WO1996030407A1

WO1996030407A1 - Bifunctional thrombin inhibitors bearing highly truncated fibrinogen recognition exosite binding component

Info

Publication number: WO1996030407A1
Application number: PCT/CA1996/000178
Authority: WO
Inventors: John Dimaio; Micheline Tarazi
Original assignee: National Research Council Of Canada
Priority date: 1995-03-27
Filing date: 1996-03-27
Publication date: 1996-10-03
Also published as: GB9506212D0; AU5097596A

Abstract

The present invention relates to peptide derivatives useful in the treatment and prophylaxis of vascular disease related to thrombosis. The peptide derivatives are based on the amino acid sequence of Hirudin having a general formula: A-L-E, wherein A is an inhibitor of the active site of the enzyme thrombin; L is a linker; and E is a fibrinogen recognition exosite binding moiety comprising 3 to 5 amino acids having binding affinity for the fibrinogen recognition exosite of thrombin.

Description

BIFUNCTIONAL THROMBIN INHIBITORS BEARING HIGHLY TRUNCATED FIBRINOGEN RECOGNITION EXOSITE BINDING COMPONENT

FIELD OF THE INVENTION

The present invention relates to peptide derivatives and their use as thrombin inhibitors, and in particular to peptide derivatives based on the amino acid sequence of hirudin.

BACKGROUND OF THE INVENTION

Platelet activation and fibrin generation are largely responsible for severe acute coronary syndromes associated with arterial thrombosis while cellular proliferation and vasoconstrietion underlying the injured vessel are recognized as processes largely responsible for latent restenosis and reclosure of such vessels following successful recanalization using balloon angioplasty procedures.

Clinical and experimental evidence suggests that thrombin generation and accretion at the site of vascular injury are critical events in the manifestation of thrombotic disorders. The use of heparin is well established in clinical practice as a means of mitigating the acute symptoms associated with thrombosis, especially venous thrombosis. However, the therapeutic use of heparin is not without limitations and the clinical benefit in platelet dependent arterial thrombosis, on its own or as an adjunct to thrombolytic agents, is not optimal. The principal antithrombotic action of heparin relies on the presence of the major endogenous cofactor antithrombin III (ATIII) and to a lesser extent on heparin cofactor II. Because of this cofactor dependency, heparin also accelerates inhibition of other proteases in the coagulation cascade, principally: factors Xa, Xlla, XI, IX and TF/VIIa complex. Therefore, the protease inhibitory activity of heparin is not specific. Heparin also requires extensive monitoring of hemostasis due to interpatient variation in anticoagulant response. The lack of full clinical benefit of standard heparin and other heparinoids can also be explained on the ground that the heparin /ATIII complex does not effectively inhibit thrombin bound to thrombus on the injured vessel wall while circulating thrombin is fully inactivated. Therefore direct and specific thrombin inhibitors, acting independent of cofactors such as ATIII, are highly desirable since they inactivate free thrombin and thrombin which is enzyme bound to the platelet rich fibrin clot.

Hirudin is known to be the most potent and specific thrombin inhibitor having an equilibrium dissociation constant of the order of 10^"12-10^~13 M range. It is a 65 amino acid residue protein originally isolated from the glandular secretions of the medicinal leech Hirudo Medicinalis. Although different forms of the protein are known, the primary and tertiary structure are highly conserved among the isolated variants; therefore no significant difference in thrombin inhibitory activity is observed. The high affinity and specificity of hirudin toward thrombin stem from a unique binding mode involving two remote regions of the molecule. Single crystal X-ray crystallography has confirmed that the N-terminal tripeptidyl sequence binds to an apolar region adjacent to the active site while the C-terminal domain of hirudin binds to a region of thrombin arbitrarily defined as the "Fibrinogen anion binding exosite" or Fibrinogen Recognition Exosite (FRE) . This thrombin domain also recognizes complementary binding sequences on fibrinogen, thrombomodulin and the thrombin receptor.

Compounds that interact with only the thrombin anion binding exosite do not mimic hirudin's binding mechanism because they lack the catalytic site-directed motif and generally have weaker inhibitory activity. In contrast, the *bifunctional" thrombin inhibitors have a catalytic site-directed segment, and are larger molecules subject to limitations associated with administration and manufacture.

It has been demonstrated that the distinctive dual binding characteristic of hirudin can be mimicked by native and modified sequences corresponding to at least the amino acid length comprising residues 45-65 of hirudin.

Krstenansky et al . in FEBS Letters (1987) 211, 10-16 and Chang et al . in FEBS Letters 164, 307 (1983) first alluded to the possibility that the sequence hirudin 45-65 could potentially contain two specific domains satisfying binding recognition to both thrombin's catalytic site as well as its FRE.

DiMaio et al . in J.Biol. Chem. (1990) 265, 21698 and then Schmitz et al . in Eur. J. Biochem (1991) 195, 251 independently found that this is indeed the case in so far as the Thr₄₅-Pro_u-Lys₄₇-Pro„ sequence is reminiscent of the prothrombin cleavage site and that the sequence Thr-Pro- Lys-Pro is inhibitory on its own as reported by Bajusz et al . in Peptides (1984) page 473.

Maraganore et al . in WO 91/02750 and DiMaio et al. in J. Biol. Chem (1990), 265:21698 optimized that sequence affording highly potent *bifunctional" thrombin inhibitors. These compounds are not proteolytically stable toward thrombin. DiMaio et al . FEBS Letters (1991), 282, 47-52, DiMaio et al . J. Med. Chem. (1992) 35:3331-3341 and DiMaio et al . PCT/CA91/00213 show that proteolytic stability is conferred through scissible bond peptidomimetics. Several efforts have been made to identify peptide fragments of hirudin capable of inhibiting thrombin. Maraganore et al. in J. Biol. Chem (1989) 264, 8692-8698 report that the dodecapeptide hirudin 53-64 is inhibitory and Krstenansky et al . in J. Med. Chem. (1987) 30, 1688- 1691, showed that the minimum active sequence required for binding to the FRE is Hirudin 56-64.

There exists the need for a molecule that exhibits thrombin inhibitory activity similar to hirudin, is stable to proteolysis and is of sufficiently low molecular weight to permit commercially feasible quantities to be produced inexpensively.

SUMMARY OF THE INVENTION

The present invention provides bivalent thrombin inhibitors having binding affinity for both the active site and fibrinogen recognition exosite (FRE) of thrombin. In one aspect, there is provided a bivalent thrombin inhibitor of the formula (I) and pharmaceutically acceptable salts and derivatives thereof:

A— L — E (I) wherein

A is an inhibitor of the active site of thrombin; L is a linker; and

E is a fibrinogen recognition exosite moiety comprised of 3 to 5 amino acids in length which maintains the binding affinity for the fibrinogen recognition exosite of thrombin.

In another aspect, there is provided pharmaceutical compositions useful for the treatment of thrombotic disorders which comprise an effective amount of a thrombin inhibitor of the present invention in combination with a pharmaceutically acceptable carrier.

In yet another aspect, there is provided a method for the treatment or prophylaxis of vascular diseases related to thrombosis which comprises administering to a patient an effective amount of a composition of the present invention.

DESCRIPTION OF THE INVENTION

For convenience, the peptide derivatives of this invention are designated hereinafter simply as peptides.

The term " amino acid" used herein includes nattirally- occurring amino acids as well as non natural analogs commonly used by those skilled in the art of chemical synthesis and peptide chemistry. A list of non natural amino acids may be found in "The Peptides", vol. 5, 1983, Academic Press, Chapter 6 by D.C. Roberts and F. Vellaccio which is incorporated herein by reference. Amino acids are specified herein by their established three letter and/or single letter codes.

The term "conservative substitution" means a modification or substitution to the native amino acid having minimal influence on the secondary structure and hydropathic nature of the amino acid or peptide. These include substitutions such as those described by Dayhoff in the Atlas of Protein Sequence and Structure 5, 1978 and by Arhos in EMBO J. 8, 779-785, 1989. For example, amino acids belonging to one of the following groups represent conservative changes:

Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr; Cys, Ser, Tyr, Thr; Val , lie , Leu, Met , Ala, Phe ; Lys , Arg , His ; and Phe , Tyr , Trp, His .

In like manner, methionine, an amino acid which is prone to oxidation may be replaced by norleucine. Substitutions also include substitutions of D-isomers for the corresponding L-amino acids.

By the term " hydrophilic amino acid" is meant an amino acid that bears a water-solubilizing substituent such as OH, COOH and NH2- Examples of hydrophilic amino acids include natural amino acid residues such as asparagine, gluta ine, arginine, and glutamic acid and unnatural amino acids such as those described in "The Peptides", Vol. 5, 1983, Academic Press, Chapter 6 by D.C. Roberts and F. Vellaccio incorporated herein by reference.

By a " hydrophobic amino acid" is usually meant an amino acid that bears an alkyl or aryl group attached to the α- carbon atom. Thus glycine, which has no such group attached to the α-carbon atom, is not a hydrophobic amino acid. The alkyl or aryl groups impart hydrophobic character to the amino acid. The alkyl or aryl group can be substituted, provided that the amino acid retains overall hydrophobic character. Examples of hydrophobic amino acids include natural amino acid residues such as alanine, histidine, isoleucine, and phenylalanine and unnatural amino acids such as those described in "The Peptides", Vol. 5, 1983, Academic Press, Chapter 6 by D.C. Roberts and F. Vellaccio incorporated herein by reference. For example, one may cite β-(2-and 3-thienyl)alanine, cyclohexylalanine and SubPhe. SubPhe represents the phenylalanine residue bearing substituentε on the aromatic ring. The term "residue", when applied to an amino acid, means a radical derived from the corresponding amino acid by removing the hydroxy1 of the carboxy1 group and one hydrogen from the amino group.

The term " alkyl " represents a saturated or unsaturated, substituted (for example, by a halogen, hydroxyl, amino, oxygen, sulfur, or C_s.₂₀ aryl) or unsubstituted, straight chain, branched chain hydrocarbon moiety having 1 to 10 carbon atoms and preferably from 1 to 6 carbon atoms. This chain may be interrupted by one or more heteroatom such as N, 0, or S. The term "alkylene" refers to a divalent alkyl chain as defined above.

The term " aryl " represents an unsaturated carbocyclic, benzenoid-type ring preferably containing from 6 to 15 carbon atoms (for example phenyl and naphthyl) which is optionally substituted. The carbocycle is optionally interrupted by one or more heteroatom such as N, O or S.

The term " aralkyl " represents an alkyl group being uninterrupted or interrupted , unsubstituted or substituted by an aryl substituent (for example benzyl), preferably containing from 6 to 30 carbon atoms in total.

The term " cycloalkyl " represents a saturated carbocycle containing 3 to 12 carbon, preferably 3 to 8 carbon, which includes for example cyclopropyl, cyclobutyl, cyclohexyl, and cyclodecyl, which may be substituted with substituents such as halogen, amino, alkyl, and/or hydroxy.

The present invention provides bivalent thrombin inhibitors of formula (I) as herein defined and as defined in application GB 9506212 (filed on 27 March 1995) incorporated herein by reference. In a particular embodiment of the invention, there is provided compounds according to formula (I) wherein the E exosite portion is represented by formula (II) :

⁽II⁾ -R₁-R₂-R3-(R₄)_m-(R5)n-Y wherein m and n are each independently 0 or 1.

R_j is substituted or unsubstituted Asp or another ^~ hydrophilic amino acid.

R_j is substituted or unsubstituted Phe or another hydrophobic amino acid. Preferably P^ is Phe or Tyr. Most preferably i is Phe.

R, is substituted or unsubstituted Glu or another hydrophilic amino acid. Preferably R₃ is Glu, Glu(OMe), or

N-methyl-Glu. Most preferably R₃ is Glu.

R₄, when present, is substituted or unsubstituted Pro, pipecolic acid (Pip) , 3-carboxytetrahydroisoquinoline, or lie or conservative substitution thereof. Preferably R₄, if present, is proline.

R_s, when present, is substituted or unsubstituted lie or another hydrophobic amino acid. Y is hydroxyl radical of the COOH terminus of the preceding amino acid, or an amine group -NIR^) (R₃₀) wherein

R_gQ and R_ββ are each independently hydrogen, alkyl or aryl.

Preferably, Y is hydroxyl radical of the COOH terminus of the preceding amino acid, or NH_j.

In particularly preferred embodiments, E is Asp-Phe-Glu-

Pro-Ile-NH,, Asp-Phe-Glu-Pro-Ile-OH, Asp-Phe-Glu(OMe)-Pro-

Ile-OH; and Asp-Tyr-Glu-Pro-Ile-NH_j.

In a more preferred embodiment, E is Asp-Phe-Glu-Pro-Ile- NH..

In another embodiment, E is the group Asp-R_-Glu-(Ile)_0.,-Y wherein:

R, is Phe or Tyr or conservative substitutions thereof; and Y is OH or NH,. In a more preferred embodiment, E is Asp-Phe-Glu-OH, Asp- Tyr-Glu-OH, or Asp-Tyr-Glu-NJL_^.

Preferred compounds of formula (I) include those compounds where the A portion is a bulky hydrophobic portion comprising a hydrophobic radical capable of binding to a complementary hydrophobic region at the catalytic site of thrombin responsible for proteolysis as are well known in the art.

Preferably A is an active site inhibitor of thrombin including substrate or non substrate type inhibitors. Examples of substrate inhibitors include (D-Phe)-Pro-Arg- Pro, D-Phe-Pro-Arg chloromethylketone (PPACK) , and (D- Phe)-Pro-Arg analogues. Examples of non substrate type inhibitors include dansyl-arginyl-pipecolic acid, and those derived from arginine and benzamidine to give, for example, (2R,4R)-4-methyl-l-[N^*-(3-methyl-l,2,3,4- tetrahydro-8-quinolinesulphonyl)-L-arginyl]-2-piperidine carboxylic acid (MD-805) , N^*-(4-toluene-sulphonyl)-D,L- amidinophenylalanyl-piperidine (TAPAP), and N^*- (2-naphthyl- sulphonyl-glycyl)-D-L,p-amidinophenylalanyl-piperidine (NAPAP) .

In one embodiment, A is represented by formula (IV) :

(IV) wherein

X_j is a hydrophobic group; sulfonyl substituted with alkyl, aryl, or aralkyl; or carbonyl substituted with alkyl, aryl, or aralkyl. X, is an amino acid residue that is linear or substituted at the α-position, alkyl, or X_a is a bond when X_x is a sulfonyl or carbonyl group.

X, is hydrogen, or branched or straight chained alkyl, aryl, or aralkyl with the proviso that when X_a is a bond, X, is not present. ~

X₄ is hydrogen, or alkyl.

X, is selected from the group consisting of alkyl, aryl, and aralkyl. X, is guanidinyl, amidino, or hydrogen.

In an embodiment, A is a compound of formula (IV) wherein X_x is a hydrophobic α-amino acid in the D-configuration, where the α-amino group is optionally neutralized, attached by a peptide linkage to X..

Preferably, X_j is cyclohexylalanine, D-Phe, D-4FPhe, or D- 4ClPhe wherein the α-amino group is optionally neutralized by acetylation or benzoylation. More preferably, J^ is D-cyclohexylalanine or D-Phe wherein the α-amino group is optionally neutralized by acetylation or benzoylation. Most preferably, X_x is D-cyclohexylalanine.

Preferably, Z, is a residue of a hydrophobic α-amino acid of the L-configuration or a cyclic imino acid which can bear one or more alkyl substituents attached to the ring, wherein said substituents may bridge to form a cyclic structure.

More preferably, X, is valine, pipecolic acid, or proline. Most preferably, X, is proline.

Preferably, X, and X₄ are hydrogen.

Preferably, X, is phenylmethylene, phenylethylene, ethylene, butylene, or propylene. More preferably X, is propylene or phenylmethylene. More preferably X, is propylene.

Preferably X, is NHC(NH)NH_j.

In an alternative embodiment, X_x is preferably selected from the group consisting of:

wherein k is an integer between 0 and 15;

M is a C,_, alkyl or hydrogen; and

X_lβ is an halogen (e.g., Cl, Br or F) .

More preferably, X^ is selected from the group consisting of:

wherein X_l is an halogen (e.g., Cl, Br, or F) ; Most preferably, 7^ is:

X₄ is preferably hydrogen.

X_a is preferably a bond and X, is not present. X, is preferably phenylethylene, phenylmethylene, ethylene, butylene, or propylene. Most preferably, X, is propylene

X, is preferably guanidyl or hydrogen. Most preferably, X, is NHC(NH)NH₂.

Examples of A portions include but are not limited to Bzs- Arg-(D-Pip); dansyl-Arg-(D-Pip) ; dansyl-Arg-(L-Pip) ; dansyl-Nle-(D-Pip) ; (D-Phe)-Arg-(D-Pip) ; Fmoc-Arg-(D-Pip) ; dansyl-Arg-(D-Tic) ; dansyl-(D-Arg)-(D-Pip) ; dansyl-Phe-(D- Pip) ; dansyl-Cha-(D-Pip) ; (D-Cha)-Arg-(D-Pip) ; α-naphthyl sulfonyl-Arg-(D-Pip) ; β-naphthyl sulfonyl-Arg-(D-Pip) ; 4- tert-Butyl-benzene sulfonyl-Arg-(D-Pip) ; dansyl-Arg-(D- Cha) ; dansyl-Arg-Acha; phenyl ethyl sulfonyl-Arg-(D-Pip) ; β-dihydroanthracenyl-β-sulfonyl-Arg-(D-Pip) ; (+)- ca phorsulfonyl-Arg-(D-Pip) ; 4-bromobenzenesulfonyl-Arg- (D-Pip) ; 2,4,6 triisopropylbenzenesulfonyl-Arg-(D-Pip) ; Ac(D-Phe)-Pro-Arg-; Ac(D-Phe)-thioPro-Arg-H; Ac(D-Cha)- Pro-Arg-; (D-Cha)-Pro-Arg-; (D-Phe)-Pro-Arg-; succinyl(D-Phe)-Pro-Arg-; and alpha-N-(Ac) (D-Phe)-Pro- Arg-.

Alternatively, A may be a peptidomimetic group as ,. described in PCT application CA95/00708 (filed on 21 December 1995) of the formula:

wherein:

A' is selected from (CH-Rg'),,., , S, SO, S0₂, 0 and NR„' wherein R,' is hydrogen, C,_₆ alkyl optionally interrupted with 1 or 2 heteroatoms; C_t._u aryl, C₃.₇ cycloalkyl or heterocyclic ring or a hydrophobic group; B' is selected from S, S0₂, 0, -N=, NH, -CH= and CR^'R wherein R,' and R,' are independently selected from hydrogen and C,_₆ alkyl provided that when A' is S, SO, S0₂, 0, or NR₈', then B' is CR/R,'; D' is selected from (CH-R,* )₀_₂ wherein R,' is hydrogen, C,.₄ alkyl or -C(0)R_j'; and CH with a double bond to B' when B' is -N= or -CH=; E* is selected from CH_j and CH substituted with the - CfOjR/, provided that only one of D' and E' is substituted with -C(0)R,'; X' is selected from 0, N- , ^or CH-R,';

Y' is selected from 0, S, SO, S0₂, N-R_b' and CH-R,' provided that when X is N-R_s' then Y is CH-R,' or 0, and when X is 0 then Y is CH-R,' ; Z' is selected from 0, S and H_j,- R_j' is a bond or an arginyl residue or an analog or derivative thereof; R_j' is selected from H and C,_₆ alkyl optionally substituted with C₆ aryl, a 6 member heterocycle or a C₃_₇ cycloalkyl ring;

R,' is selected from H, NRj'R,' and C^ alkyl; and R₄'and R,' are independently selected from H; NR R,'; C₆. aryl or C₃_₇ cycloalkyl optionally substituted with C,_₆ alkyl; C_j. alkyl optionally interrupted by one or more heteroatom or carbonyl group and optionally substituted with OH, SH, NRj'R,' or a C_<_₁₆ aryl, heterocycle or C₃.₇ cycloalkyl group optionally substituted with halogen, hydroxyl, C,_₆ alkyl; an amino acid side chain; and a hydrophobic group.

In a particular embodiment, A is a peptidomimetic group of formula:

wherein R_t' to R₅' are as previously defined.

Preferred compounds of formula (I) may also include those compounds where L is a divalent straight chained link moiety that has a chain length of at least about 10 atoms.

The linker L to be used in the context of the present invention is required to have sufficient length to permit portions A and E of the peptides of formula (I) to interact with two different and independent binding sites separated by a critical distance (approximately 15 A) from each other on the thrombin surface. Preferably, L is a divalent straight chained linker moiety having a chain length of at least about 10 atoms. L may be a straight or branched carbon chain that can be interrupted or substituted by one or more hydroxy, oxygen, sulfur, amino, alkyl, alkoxy, aryl, and aralkyl groups that may be substituted by hydroxy, amide, hydroxy, imidazol, carbonyl, or halogen groups.

In some embodiments, L may be composed, at least in part, of natural or non natural amino acids. L can be amino acids 49 to 54 of native hirudin.

Preferably L is represented by the formula (V) :

-<CH₂)_n-(CO),-Z.

(V) wherein m is 0 or 1. n is an integer ranging from 0 to 4. Z is a straight or branched carbon chain that can be interrupted or substituted by one or more 0, S, NH, alkyl, alkoxy, aryl, aralkyl, imidazol, carbonyl, or halogen groups.

Preferably, Z can be a synthetic spanner of the general formula (VI) :

-[NH-R,-CO]_m-(R₁₀-)_n (VI) wherein m is an integer ranging from 1 to 4. n is an integer ranging from 0 to 4.

R, is a hexapeptide, or saturated or unsaturated alkyl chain corresponding to 18 atoms or less.

R_jβ, if present, is one or more amino acids.

More preferably R, is - (CHQ_2) I-H~ • Further preferably R, is - (CHQ_₂) 1-4"• Even more preferably R, is -(CHQ_2)4"- Most preferably R, is also -CH_g-CH_g-C^-CH_g-.

More preferably R_lβ, if present, is one to four amino acids in length. Even more preferably, R^, if present, is one amino acid in length.

Even further preferably, R₁₀, if present, is Gly. Most preferably, R^ is not present. Most preferably Z is [5-aminovaleryl]₂. Most preferably L is -(CH_j),-(CO)-[5-aminovaleryl]₂.

In an alternative embodiment, Z is preferably (R)- pipecolic acid-R^ls- wherein R^1S is one or more amino acids. Preferably, R^1S is selected from the group consisting of Ada-Abu, Ada-Aca, Ada-Asn-Gly-, β-Ala-Gly-Gly-Ava, Ada- Gly, Gly-Ada, Aua-Gly, and Aca-Abu. Ada is 12- aminododecanoic acid, Abu is 4-aminobutyric acid, Aca is 6-aminocaproic acid, Aua is 11-aminoundecanoic acid and Ava is 5-aminovaleric acid More preferably R¹⁵ is Ada-Abu, or Ada-Asn-Gly-.

Examples of the L portion include but are not limited to (12-aminododecanoic acid) -4-aminobutyric acid)-; (12- aminododecanoic acid) -6-aminocaproic acid); (8- aminocapylic acid) -4-aminobutyric acid)-; (12- aminododecanoic acid) -asparagyl-glycyl) ; (4-aminobutyric acid-glycyl) ; (5-amino valeric acid) -glycyl); (6- aminocaproic acid) -glycyl) ; (7-aminoheptanoic acid) - glycyl) ; (8-aminocaprylic acid) -glycyl); (12- aminododecanoic acid); (11-aminoundecanoic acid) -glycyl); (Glycyl)-12-aminododecanoic acid); (12-aminododecanoic acid)-glycyl) ; (β-Alanyl-glycyl-glycyl-5-aminovaleric acid); (CH₂)!_₄(CO)-QSHNDG; CH₂ (CO)-GSHNDG; (CH₂)₄(CO)- [NH-CH₂-CH=CH-CH₂-(CO)],._j; CH₂N(COCH₃)CH₂(CO)-(Gly)₄-; CH₂-(4-pyridylacetyl)-(Gly)₂_₄-; CH₂-(4-pyridylacetyl)- Abu-Gly-; CH₂-(4-pyridylacetyl)-Ava-Gly-; CH₂-(4- pyridylacetyl)-Aca-Gly-; CH₂-(4-pyridylacetyl)-Aha-Gly-,- CH₂SCH₂(CO)-(Gly) -; CH₂SCH₂(CO)-(Gly) -; CH₂~(4- pyridylacetyl)-NH(CH₂)₃CO-Gly-; CH₂-(4-pyridylacetyl)- NH(CH_a)₁_₃CO-; (CH₂)₄(CO)-[NH(CH₂)₄(CO) ]₂~; and (CH^fCO) [NH(CH₂)₄(CO)]-NG;

Preferred compounds of formula (I) are selected from the group consisting of:

(1) alpha-(4-t-butylbenzenesulphonyl)-Arg-(R)-Pip-Ada- Abu-DFEPI-OH;

(2) alpha-N-(4-t-butylbenzenesulphonyl)-Arg-(R)-Pip-Ada- Asn-Gly-DFEPI-OH; (3) alpha-N-(4-t-butylbenzenesulphonyl)-Arg-(R)-Pip-Ada- Abu-DF-Glu(OMe)-PI-OH;

(4) alpha-N-(4-t-butylbenzenesulphonyl)-Arg-(R)-Pip-Ada- Asn-Gly-DFE-OH;

(5) H-(R)-Phe-Pro-AOGD-(5-aminovaleryl)₂-DFEPI-OH; (6) Ac-(R)-Phe-Pro-AOGD-(5-aminovaleryl)₂-DFEPI-OH;

(7) H-(R)-Phe-Pro-AOGD-(5-aminovaleryl)₂-DFE-OH;

(8) H-(R)-Phe-Pro-AOGD-(5-aminovaleryl)₂-DFEI-OH;

(9) H-(R)-Phe-Pro-AOGD-(5-aminovaleryl)₂-DF-Glu(OMe)-PI- OH; (10) Ac-(R)-Phe-Pro-AOGD-(5-aminovaleryl)₂-DF-Glu(OMe)-PI- OH;

(11) H-(R)-Phe-Pro-AOGD-(5-aminovaleryl)₂-DF-N-Methyl-Glu- PI-OH;

(12) Ac-(R)-Phe-Pro-AOGD-(5-aminovaleryl)₂-DF-N-Methyl-Glu- PI-OH;

(13) H-(R)-Phe-Pro-AOGD-(5-aminovaleryl)_j-DFEPI-NH,;

(14) H-(R)-CyclohexylAla-Pro-AOGD-(5-aminovaleryl),-DFEPI-

NH,; (15) H-(R)-CyclohexylAla-Pro-AOGD-(5-aminovaleryl).-DYE- NH,; (16) H- (R)-CyclohexylAla-Pro-AOGD- (5-aminovaleryl)₂-DYE-OH;

(17) H- (R)-CyclohexylAla-Pro-AOGD- (5-aminovaleryl)₂-DFE-OH;

(18) H- (R) -CyclohexylAla-Pro-AOGD- (5-aminovaleryl) -G- DYEPI-NH_j,- and (19) H- (R)-CyclohexylAla-Pro-AOGD- (5-aminovaleryl)₂- DYEPI-NH_j.

AOGD represents the group -Arg- (CH,^(CO)- .

More preferred compounds of formula (I) are selected from the group consisting of

(1) alpha- (4-t-butylbenzenesulphonyl) -Arg- (R) -Pip-Ada- Abu-DFEPI-OH;

(2) alpha-N- (4-t-butylbenzenesulphonyl) -Arg- (R) -Pip-Ada- Asn-Gly-DFEPI-OH;

(5) H- (R)-Phe-Pro-AOGD- (5-aminovaleryl)₂-DFEPI-OH;

(6) Ac- (R) -Phe-Pro-AOGD- (5-aminovaleryl)₂-DFEPI-OH;

(9) H- (R) -Phe-Pro-AOGD- (5-aminovaleryl)₂-DF-Glu(OMe) -PI- OH; (13) H-(R) -Phe-Pro-AOGD- (5-aminovaleryl)_J-DFEPI-NH_J,- and

(14) H-(R)-CyclohexylAla-Pro-AOGD- (5-aminovaleryl)₂-DFEPI- NH_j.

A most preferred compound of formula (I) is (14) H-(R)- CyclohexylAla-Pro-AOGD- (5-aminovaleryl)_j-DFEPI-NH_j;

While it may be possible that, for use in therapy, a compound of the invention may be administered as the raw chemical, it is preferable to present the active ingredient as a pharmaceutical formulation.

The invention thus further provides a pharmaceutical formulation comprising a compound of formula (I) and pharmaceutically acceptable acid addition salt thereof together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

In a further embodiment of the present invention is provided the use of a compound of formula (I) in the manufacture of a medicament for the treatment of vascular diseases in a mammal including human.

In an alternative aspect of the present invention is provided a method for the treatment of vascular diseases in a mammal including human, comprising the administration of an effective amount of a compound of formula (I) .

It will be appreciated by people skilled in the art that treatment extends to prophylaxis as well to the management of established vascular disease.

The compounds of the present invention are useful in combinations, formulations and methods for the treatment and prophylaxis of vascular diseases. These diseases include myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, restenosis following arterial injury or invasive cardiological procedures, acute or chronic atherosclerosis, edema and inflammation, cancer and metastasis.

The term "combination" as used herein, includes a single dosage form containing at least one compound of this invention and at least one thrombolytic agent, a multiple dosage form, wherein the thrombin inhibitor and the thrombolytic agent are administered together or separately, but concurrently, or a multiple dosage form wherein the two components are administered separately, but sequentially. In sequential administration, . The thrombin inhibitor may be given to the patient during the time period ranging from about 5 hours prior to about 5 hours after administration of the thrombolytic agent. Preferably, the thrombin inhibitor is administered to the patient during the period ranging from 2 hours before, to 2 hours following administration of the thrombolytic agent.

Thrombolytic agents which may be employed in the combinations of the present invention are those known in the art. Such agents include, but are not limited to, tissue plasminogen activator (TPA) purified from natural sources or recombinantly produced, streptokinase, urokinase, purokinase, anisolated streptokinase plasminogen activator complex (ASPAC) , animal salivary gland plasminogen activators and known, biologically active derivatives of any of the above.

The dosage and dose rate of the compounds of this invention will depend on a variety of factors, such as the weight of the patient, the specific pharmaceutical composition used, the object of the treatment, i.e., therapy or prophylaxis, the nature of the thrombotic disease to be treated, and the judgment of the treating physician.

According to the present invention, a preferred pharmaceutically effective daily dose of the compounds of this invention is between about O.lμg/kg body weight of the patient to be treated ("body weight") and about 20 mg/kg body weight.

Most preferably, the therapeutic and prophylactic compositions of the present invention comprise a dosage of between about 10 μg/kg body weight and about 500 μg/kg body weight of the compounds of this invention. It should also be understood that a daily pharmaceutically effective dose of either the compounds of this invention or the thrombolytic agent present in combinations of the invention, may be less than or greater than the specific ranges cited above.

According to an alternate embodiment of this invention, compounds may be used in compositions and methods for coating the surfaces of invasive devices, resulting in a lower risk of clot formation or platelet activation in patients receiving such devices. Surfaces that may be coated with the compositions of this invention include, for example, prostheses, artificial valves, vascular grafts, stents and catheters. Methods and compositions for coating these devices are known to those of skill in the art. These include chemical cross-linking or physical adsorption of the compounds of this invention-containing compositions to the surfaces of the devices.

According to a further embodiment of the present invention, compounds may be used for ex vivo thrombus imaging in a patient. In this embodiment, the compounds of this invention are labeled with a radioisotope. The choice of radioisotope is based upon a number of well- known factors, for example, toxicity, biological half-life and detectability. Preferred radioisotopes include, but are not limited to ^l2SI, ¹²³I and I. Techniques for labeling the compounds of this invention are well known in the art. Most preferably, the radioisotope is '"i and the labeling is achieved using ^,2'l-Bolton-Hunter Reagent. The labeled thrombin inhibitor is administered to a patient and allowed to bind to the thrombin contained in a clot. The clot is then observed by utilizing well-known detecting means, such as a camera capable of detecting radioactivity coupled to a computer imaging system. This technique also yields images of platelet-bound thrombin and meizothrombin.

This invention also relates to compositions containing the compounds of this invention and methods for using such compositions in the treatment of tumor metastases—The efficacy of the compounds of this invention for the treatment of tumor metastases is manifested by the inhibitors ability to inhibit thrombin-induced endothelial cell activation. This inhibition includes the repression of platelet activation factor (PAF) synthesis by endothelial cells or the inhibition of cellular adhesion molecular (CAM) expression. These compositions and methods have important applications in the treatment of diseases characterized by thrombin-induced inflammation and edema, which is thought to be mediated be PAF. Such diseases include, but are not limited to, adult respiratory distress syndrome, septic shock, septicemia and reperfusion damage. Early stages of septic shock include discrete, acute inflammatory and coagulopathic responses.

This invention also relates to the use of the above- described compounds, or compositions comprising them, as anticoagulants for extracorporeal blood. As used herein, the term "extracorporeal blood" includes blood removed in line from a patient, subjected to extracorporeal treatment, and then returned to the patient in such processes as dialysis procedures, blood filtration, or blood bypass during surgery. The term also includes blood products which are stored extracorporeally for eventual administration to a patient and blood collected from a patient to be used for various assays. Such products include whole blood, plasma, or any blood fraction in which inhibition of coagulation is desired.

The amount or concentration of compounds of this invention in these types of compositions is based on the volume of blood to be treated or, more preferably, its thrombin content. Preferably, an effective amount of a compound of this invention of this invention for preventing coagulation in extracorporeal blood is from about 1 μg/60 ml of extracorporeal blood to about 5 mg/60 ml of extracorporeal blood.

The compounds of this invention may also be used to inhibit clot-bound thrombin, which is believed to contribute to clot accretion. This is particularly important because commonly used anti-thrombin agents, such as heparin and low molecular weight heparin, are ineffective against clot-bound thrombin. Finally, the compounds of this invention may be employed in compositions and methods for treating neurodegenerative diseases. Thrombin is known to cause neurite retraction, a process suggestive of the rounding in shape changes of brain cells and implicated in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

Synthesis of the peptide derivatives of the present invention

The peptides of the present invention may be synthesized using a variety of methods which are well known to those skilled in the art. For example, the peptides may be synthesized by the solid phase method on a suitable peptide synthesizer such as that described by Stewart et al. in "Solid phase peptide synthesis", Freeman & Co., San Francisco, 1969 or in Szewczuk, Z., Gibbs, B. F., Yue, S.- Y., Purisi a, E., & Konishi, Y. (1992) Biochemistry 31, 9132-9140 which are hereby incorporated by reference. Final products were obtained as lyophilizates with 98% or higher purity estimated by analytical HPLC. The purified peptides were identified by amino acid analysis on a Beckman Model 6300™ high performance analyzer and by molecular mass analysis using a SCIEX API III™ mass spectrometer. Peptide contents in lyophilizates were determined by the amino acid analysis.

In some instances however, regions A, L, and E of the peptides of the present invention (formula I) may be a synthetic moiety for which chemical synthesis is required prior to linking this moiety with other amino acids to yield the desired peptide through conventional solid phase synthesis. Examples 1 to 7 appearing further in the specification illustrate the procedure required to synthesize embodiments in region A while examples 8 and 9 describe the synthesis of a synthetic linker to be used in region L. It will be appreciated by those skilled in the art that a skilled organic chemist can readily prepare the chemical moiety which may be required for regions A, L, and E of the peptides of the present invention.

Some of the peptides of the present invention were specifically synthesized on an Applied Biosystems 430A peptide synthesizer. BOC-GlnPAM resin (Applied

Biosystems; 0.64 mmol/gram) was used as the solid phase support. Amino acid coupling was mediated by dicyclohexylcarbodiimide/N-hydroxybenzoyltriazole and deprotection was carried out with 50% trifluoroacetic acid (TFA) in methylene chloride for 3 minutes followed by an additional 20 minute cycle. Side chain protecting groups were as follows: Asp(Chx), Glu(Bzl), His(Bom), Arg(Tos) , Tyr(2-BrZ), Ser(Bzl). The fully protected peptide resin was then treated with liquid hydrogen fluoride containing anisole and dimethyl sulfide (10% by volume) at -5°C for 60 minutes. Excess HF was removed under a stream of nitrogen and the residual solid was extracted with ether and filtered. The resin was extracted three times with glacial acetic acid and water followed by lyophilization. Purification and analysis of the synthetic peptides The resulting lyophilized crude peptides may be purified to homogeneity by using generally accepted peptide purification techniques. One suitable technique is reverse phase chromatography on a Vydac octadecyl silica glass column (15 A, 1.5 X 30 cm, 40 psi) using a linear gradient of the solvent system: A, 500 ml 500 ml 0.1% TFA/H20 and B, 1L 60% Acetonitrite/H20 containing 0.1% TFA. The fractions are analyzed by reverse phase HPLC on a Varian LC using a Vydac C18 analytical column and 215 nm detection. Fractions corresponding to greater than 99% purity may be pooled and lyophilized. Peptide content is determined by amino acid analysis on a Beckman model 6300 amino acid analyzer. Samples are then dried in a Waters Pico-Tag Work Station. Constant boiling HCI (200 μl) containing 1% phenol was added to the vial and alternatively purged (with dry nitrogen) and evacuated after three purges. Finally, the vial containing the sample is heated at 150°C for 1 hour under vacuum. Mass spectral analyses were carried out on a SCIEX® API III spectrometer equipped with an ionspray inlet source.

Thus, the structure and sequence of the peptides synthesized in the context of the present invention may be confirmed by correct amino acid composition and mass spectra in order to show agreement with the calculated molecular weights.

The following examples are provided to further illustrate rather than limit the scope of the present invention.

Abbreviations used in the examples include BOC: tert- butoxycarbonyl; Tos: p-toluene sulfonyl; CH2CI2: methylene chloride; TEA: triethylamine; BOP: benzotriazolyl N- oxytrisdimethylamino phosphonium hexafluorophosphate; DMF: dimethyl formamide; EtOAc: ethyl acetate; DCC: N,N'- dicyclohexylcarbodiimide; DPPA: diphenyl-phosphoryl azide; THF: tetrahydrofuran; HF: hydrogen fluoride, CBZ: benzyloxycarbonyl.

EXAMPLES

Peptide Synthesis

Materials and Methods

Human a-thrombin (3,000 NIH units/mg) , bovine fibrinogen

(-70% of protein, 85% of protein clottable) , Tos-Gly-Pro- Arg-AMC^'HCl salt, poly(ethylene glycol) 8000™, Ada and

Tris were purchased from Sigma Inc. AMC dansyl chloride, 1-naphthalenesulfonyl chloride, 2-naphthalenesulfonyl chloride, 4-tert-butylbenzenesulfonyl chloride, Ada, Ava and D,L-Pip were obtained from Aldrich. Boc-Abu, Boc- Bal, Boc-Aca, Boc-Aha, Boc-Cha, Boc-D-Cha, Boc-L-Pip, Boc- D-Pip, and Boc-D-Tic were purchased from BaChe . Acha was obtained from Fluka Inc. Boc-Ada, Boc-D,L-Pip, and Boc- Acha were prepared according to the procedure described by Chaturvedi, D. N., Knittel, J. J., Hruby, V. J., Castrucci, A. M., & Hadley, M. E. (1984) J. Med. Chem. 27, 1406-1410 which is hereby incorporated by reference. All other amino acid derivatives for peptide synthesis were purchased from Advanced ChemTech except Boc-Glu(OBzl)-OH, which was obtained from Sigma. The side chain protecting groups for Boc-amino acids were benzyl for glutamic acid (Glu) and aspartic acid (Asp) , tosyl (Tos) for arginine (Arg) and 2-bromobenzyloxycarbonyl for tyrosine (Tyr) . Boc-Gln-OCH_j-phenylacetylamidomethyl resin (0.714 mmol/g) and p-methyl-benzhydrylamin resin (0.770 mmol/g) were purchased from Applied Biosystems Inc. Boc-D-Glu(OBzl( )- OCH2-pheynylacetylamidomethyl resin (0.31 mmol/g) was purchased from Peninsula Laboratories, Inc. The solvents for peptide synthesis were obtained from B&J Chemicals and Applied Biosystems Inc. Citric acid was purchased from Anachemia. HF and TFA were purchased from Matheson and Halocarbon Products Co., respectively. The peptides were prepared according to the method described in Szewczuk, _ . , Gibbs, B. F., Yue, S.-Y., Purisima, E., & Konishi, Y. (1992) Biochemistry 31, 9132- 9140 which is hereby incorporated by reference. Final products were obtained as lyophilizates with 98% or.higher purity estimated by analytical HPLC. The purified peptides were identified by amino acid analysis on a Beckman Model 6300™ high performance analyzer and by molecular mass analysis using a SCIEX API III™ mass spectrometer. Peptide contents in lyophilizates were determined by the amino acid analysis.

EXAMPLE 1 (2S)-2-(BOC)-N-methoxy-N-methyl-5- tosylguanadinopentanamide To a solution of Nα-BOC-NG-Tosyl Arginine (428 mg, 1 mmol) in 30 ml of DMF, at 0°C in an ice bath, containing TEA (0.4 ml, 3 mmol) and N,0-dimethylhydroxyl-amine hydrochloride (146 mg, 1.5 mmol) was added BOP reagent (500 mg, 1.1 mmol) (B. Castro, J.R. Dormoy, G. Elvin, C. Selve, Tetrahedron Letters # 14, pp. 1219-1222, 1975). The reaction was stirred for 15 hours at 4°C after which the solvent was evaporated under high vacuum. The residue was dissolved in 50 ml of EtOAc and washed with H.O. The organic phase was extracted further with 5% NaHCO, (3 times), IN HCI (3 times) and dried over Na₂S0₄. The solvent was filtered over celite and concentrated in vacuo. Addition of a small amount of hexane to the concentrate deposited a white solid (500 mg) corresponding to the title compound. Mass spectral analysis: M/Z = 472 (M+H)+.

∑∑_____l___ 6-BOC-9-tosylguanidino-l-nonen-5-one To a solution of the product from example 1 (600 mg, 1.3 mmol) in 25 ml of THF was added 10 equivalents of the Grignard reagent prepared from 4-bromo-l-butene (Note on preparation: 312 mg of Magnesium turnings (13 mmol) in 50 ml of anhydrous ether was treated with 1.75 g of 4-bromo- 1-butene dropwise to maintain a gentle reflux) after total consumption of the metal the Grignard solution was transferred by syringe under argon to the THF mixture. The entire THF mixture was quenched with aqueous NH4CI after TLC showed disappearance of starting material (TLC

was performed on Kieselgel^ 60F 254, Merck, glass plates) . The phases were separated and the organic phase was washed further with IN HCI and H2O, dried ( a2Sθ4> and evaporated under vacuum. Chromatography on silica gel (eluting with 4:1 EtOAc/hexane afforded a clear oil corresponding to the title compound. Mass spectral analysis M/Z = 469 (M+H)+.

EXAMPLE 3

5-BOC-4-oxo-8-tosylguanidinooctanoic acid The product from example 2 (2.5 g, 5.3 mmol) was dissolved in 50 ml of acetonitrile followed by addition of sodium periodate (8 g, 37.5 mmol) dissolved in 50 ml of water.

The whole mixture was treated with 100 mg of ruthenium chloride. After one hour vigorous stirring at room temperature, no starting material was observed by TLC.

The mixture was diluted with 100 ml of H2O and 100 ml of ether. The phases were separated and the aqueous phase was extracted further with ether. The combined organic extracts were washed with H2O, dried ( (Na2Sθ > and evaporated to dryness affording 1.5 g of a foam corresponding to the title compound. M/Z = 485 (M+H)+.

Example 4 Synthesis of 6-BOC-5-oxo-9-tosylguanidinononanoic acid. The title compound of this step was synthesized in a manner analogous to examples 1 to 3. Briefly the product from example 1 was reacted with a Grignard reagent prepared from Magnesium and 5-bromo-l-pentene. The resulting adduct isolated as an oil analogous to example 3 was subsequently treated with a combination of sodium periodate and ruthenium chloride to afford the title homologue of this example. M/Z = 499 (M+H)+.

Example 5

7-BOC-6-oxo-10-tosylguanidinodecanoic acid. The title compound of this example was prepared in a manner analogous to examples 1 to 4. In this example, the product from example 1 was reacted with the Grignard reagent prepared from Magnesium and 6-bromo-l-hexene. Following isolation of the adduct by silica gel chromatography as described in example 2, the adduct was reacted with sodium periodate and ruthenium chloride. Isolation of the product afforded the title compound as an oil. M/Z = 513 (M+H)+.

EXAMPLE 6

Ethyl; 4N-t-BOC-3-oxo-7-tosylguanidine thioheptanoate (mixed anhydride method) .

Formation of mixed euihydride: To a stirring solution of g(2.4mmol) of (L) -Nα-BOC-Arg(Nw)TOS)OH and 0.66 ml(0.48 mmol) of triethylamine in 15 ml of anhydrous tetrahydrofuran at -20°C was added 0.40ml(0.3 mmol) of isobutylchlorofor ate dropwise over 15 minutes. After 1 hour the mixture was diluted with 15 ml of ether -and the precipitated solid was filtered. The filtrate containing the mixed anhydride was stored at 0°C.

Meanwhile, to a stirred solution of diisopropylamine (3.4 ml, 24 mmol) in 25 ml of anhydrous ether under argon at 0°C was treated with one equivalent of N-But Li in THF dropwise over 30 minutes. After, the reaction mixture was cooled to -60°C and treated with 2.5 ml of ethyl thioacetate. After stirring at -60°C for 30 minutes, the mixture was treated with 6 g of MgBr2 etherate and stirred for an additional 30 minutes. Finally, this mixture was treated with the preformed mixed anhydride and stirring was continued for 5 hours until reaction was complete by HPLC.

The reaction mixture was treated dropwise with 6 M NH4CI and the phases were separated. The organic phase was diluted with 50 ml of EtOAc and extracted with IN HCI (3 X) , H2O (3 X) dried with a2S04 and evaporated under high vacuum affording the title compound as an oil M/Z = 515 (M + H)+.

EXAMPLE 7

Coupling of the thioester from example 6 to α-amino acid esters and deprotected amino acyl polystyrene resins.

The protected arginyl statone from example 6 (2 equivalents) was dissolved in CH2CI2 and added to a mixture of α-amino acid ester (1 equivalent) or polystyrene resin containing the growing polypeptide chain. To this mixture was added Cuprous Iodide (2 equivalents) and triethyl amine (2 equivalents) . The reaction is monitored by HPLC in the case of amino acid ester or by conventional ninhydrin test in the case of polystyrene bound peptides.

EXAMPLE 8

Preparation of the subunit of the synthetic spacer_pf formula II: -[NH-CH2-CH=CH-CH₂-(CO) 33-

The synthesis was modeled upon Cox M.T., Heston D.W. and Horbury J., "J. Chem. Soc. Chem. Comm. , 1980, 799-800 with major modifications. The complete process is outlined as follows:

a) Synthesis of trans-β-hydromuconic acid dimethyl ester. 22 g (153 mmol) of trans-β-hydromuconic acid was dissolved in 200 ml of benzene containing 500 mg of p-toluene sulfonic acid and 100 ml of methanol. The solution was maintained at reflux for 6 hours and treated with 100 ml of water. The phases were separated and the organic layer was extracted further with 5% aHCθ3 and H2O. After drying ( a2Sθ4) , the solvent was evaporated under vacuum and the residue was distilled (83-85°C) 0.5 mm Hg) affording 19 g of the title compound.

b) Synthesis of trans-β-hydromuconic acid monomethyl ester.

5 g (27.5) mmol) of the product from step a) was suspended in 100 ml of a solution of 0.1 M KH2PO4 followed by addition of 20 mg of pig liver esterase. The pH of the solution was maintained at 7 by dropwise addition of a solution of 1 M NaOH. Following the addition of 1 M NaOH corresponding to 1 mole equivalent of the diester, the solution was treated with charcoal, stirred for 5 minutes and filtered over celite. The filtrate was extracted with ether and the combined organic extracts were discarded. The aqueous phase was made acidic with 3 N HCI and reextracted with ether. The combined ethereal extracts were dried ( a2Sθ4) and evaporated in vacuo. The residue was distilled under reduced pressure (105-110°C, 0.5 mmHg) leaving 4 g of an oil corresponding to the title compound.

c) Synthesis of 4-methoxycarbonyl-2-dehydro butyl isocyanate.

1.22 g (7.3 mmol) of the mono ester from step b) was dissolved in 25 ml of benzene. 0.76 ml (8.7 mmol) of oxalyl chloride was added dropwise over 15 minutes and the solution was stirred vigorously for 3 hours. The solution was evaporated under vacuum. The residue dissolved in 10 ml of acetone was added to a precooled solution (0°C) of sodium azide 1 g in 20 ml 50% water/acetone. After 30 minutes the mixture was diluted with water (50 ml) and extracted 3 times with 20 ml portions of benzene. The combined organic extracts were dried (Na2S04) and filtered. The filtrate was heated in an oil bath at 80°C until no further nitrogen evolution was observed. The solvent was evaporated under vacuum and the residue distilled under reduced pressure (80-85°C, 0.5 mHg) affording 700 mg of the title compound.

d) Synthesis of 4-N-Butyloxycarbonyl-pent-3-en-oic acid. Tert-butanol 890 mg (12.2 mmol) was added to a solution containing the product from step c) (1 g, 6.1 mmol) in 25 ml of benzene. The whole solution was refluxed for 10 hours after which it was evaporated under vacuum. The residue was treated with pig liver esterase as described in step b) and work up as described in that step afforded 700 mg of the title compound.

The product from step d) is then used as a unit in the preparation of synthetic spacer II. These units are assembled to form spacer (II) using techniques that are well known to those skilled in the art.

EXAMPLE 9

Determination Of Thrombin Inhibitory Dissociation

Constants K_α

K_j was determined with a Hitachi F2000^* or a Perkin-Elmer^* fluorometer using the fluorogenic substrate Tos-Gly-Pro- Arg-AMC. The assay was carried out in 50 mM Tris-HCl buffer (pH 7.8) containing 0.1 M NaCl and 0.1% poly(ethylene glycol) 8000^* at room temperature. Buffer, substrate and inhibitor were mixed together and the reaction was initiated with the enzyme solution. The initial velocities were recorded at several inhibitor and substrate concentrations. The kinetic data (the steady- state velocity at various concentrations of the substrate and the inhibitors) of the competitive inhibition was analyzed using the methods described by Segel (1975) . Dixon and Lineweaver-Burk plots were used to determine K,,, V_M, and K. .

Binding is the establishment of the equilibra between enzyme, inhibitor, and enzyme-inhibitor complexes.

Doubling of Thrombin Time (dTT) assay dTT was determined in a fibrinometer (STAGO ST4^") . Cuvettes containing Tris buffer(75μl) , test inhibitor solution (50μl), fibrinogen solution (50μl, 0.12%), and a metal ball bearing were prewarmed and transferred to the test chamber of the fibrinometer. Thrombin solution (25μl) was added using a multipipette and the timer was started to activate the ball movement. When ball movement ceased indicating clotting had occurred, the time was recorded.

denotes Trademark A time versus inhibitor concentration curve was constructed and dTT values were extrapolated from the inhibitor concentration curve. The dTT is defined as the dose required to double the coagulation time compared to control without inhibitor compound.

Fibrin Clot Assay:

The assay was performed according to Krstenansky et al. , FEBS 21:10,1987). The wells in a 96 well microplate contained inhibitor test compound (lOOμl) in Tris buffer, human plasma (60μl) and the coagulation reaction was initiated by addition of human thrombin (50μl, 4.25nM). Optical densities of the wells were read at 405nM each minute for 60 minutes using a microplate reader (Dynatek^' MR5000) . A curve of optical density versus concentration of inhibitor was plotted and the IC₅₀ value was calculated at 30 minutes as the inhibitor concentration that gives half of the optical density of the control without inhibitor added.

Results for K,, dTT, and IC₅₀ (fibrin clot) are shown in Table I.

TABLE I

ND - not done

* - Literature value

Arterial Thrombosis Model

FeC^ Induced Carotid Arterial In-iurv Model

The FeCl₃ induced injury to the carotid artery in rats was induced according to the method described by Kurz, K.D.,

Main, R.W., Sandusky, G.E., Thrombosis Research 60; 269-

280, 1990 and Schumacher, W.A. et al. J. Pharmacology and

Experimental Therapeutics 267; 1237-1242, 1993.

Male, Sprague-Dawley rats ( 375- 410 g) were anesthetized with urethane { 1500 mg\kg ip) . Animals were laid on a 37°C heating pad. The carotid artery was exposed through a midline cervical incision. Careful blunt dissection was used to isolate the vessel from the carotid sheath. Using forceps, the artery was lifted to provide sufficient clearance to insert two small pieces of polyethylene tubing (PE-205^*) underneath it. A temperature probe (Physitemp^* MT23/3) was placed between one of the pieces of tubing and the artery. Injury was induced by topical application on the carotid artery above the temperature probe of a small disc (3 mm dia.) of Whatman^" No.l filter paper previously dipped in a 35% solution of FeCl3. The incision area was covered with aluminum foil in order to protect the FeCl₃ from degradation by light. The vessel temperature was monitored for 60 minutes after application of FeCl3 as an indication of blood flow. Vessel temperature changes were recorded on a thermister (Cole- Palmer^* Model 08533-41) .

The time between the FeCl₃ application and the time at which the vessel temperature decreased abruptly (>2.4°C) was recorded as the time to occlusion of the vessel. Inhibitor compounds were given either as an iv bolus (via jugular vein) (Table III) or an iv bolus followed immediately by an iv infusion (via femoral vein) (Table IV) . For iv bolus, the dose of inhibitor needed to double the time to occlusion in comparison to control animals in which injury was induced in the absence of inhibitor was determined. When compounds were given by infusion, a prestudy was carried out to determine the dose needed to increase the APTT by 2-4 times control values. The antithrombotic effect of this infusion dose was then determined.

Representative injured arteries were observed under a light microscope at 10X (Leica^*) for indication of degree of occlusion ( complete, partial, no occlusion) . The biological data is reported in Table II.

TABLE II

Effect of iv Bolus Doses of Compounds on FeCl_j-Induced Thrombosis iv bolus dose needed to double occlusion time

Compound No. (mcr/kg)¹

1 >1

2 >2

4 >2

5 >2

6 >4

7 >2

8 >2

9 >4

10 >4

11 >4

12 >4

13 >4

14 >4

15 >2

18 >

Heparin 200U/Kg 1

Control occlusion time was 19 ± 1 min (N=ll)

Activated Partial Thromboplastin Time Test (APTT

APTT and TT were determined on infused doses. In a prestudy to determine infused doses needed to increase APTT 2-4 times normal, blood samples (1.0 ml) were taken from a cannulated right jugular vein into sodium citrate buffer (lOOμl, 0.105M) at specific times: 0 (pre), 1, 5, 15, 30, 40, 50, 60 and 90 minutes. The blood samples were centrifuged and APTT and TT measured on plasma using a fibrinometer (ST4 BIO DIAGNOSTICA STAGO^*) . After establishing the dose needed to increase the APTT 2-4 times normal, the antithrombotic effect of this dose was tested. When the antithrombotic effect of this infusion was tested, blood samples were taken to determine APTT, pre-infusion times (-30min) , 30 minutes after starting the infusion, immediately before inducing thrombosis (0 time) , and 60 minutes after inducing thrombosis (60min) .

Cuvette-strips with a metal ball bearing in each cuvette were prewarmed to 37°C in an incubation chamber of the fibrinometer for at least 3 minutes. The plasma and the PTT Automate^* were combined in the cuvette for an incubation of 170 sec. After the incubation, the cuvettes were transferred in the test chamber area and the calcium chloride solution (0.025M) prewarmed at 37°C was added.

The results are reported in Table III.

TABLE III Effect of Intravenous Dose (bolus followed by infusion) of Thrombin Inhibitors needed to cause 2-4 fold increase in APTT on FeCl₃-induced Carotid Artery Thrombosis

Mean Time

Bolus Infusion APTT (sec)* to

Compound dose dose Time Thrombosis Occlusion No. ( σ/kσ) (uσ/kcr/min) induced (min) (min)

5 1.00 100 -30 0 60 54 19sec 50s 39s

Heparin 29

* Time Thrombosis induced (min) Platelet Aggregation Assay

Preparation of washed rat and human platelet Rat or human blood is collected into ACD anticoagulant. The blood is centrifuged and the supernatant, platelet rich plasma (PRP) is removed.

The PRP from rat blood is centrifuged and the platelet pellet is resuspended and washed twice with calcium free hepes Tyrode's buffer (pH 6.4) and resuspended in Tyrode's buffer containing calcium (pH7.4). The PRP from human blood is centrifuged and the platelet is resuspended and washed twice in Tyrode's buffer containing calcium and hepes (pH7.4) .

Platelet α-granule contents are radiolabeled with C¹⁴ serotonin (15-20μl of a 50 μCi/μl solution) in the first wash.

Measurement of Platelet Aggregation and Release of α- granule contents

The aggregation is measured in an aggregometer. 400μl of labeled platelet suspension is added to a prewarmed cuvette (37°C) . After 30 seconds of preincubation, inhibitor test compound (50μl) was added. After a further minute, buffer (hepes tyrode with albumin, glucose, Ca, and Mg) and agonist (thrombin or ADP) in a total volume of

50μl was added. Aggregation is recorded and the reaction stopped with 125μl of ice cold EDTA 16 mM in 1% formaldehyde. The sample is centrifuged at 12000g for 2-3 minutes in Eppendorf microcentrifuge. An aliquot (lOOμl) of supernatant is counted with a beta counter and the radioactivity is expressed as the percentage of the radioactivity in an aliquot of the total platelet suspension. Aggregation results are expressed as the percentage of the maximum aggregation in the absence of inhibitor compound.

IC₅₀ values are determined as the concentration necessary to inhibit maximum platelet aggregation or release in the absence of inhibitor by 50%.

Results are shown in Table IV.

Table IV

Inhibitory Effect of Compounds on Aggregation and Secretion from Washed Platelets from Rats and Humans

Claims

WE CLAIM:

1. A thrombin inhibitor of the formula (I) and pharmaceutically acceptable salts and derivatives thereof:

(I) A-L-E wherein

A is an inhibitor of the active site of thrombin;

L is a linker; E is a fibrinogen recognition exosite binding moiety comprising 3 to 5 amino acids having binding affinity for the fibrinogen recognition exosite of thrombin.

2. A thrombin inhibitor according to claim 1, wherein the E exosite portion is represented by formula (II) :

⁽ID -RpR Rs-CR m-CRs Y

wherein m and n are independently 0 or 1;

R_j is substituted or unsubstitμted Asp or another hydrophilic amino acid; R, is substituted or unsubstituted Phe or another hydrophobic amino acid;

R, is substituted or unsubstituted Glu or another hydrophilic amino acid; R₄ is substituted or unsubstituted Pro, pipecolic acid, 3-carboxytetrahydroisoquinoline, lie or a conservative substitution thereof;

R, is substituted or unsubstituted lie or e iother hydrophobic amino acid; and Y is a hydroxyl radical of the COOH terminus of the preceding amino acid, or an amine group -N(R_jt)(R,. > --• wherein R₂₀ and R₃ are each independently hydrogen, alkyl, or aryl.

3. A thrombin inhibitor according to claim 2, wherein the E exosite portion is selected from: Asp-Phe-Glu- Pro-Ile-NH_j, Asp-Phe-Glu-Pro-Ile-OH, Asp-Phe-Giu(OMe)- Pro-Ile-OH; and Asp-Tyr-Glu-Pro-Ile-NH_j.

4. A thrombin inhibitor according to claim 1, wherein the E exosite portion is represented by formula:

Asp-R,-Glu-(He)_β.,-Y wherein:

R, is Phe or Tyr or a conservative substitution thereof; and Y is OH or NH,.

5. A thrombin inhibitor according to claim 4, wherein the E exosite portion is selected from: Asp-Phe-Glu- OH, Asp-Tyr-Glu-OH, and Asp-Tyr-Glu-NK_j.

6. A thrombin inhibitor according to claim 1, wherein A is a group of formula (IV) :

(IV) wherein

X_j is a hydrophobic group; sulfonyl substituted with alkyl, aryl, or aralkyl; or carbonyl substituted with alkyl, aryl, or aralkyl; X, is an amino acid residue, alkylene, or X. is a bond when X, is either sulfonyl or carbonyl; X, is hydrogen, a straight or branched alkyl, aryl, or aralkyl provided that when X, is a bond, X₃ is not present;

X₄ is hydrogen, or alkyl; X_s is selected from the group consisting of alkyl, aryl, and aralkyl; and

X, is hydrogen, guanidinyl or amidinyl.

7. A thrombin inhibitor according to claim 6, wherein 7^ is selected from cyclohexylalanine, D- cyclohexylalanine, Phe, D-Phe, D-4F-Phe, or D-4ClPhe wherein the α-amino group is optionally acetylated or benzoylated and X, and X₄ are both hydrogen.

8. A thrombin inhibitor according to claim 6, wherein X₃ is selected from valine, pipecolic acid and proline and X, and X₄ are both hydrogen.

9. A thrombin inhibitor according to claim 6, wherein X₅ is phenylmethyl, phenylethyl, ethylene, butylene, or propylene.

10. A thrombin inhibitor according to claim 6, wherein X, is guanidinyl.

11. A thrombin inhibitor according to claim 1, wherein A is selected from: Bzs-Arg-(D-Pip) ; dansyl-Arg-(D- Pip) ; dansyl-Arg-(L-Pip) ; dansyl-Nle-(D-Pip) ; (D- Phe)-Arg-(D-Pip) ; Fmoc-Arg-(D-Pip) ; dansyl-Arg-(D- Tic); dansyl-(D-Arg)- (D-Pip) ; dansyl-Phe-(D-Pip) ; dansyl-Cha-(D-Pip) ; (D-Cha)-Arg- (D-Pip) ; α-naphthyl sulfonyl-Arg-(D-Pip) ; β-naphthyl sulfonyl-Arg-(D- Pip) ; 4-tert-Butyl-benzene sulfonyl-Arg- (D-Pip) ; dansyl-Arg-(D-Cha) ; dansyl-Arg-Acha; phenyl ethyl sulfonyl-Arg-(D-Pip) ; β-dihydro-anthracenyl-β- sulfonyl-Arg- (D-Pip) ; (+)-camphorsulfonyl-Arg- (D- Pip) ; 4-bromobenzenesulfonyl-Arg- (D-Pip) ; 2,4,6 triisopropylbenzenesulfonyl-Arg- (D-Pip) ; Ac(D-Phe) - Pro-Arg-; Ac(D-Phe) -thioPro-Arg-H; Ac (D-Cha) -Pro-Arg- ; (D-Cha)-Pro-Arg-; (D-Phe)-Pro-Arg-; succinyKD- Phe)-Pro-Arg-; and alpha-N-(Ac) (D-Phe) -Pro-Arg_-.

12. A thrombin inhibitor according to claim 1, wherein L is a straight or branched carbon chain optionally interrupted by one or more carbonyl or heteroatom selected from N, 0 and S; said chain optionally substituted by one or more hydroxy, oxygen, sulfur, amino, alkyl, alkoxy, aryl, and aralkyl groups optionally substituted by hydroxy, amino, hydroxy, imidazole or halogen groups.

13. A thrombin inhibitor according to claim 1, wherein L is amino acids 49 to 54 of native hirudin.

14. A thrombin inhibitor according to claim 1, wherein L is a group of formula (V) :

(V) -(CH₂)_n-(CO)_m-Z wherein m is 0 or 1; n is an integer from 0 to 4;

Z is a straight or branched carbon chain that can be interrupted by carbonyl or a heteroatom selected from O, N or S; or substituted by one or more alkyl, alkoxy, aryl, aralkyl, imidazol, carbonyl, or halogen groups.

15. A thrombin inhibitor according to claim 14, wherein Z is a group of formula (VI) :

(VI) -[NH-R,-CO]_m-(R_l0-) wherein is an integer ranging from 1 to 4; n is an integer ranging from 0 to 4;

R, is a hexapeptide, or a saturated or unsaturated alkylene chain of 18 carbon atoms or less; and R₁₀ is one or more amino acids.

16. A thrombin inhibitor according to claim 14, wherein L is selected from: (12-aminododecanoic acid) -4- aminobutyric acid)-; (1 -aminododecanoic acid) -6- aminocaproic acid) ; (8-aminocapylic acid) -4- aminobutyric acid)-; (12-aminododecanoic acid) - asparagyl-glycyl) ; (4-aminobutyric acid-glycyl) ; (5- amino valeric acid) -glycyl); (6-aminocaproic acid) - glycyl) ; (7-aminoheptanoic acid) -glycyl) ; (8- a inocaprylic acid) -glycyl) ; (12-aminododecanoic acid); (11-aminoundecanoic acid) -glycyl); (Glycyl) - 12-aminododecanoic acid); (12-aminododecanoic acid)- glycyl) ; (β-Alanyl-glycyl-glycyl-5-aminovaleric acid); (CH₂) χ_ (CO) -QSHNDG; CH₂ (CO) -GSHNDG; (CH₂)4(CO)-[NH-CH₂-CH=CH-CH2-(CO) ],_.,; CH₂N(COCH₃)CH₂(CO)-(Gly) -; CH₂-(4-pyridylacetyl) - (Gly)₂_₄-; CH₂-(4-pyridylacetyl)-Abu-Gly-; CH₂-(4- pyridylacetyl) -Ava-Gly-; CH₂- (4-pyridylacetyl) -Aca- Gly-; CH₂-(4-pyridylacetyl)-Aha-Gly-; CH₂SCH₂(CO)- (Gly)₄-; CH₂SCH₂(CO)-(Gly) -; CH₂-(4- pyridylacetyD-NHtCH_jJ_jCO-Gly-; CH₂-(4- pyridylacetyD-NHtCH_jJ_j._jCO-; (CH₂)₄(CO)- [NH(CH₂)₄(CO)]₂-; and (CH₂)₄ (CO) -[NH(CH₂)₄ (CO) ] -NG.

17. A thrombin inhibitor according to claim 1 selected from:

(1) alpha-(4-t-butylbenzenesulphonyl) -Arg- (R) -Pip- Ada-Abu-DFEPI-OH;

(2) alpha-N-(4-t-butylbenzenesulphonyl) -Arg- (R) -Pip- Ada-Asn-Gly-DFEPI-OH; (3) alpha-N-(4-t-butylbenzenesulphonyl) -Arg- (R) -Pip- Ada-Abu-DF-Glu(OMe) -PI-OH;

(4) alpha-N-(4-t-butylbenzenesulphonyl)-Arg- (R) -Pip- Ada-Asn-Gly-DFE-OH; (5) H-(R)-Phe-Pro-AOGD-(5-aminovaleryl)₂-DFEPI-OH;

(6) Ac-(R)-Phe-Pro-AOGD-(5-aminovaleryl)₂-DFEPI-OH;

(7) H-(R)-Phe-Pro-AOGD-(5-aminovaleryl)₂-DFE-OH;

(8) H- (R) -Phe-Pro-AOGD- (5-aminovaleryl)₂-DFEI-OH;

(9) H- (R) -Phe-Pro-AOGD- (5-aminovaleryl)₂-DF-Glu(OMe) - PI-OH;

(10) Ac-(R) -Phe-Pro-AOGD- (5-aminovaleryl)..-DF- GlulOMe) -PI-OH;

(11) H- (R)-Phe-Pro-AOGD- (5-aminovaleryl)₂-DF-N-Methyl- Glu-PI-OH; (12) Ac-(R)-Phe-Pro-AOGD- (5-aminovaleryl)₂-DF-N- Methyl-Glu-PI-OH; (13) H- (R) -Phe-Pro-AOGD- (5-aminovaleryl)_j-DFEPI-NH,; (14) H-(R) -CyclohexylAla-Pro-AOGD- (5-aminovaleryl)₂- DFEPI-N^; (15) H-(R)-CyclohexylAla-Pro-AOGD-(5-aminovaleryl)₂- DYE-NH,;

(16) H- (R)-CyclohexylAla-Pro-AOGD- (5-aminovaleryl)₂- DYE-OH;

(17) H-(R)-CyclohexylAla-Pro-AOGD- (5-aminovaleryl)₂- DFE-OH;

(18) H-(R)-CyclohexylAla-Pro-AOGD- (5-aminovaleryl) -G- DYEPI-NH_j,- and

(19) H-(R) -CyclohexylAla-Pro-AOGD-(5- aminovaleryl)₂-DYEPI-NH_j.

18. A composition useful for the treatment of thrombotic disorders comprising a thrombin inhibitor according to claim 1 in combination with a pharmaceutically acceptable carrier.

19. A composition useful for the treatment of thrombotic disorders comprising a thrombin inhibitor according to claim 2 in combination with a pharmaceutically acceptable carrier.

20. A method for the treatment or prophylaxis of vascular diseases related to thrombosis which comprises administering to a patient an effective amount of a composition according to claim 17.