trisodium citrate (1 vol/9 vol of blood) . It is centrifuged at 250 g for 10 minutes- The platelet-rich plasma (P3P) thus 20 obtained is removed and the platelets are counted. 2 ng/ml of prostacyclin dissolved in chilled tris buffer at pH 9.0 are added to the P3P. The mixture is centrifuged at 110 g for 10 minutes and decanted. Prostacyclin, dissolved in 50 mM sodium hydroxide at pH 12, is again added, 25 so as to have a final concentration of 200 ng/ml. The P3P is centrifuged again at BOO g for 10 minutes. The platelet-poor plasma is removed and the pellet is suspended in one volume of Tyrode's solution containing 200 ng/ml of prostacyclin, this volume being equal to the initial volume of P3P. This 270913 suspension is centrifuged at 800 g for 10 minutes. The placing in suspension of the pellet and the centrifugation are repeated a second time under the same conditions. The final pellet is 5 resuspended in prostacyclin-free Tyrode's solution and is left to stand for 2 hours in order to allow the complete removal of the prostacyclin. The aggregation of these platelets is induced with hr'man thrombin to the final concentration of 0.3 NIH units/ml. The variations in optical density are recorded using 10 a 4-chann. 7 aggregometer. The compound to be tested or the vehicle thereof is added to the platelet suspension (maximum volume of 3 fil added), 2 minutes before the addition of thrombin. The concentration which inhibits the aggregation by 50 % (ICjq) is determined. 15 5. Activity with respect to bovine trypsin. The compound to be tested or the vehicle thereof (50 iil) is incubated, for 5 minutes at room temperature, with 50 fil of bovine trypsin dissolved in tris HCl buffer at pH 8.0. 20 The final concentration of trypsin is 229 units/ml. The reaction is triggered by addition of the substrate, N a-benzoyl-L-arginine-4-nitroaniline (50 /il, final concentration 50 /M) . The mixture is incubated for 20 minutes at room temperature and the optical density of the 4-nitroaniline 25 released is measured at 405 nm. The 4-nitroaniline concentration is calculated using a calibration curve, after subtraction of the optical density from the "blanks'* (100 fi 1 of buffer +.50 fil of substrate). The concentration which inhibits the enzymatic activity by 50 * (IC;,,) is determined. 2709 - 29 - In parallel, the compounds of the invention were tested with regard to the coagulation of rat plasma ex-vivo. The animals are treated with the compound to be tested or with the vehicle thereof, via the i.v. route, orally or 5 subcutaneously, before the blood is withdrawn. The thrombin time is measured as described at 3. The compounds of the invention are thrombin inhibitors with CA^ and ICs0 values between 10~B and 10~6 M. They show no or very little inhibitory activity with respect to 10 bovine trypsin, which demonstrates their specificity. They inhibit the coagulation of rat plasma at doses below 1 trig/kg i.v. and are also active via the oral and subcutaneous routes. The compounds of the invention may be useful in all 15 the clinical indications associated with thrombosis or in those in which thrombotic complications may be involved. To this end, they may take all the forms suitable for i oral, parenteral or intravenous administration, such as tablets, dragees, capsules including gelatin capsules, 20 drinkable or injectable suspensions or solutions, in combination with suitable excipients, and may be in doses to enable an administration of 1 to 1000 mg daily per patient, taken in one or more doses. The present invention provides a pharmaceutical 25 composition which comprises a compound or a salt thereof of the present invention and a suitable excipient. The present invention also provides a compound of the invention for use in a method of treatment of the human or animal body. </div>

Claims

<div class="application article clearfix printTableText" id="claims"> The present invention further provides a compound of the present invention for use in a method of treatment of thrombosis. The present invention provides the use of a compound 5 of the present invention in the manufacture of a medicament for the treatment of thrombosis. The compounds of the present invention can be used in a method for treating or preventing thrombosis in a subject which comprises administering to that subject an effective 10 amount of a compound of the present invention or a salt thereof. 270913 il - 31 - WHAt^WE CLAIM 13

1. A compound of formula (I) O

(I)

C

in which

R represents a hydrogen atom, a straight or branched (C,-C4) alkyl group, a straight or branched - CO (C1 - C4) alkyl group or a straight or branched -COz(Cy-Cu) alkyl group,

R, represents a phenyl group or a cyclohexyl group,

Rj represents a group 1 • or a group \ / * where;R* >

R'5 is a hydrogen atom or a (C1-C4) alkyl group and R3 and R4 either each represent a hydrogen atom or together represent the residue of a dihydroxy compound, in the form of optical or geometric isomers, which are pure or in the form of mixtures^or an addition salt thereof with a pharmaceutically acceptable acid or base.

2. A compound according to claim 1 or a salt thereof in which R3 and R^ together represent the residue of 2,3-butanediol, 2,3~dimethyl-2,3-butanediol or {lot,. 2,6,6-trimethylbicyclo-[3.1.1]heptane-2,3-diol [(+] pinanediol].

il - 32 -

3 - A compound according to claim 1 or a salt thereof, in which the configuration is [2(R>] or [3aS,

[2 (R) , 3aa, 46, 6&, 7aa] ] .

4. A compound according to claim 1 or a salt thereof, in which R represents a hydrogen atom or a straight or branched (Cj-C^) alkyl group, R1 represents a phenyl group and R, and R4 each represent: a hydrogen atom.

5. A compound according to claim 1 or a salt thereof, in which Rj represents a group

H^^MH

, where Rj is ais defined in claim 1.

6. (R) - tl- [ (D-Phenylalanyl-L-prolyl) amino] -4- (1H-imidazol-4-yl)butyl] boronic acid or an addition salt thereof with a pharmaceutically acceptable acid or base.

7. Process for the production of a compound of formula (I) as defined in claim 1 in which Rg represents a group V / /Where Rj is as defined in claim l, which process conqprises reacting a boronic tripeptide of formula (II)

IL

- 33 -

13

o

(II)

in which R, is as defined in claim 1, Rj and R4 together represent a residue of a dihydroxy compound, R6 represents either a hydrogen atom when R7 represents a straight or branched -CO (C^-C^) alkyl group, or a straight or branched -C02 (Cj-C^) alkyl group when R7 represents a hydrogen atom or a straight or branched (C,—CA)alkyl group, with a compound of formula (III)

in which Rj is as defined in claim 1, to give a compound of formula (la)

(III)

IL

- 34 -

2?0913

ReRyN

NHL &

PR3\

^orJ

(la)

N^N

Rs

in which R,,, Rg, R4, Rg, R6 and R7 are as defined above followed, if desired, by reacting the compound of formula (la) with hydrochloric acid or with boron trichloride to obtain a compound of formula (lb)

0

C

_w

HQ

(lb)

il - 35 -

in which R, R, and R; are as defined in Claim 1, and/or optionally converting the resulting compound into a free base or an addition salt thereof.

8. A process for the production of a compound of formula (I) as defined in Claim 4, in which Rg represents a group

N^^NH

\ / where Re is as defined in claim 1, which process comprises coupling a compound of formula (XII)

(XII)

or4

or an addition salt thereof in which Rj and R4 together represent a residue of a dihydroxy compound and Rj is as defined in Claim 1 with an activated form of a dipeptide of formula (XIII)

P

V

(XIII)

o—x in which R1 is as defined in Claim 1, R6 and R7 are as defined in claim 7 and X represents a pyrrolidin-l-yl-2,5-dione group or a 2-methylpropyloxycarbonyl group, to give a compound of formula (Ic)

il

36

1

0

(ic)

or an addition salt thereof in which R,, Rj, R4, Rj, R6 and R7 are as defined above and, where appropriate, optionally converting the compound of formula (Ic) into the free base or other addition salt thereof and/or optionally reacting with hydrochloric acid or with boron trichloride to give a compound of formula (Id)

0

OH

(Id)

HZ, za - 37 -

in which R, R1 and Rg are as defined in Claim 1 and, if desired, converting the resulting compound into the free base or an addition salt thereof.

5

9. A pharmaceutical composition which comprises a compound or a salt thereof as defined in claims 1, in combination with any suitable excipient.

10. A process according to claim 7 substantially as described in Example 1 or 2.

10 11. A process according to claim 8 substantially as described in any one of Examples 3 to 7.

12. A compound or a salt thereof as defined in any one of claims 1 to 6 for use in a method of treatment of the human or animal body.

15 13. A compound or a salt thereof as defined in any one claims 1 to 6 for use in a method of treatment of thrombosis.

14. Use of a compound or a salt thereof as defined in any one of claims 1 to 6 in the manufacture of a medicament

20 for the treatment of thrombosis.

15. A compound or a salt thereof as defined in any one of claims 1 to € when obtained by a process as defined in any one of claims 7, 8, 10 or 11.

16. A compound specifically mention^

; \ ^

$y Mif / th&rtfuthorised Agents A J. PARK « SON.

(p(ZS~~

</div>