WO1999013062A1 - Factors vii fragments and analogs thereof and their use in the treatment of blood clotting disorders - Google Patents

Abstract

This invention provides a compound which is capable of interacting with the internal receptor in the catalytic domain of FIXa or FX defined by the residues Ile?290, Ala291, Asp292, Tyr293, Thr294, Glu374¿, and Phe378 in FIXa and Leu?300, Pro301, Glu302, Trp305, Ala306, Lys385¿ and Phe389 in FXa or the ligand defined by residues Cys?95 to Cys99¿ in FIXa or Cys?96 to Cys100¿ in FXa to prevent the formation of a functional FVIIIa/FIXa complex or FXa/FV complex respectively.

Description

FACTORS VII FRAGMENTS AND ANALOGS THEREOF AND THEIR USE IN THE TREATMENT OF BLOOD CLOTTING DISORDERS

The present invention is concerned with reagents and compositions thereof which reduce blood clot formation.

Blood clotting relies upon a series or cascade of activating reactions to produce the ultimate fibrin clot . The cascade leading to fibrin formation may be triggered initially in two different ways - by contact with abnormal surfaces (the "intrinsic pathway") or by traumatization of blood vessels which causes secretion of the lipoprotein known as "tissue factor" or TF (the "extrinsic pathway") . The present invention is primarily concerned with the extrinsic blood clotting pathway .

Coagulation factor VII (FVII) is a vitamin Independent plasma glycoprotein of M_r = 50,000 consisting of a single polypeptide chain with 406 amino acid residues (F.S. Hagen et al . , Proc . Natl . Acad. Sci. USA, 1986, 83: 2412-2416; P.J. 0 'Hara et al . , Proc. Natl. Acad. Sci. USA, 1987, 84: 5158-5162) and is involved in the extrinsic blood clotting pathway. The zymogen form is converted to the fully enzymatically active form FVIIa by factor X and other coagulation proteases through hydrolysis of a single peptide bond Arg¹⁵²-Ile¹⁵³. This results in formation of an enzyme with two polypeptide chains which are held together by a single disulphide bond. The light chain of 152 amino acid residues contains at its amino terminal part the y- carboxy-glutamic acid (Gla) domain, followed by two epidermal growth factor-like domains (EGF-1 and EGF-2) . The heavy chain consists of 254 residues and contains the trypsin-like catalytic domain.

Activation of FVII to FVIIa has been reported to be markedly enhanced when FVII is bound to its co-factor tissue factor (TF) (see Nemerson, Semin. Hematol. 29(3) : 170-176 (1992)). Yamamoto et al . have also suggested that conversion of FVII to FVIIa may be autocatalytic (see J. Biol. Chem. 267 (27) : 19089-19094 (1992)).

The function of FVIIa is activation of factor X (FX) by complexation with tissue factor (TF) in the presence of Ca²⁺ on a phospholipid membrane surface. Activation of FX to FX_a leads to formation of blood clots by the extrinsic pathway. Additionally, the complex FVII_a/TF can activate factor IX to factor IX_a and lead to clotting through the intrinsic pathway.

TF is an integral membrane protein which appears on many cell types. However, cells which constitutively express TF, for example the muscle cells of vessels intima, are not normally exposed to blood (see Edgington et al., Thromb. Haemostas . 66 (1) : 67-69 (1991)). Thus initiation of the extrinsic blood clotting pathway appears to require either the disruption of blood vessel walls (see Almus et al . , Blood 7£.: 354-360 (1990)) and/or activation of endothelial cells or monocytes to express TF (see Edwards et al., Blood

359-370 (1979) and Bevilaqua et al . , PNAS USA £2: 4533-4537 (1986)). Disruption of the blood vessel wall may occur due to fissuring of an atherosclerotic plaque which exposes tissue macrophages and smooth muscle cells to the blood (see Wilcox et al . , PNAS USA : 2839-2843 (1989)). TF may also be exposed following injury to blood vessels during thrombolytic therapy, surgery for grafting, mechanical restoration of vessel patency or other similar techniques. On the other hand, TF expression in endothelial cells or in monocytes may be induced during sepsis due to production of tumour necrosis factor-α or interleukin-1 (see Edwards et al . , supra and Gregory et al., J. Clin. Invest. 7£: 2440-2445 (1985)).

Activation of the extrinsic pathway for blood clot formation is the primary event leading to fibrin formation (see Weiss et al . , Blood 21: 629-635 (1988) and Weiss et al . , Blood 23.: 968-975 (1989)) and is thus of prime importance in the pathogenesis of arteriosclerotic lesions and in reocclusion and restenosis following endarterectomy .

Blood coagulation in vivo, in both normal hemostatis and different thrombotic diseases, is generally considered to be initiated by the factor VIIa/TF complex. Under basal conditions, i.e. no thrombosis or provocative stimuli, the FVII/TF pathway is responsible for the generation of trace amounts of FIXa and FXa and thrombin found in plasma. These levels are not sufficient to precipitate a coagulation event due to the presence of natural coagulation inhibitors such as TFPI or AT- III but the levels are necessary to maintain the coagulation system in a state which can rapidly respond to a provocative stimuli.

Upon vascular injury, TF which is not normally in contact with blood becomes available to FVIIa which leads to generation of levels of FIXa, FXa and thrombin above what can be effectively handled by natural inhibitors. This in turn leads to activation of FVIII and FV which are essential cofactors for FIXa and FXa respectively and to the activation of platelets. The latter is a prerequisite for assemblage of FIXa/FVIIIa complex and FXa/FVa complex. The process culminates in thrombin generation and subsequently cleavage of fibrinogen to insoluble fibrin. Thus a coagulation/thrombotic event is precipitated.

Effective therapeutic agents able to intervene in the activation of this pathway have not been readily available, despite demand (see Shepard, TIBTECH j3: 80-85 (1991) ) .

One proposed way of providing potential therapeutic agents capable of preventing the primary event in blood clot formation through the extrinsic pathway would thus be to identify peptides derived from the primary structure of FVII which are capable of inhibiting the complex formation between FVII, TF and FX, which is necessary for FX activation.

Peptides corresponding to sequence portions between the Gla and EGF-l domains as well as from the catalytic domain, were disclosed in WO91/07432 (Board of Regents, The University of Texas System) as being useful in the treatment of blood clotting disorders. Although inhibition of the FVIIa/TF complex is discussed, those peptides which cause an effect do so by inhibition of the Gla function. Such peptides are therefore unspecific in their action since other physiological proteins have Gla domains. Hence the function of proteins with Gla domains would be disturbed by administration of the disclosed peptides.

A particular peptide from the EGF-2 domain was disclosed in WO90/03390 (Corvas, Inc.) as having potential uses in preventing the formation of the fully formed FVIIa/TF complex. The sequence -SDHTGTKRSCR- which is located at amino acids 103-113 of FVII or analogues thereof is said to inhibit the cascade reaction initited by FVIIa/TF complex. Other regions, namely from amino acids 50 to 101 and 114 to 127 in the EGF-2 domain were shown to be inactive in the inhibition of FX activation by FVIIa/TF.

However, in W096/18654 (Nycomed Pharma AS) it was shown that the sequence -SDHTGTKRSCR- is in fact a poor inhibitor and that peptides possesing the FVII amino acid sequence 82-128 when in linearised form are much more active inhibitors.

In WO95/00541 (Nycomed Pharma AS) a number of small peptides from the EGF-l and EGF-2 domains are disclosed which may be useful in treating blood clotting disorders. Particularly, peptides corresponding to the amino acids 91 to 104 and 114-127 of the FVII sequence are said to be active.

In W096/18653 (Nycomed Pharma AS) a specific cyclic peptide is disclosed, disulphide-cyclo- [H-Cys-Glu-Gln- Tyr-Cys-OH] , which is said to inhibit the formation of an FVIIa and TF complex.

It is now proposed that an alternative to inhibiting FVII/TF complex formation would be to prevent the formation of a functional FXa/FVa complex or FIXa/FVIIIa complex.

The function of the FVIIa/TF complex is to initiate coagulation whereas the function of FIXa/FVIIIa complex and the FXa/FVa complex is to propagate coagulation. In particular, FIXa is important in the propagation process since it is the FIXa/FVIIIa complex that generates high levels of FXa which in turn activates thrombin. The FXa/FVa complex is the sole physiological activator of prothrombin to yield thrombin. Thus inhibitors of FIXa and FXa are potential alternatives to currently available anticoagulants for the treatment and prevention of thrombotic disorders.

Comparatively little is known about the mechanism and sites of interaction at the^' molecular level. However, it has been proposed that the procoagulant proteases FVIIa, FIXa and FXa have highly similar architectures and mechanisms of actions. Each is composed of the same domains; an amino-terminal carboxy y-glutamic acid-rich domain (Gla) , two epidermal growth factor (EGF) like domains and a trypsin-like catalytic domain .

The factors attach to activated cell membranes in an elongated form via their Gla domain, and complex assemblage with the protein cofactor occurs on the cell membrane. In the absence of cofactor, FVIIa, FIXa and FXa exhibit very low enzyme activity. Binding of the cofactor positions the active site and the movement of the catalytic domain dampens through interdomain interactions and interactions with the cofactor. This stabilisation is a requirement for enzyme activity.

It is also a common property of FVIIa, FIXa and FXa that upon binding to the cofactor, the EGF-2 like domain become sandwiched between the catalytic domain and the cofactor. Structural and mutational data show that the C-terminal part of the loop in the EGF-2 like domain interacts with the cofactor and the N-terminal part of the same loop, residues 95-99 in FIXa and 96-100 in FXa, makes close contact with the catalytic domain, (See figure 1) .

Basically, this is a ligand/receptor interaction. The EGF-2 loop structure is the ligand and the internal receptor in the catalytic domain is defined by residues Leu²⁶³, Pro²⁶⁴, Glu²⁶⁵, Phe²⁶⁸, Ser²⁶⁹, Tyr³⁵⁷ and Arg³⁵³ in FVIIa and He²⁹⁰, Ala²⁹¹, Asp²⁹², Tyr²⁹³, Thr²⁹⁴, Glu³⁷⁴, and Phe³⁷⁸ in FIXa and Leu³⁰⁰, Pro³⁰¹' Glu³⁰², Trp³⁰⁵, Ala³⁰⁶, Lys³⁸⁵ and Phe³⁸⁹ in FXa.

An important feature is the aromatic stacking involving the side chains of Tyr¹⁰¹ in the EGF-2 like domain and Phe²⁶⁸, Tyr³⁵⁷ in the catalytic domain in FVIIa; Phe⁹⁸, Tyr²⁹³ and Phe³⁷⁸ in FIXa and Phe⁹⁹, Trp³⁰⁵, Phe³⁸⁹ in FXa. A consequence of the vital role played by the EGF- 2 domain is the importance of the internal receptor in the catalytic domain.

This finding is supported by the fact that the TF exon 3 region - i.e. the N-terminal part of TF which therefore interacts with the EGF-2 and catalytic domains - is essential for biological activity.

Also, the finding in WO 95/00541 (Nycomed Pharma) that the biological activity in the cyclic dodecamer peptide could be localised to residues 99-101 in FVII also supports the fact that the EGF-2 catalytic domain interaction is important in the formation of a viable FVII/TF complex rather than the interaction between EGF- 2 and TF . Also, a cyclic pentamer -CEQYC-, mimicking the structure of FVII at amino acids 98-102, and an equivalent cyclic pentamer -CDQFC-, mimicking the structure of FX at amino acids 96-100, both inhibit FX activation. A cyclic pentamer with a random sequence -CVNEC- was found to be non-inhibitory (See figure 2) .

Moreover, patients who are homozygous for Gin¹⁰⁰ Arg mutation in the FVII:EGF-2 domain have significantly less specific enzyme activity and a reduced thrombus formation. In the native protein Gin¹⁰⁰ makes hydrophobic contact with His¹¹⁵ and Tyr¹¹⁸ in the EGF-2 domain and Thr²⁶⁷ in the catalytic domain. In the mutant protein the side chain of Arg¹⁰⁰ is incorrectly positioned and disturbs the important interaction between EGF-2 and the catalytic domain.

Accordingly, the EGF-2 domain appears critical in the coagulation of blood.

It has now been surprisingly found that any compound, peptide or non-peptide, natural or synthetic which is capable of interacting with the internal receptor in the catalytic domain of FIXa or FX would interfere with the conformation of the catalytic domain disturbing the interaction with the EGF-2 domain. Interfering with this interdomain docking site would allow for development of low molecular weight antithrombotic drugs, an approach that may circumvent difficulties with side effects that will always be problematic in an active site strategy.

Thus, viewed from one aspect the invention provides a compound which is capable of interacting with the internal receptor in the catalytic domain of FIXa or FX defined by the residues He²⁹⁰, Ala²⁹¹, Asp²⁹², Tyr²⁹³, Thr²⁹⁴, Glu³⁷⁴, and Phe³⁷⁸ in FIXa and Leu³⁰⁰, Pro³⁰¹' Glu³⁰², Trp³⁰⁵, Ala³⁰⁶, Lys³⁸⁵ and Phe³⁸⁹ in FXa or the ligand defined by residues Cys⁹⁵ to Cys⁹⁹ in FIXa or Cys⁹⁶ to Cys¹⁰⁰ in FXa to prevent the formation of a functional FVIIIa/FIXa complex or FXa/FV complex respectively.

Viewed from a further aspect the invention provides a pharmaceutical composition containing one or more compounds capable of interacting with the internal receptor in the catalytic domain of FIXa or FX defined by the residues He²⁹⁰, Ala²⁹¹, Asp²⁹², Tyr²⁹³, Thr²⁹⁴, Glu³⁷⁴, and Phe³⁷⁸ in FIXa and Leu³⁰⁰, Pro³⁰¹' Glu³⁰², Trp³⁰⁵, Ala³⁰⁶, Lys³⁸⁵ and Phe³⁸⁹ in FXa or the ligand defined by residues Cys⁹⁵ to Cys⁹⁹ in FIXa or Cys⁹⁶ to Cys¹⁰⁰ in FXa to prevent the formation of a functional FVIIIa/FIXa complex or FXa/FV complex respectively.

Viewed for a yet further aspect, the invention provides the use of a compound capable of interacting with the internal receptor in the catalytic domain of FIXa or FX defined by the residues lie²⁹⁰, Ala²⁹¹, Asp²⁹², Tyr²⁹³, Thr²⁹⁴, Glu³⁷⁴, and Phe³⁷⁸ in FIXa and Leu³⁰⁰, Pro³⁰¹' Glu³⁰², Trp³⁰⁵, Ala³⁰⁶, Lys³⁸⁵ and Phe³⁸⁹ in FXa or the ligand defined by residues Cys⁹⁵ to Cys⁹⁹ in FIXa or Cys⁹⁶ to Cys¹⁰⁰ in FXa to prevent the formation of a functional FVIIIa/FIXa complex or FXa/FV complex respectively..

Viewed from a still yet further aspect, the invention provides the use of a compound capable of interacting with the internal receptor in the catalytic domain of FIXa or FX defined by the residues lie²⁹⁰, Ala²⁹¹, Asp²⁹², Tyr²⁹³, Thr²⁹⁴, Glu³⁷⁴, and Phe³⁷⁸ in FIXa and Leu³⁰⁰, Pro³⁰¹' Glu³⁰², Trp³⁰⁵, Ala³⁰\ Lys³⁸⁵ and Phe³⁸⁹ in FXa or the ligand defined by residues Cys⁹⁵ to Cys⁹⁹ in FIXa or Cys⁹⁶ to Cys¹⁰⁰ in FXa for use in the manufacture of a medicament for the prevention of the formation of a functional FVIIIa/FIXa complex or FXa/FV complex respectively .

Viewed from another aspect the invention provides a method of treatment of the human or animal body to combat or prevent blood clotting disorders said method comprising administering to said body one or more compounds capable of interacting with the internal receptor in the catalytic domain of FIXa or FX defined by the residues He²⁹⁰, Ala²⁹¹, Asp²⁹², Tyr²⁹³, Thr²⁹⁴, Glu³⁷⁴, and Phe³⁷⁸ in FIXa and Leu³⁰⁰, Pro³⁰¹' Glu³⁰², Trp³⁰⁵, Ala³⁰⁶, Lys³⁸⁵ and Phe³⁸⁹ in FXa or the ligand defined by residues Cys⁹⁵ to Cys⁹⁹ in FIXa or Cys⁹⁶ to Cys¹⁰⁰ in FXa to prevent the formation of a functional FVIIIa/FIXa complex or FXa/FV complex respectively.

The compounds capable of interacting with the internal receptor in the catalytic domain or its ligand can have a wide variety of structures and will be readily determined by the person skilled in the art. In particular the compounds must be capable of interacting with the internal receptor or its ligand in order to prevent the formation of a functional FVIIIa/FIXa complex or FXa/FV complex.

Non-peptide compounds suitable for use according to the invention are those with complementary conformations to the target receptor sites or its ligand and can be readily prepared by conventional combinatorial chemical approaches .

Preferred compounds according to the invention are linear peptides of the general structure 1, disulphide- cyclic peptides of the general structure 2, lactam- cyclic peptides of the general structures 3a ("head-to- tail" amide bond) , 3b ("side chain-to-tail" amide bond) and 3c ("head-to-side chain" amide bond), as well as cyclic peptides of the general structure 4. In these structures Saa, Vaa, Waa, Xaa, Yaa and Zaa denote specified (see below) amino acid residues ( e . g. -NH-CH₂- CO- for a Gly residue) , such residues being of any stereochemistry and optionally containing Cα- and/or No - methyl substituents additional to the amino acid side chain:

R^Vaa-Waa-Xaa-Yaa-Zaa-R² (1)

R^Saa-Waa-Xaa-Yaa-Saa-R² (2) t I

— ( (Vaaaa--Wwaaaa--Xλaaaa--Yιaaaci--Z_ιactaa) _a——jr (3a)

R^x-Vaa-Waa-Xaa-Yaa-Zaa-τ (3b)

Vaa-Waa-Xaa-Yaa-Zaa-R² (3c) L i o

\

C-Waa-Xaa -Yaa-NH

R 3³ - C I A C I - R 3³ ( 4 )

Specifically, in 1-4 one of the three residues Waa, Xaa and Yaa is an amino acid residue from group I below. The remaining two residues are from groups II and III, respectively, or they are both from group III.

Group I denotes aromatic amino acid residues, including Tyr, Aph and Phe. Also included are side-chain constrained analogues of Tyr such as Tic (OH) and Hat. Aromatic amino acids containing a phenol function may be O-alkylated.

Group II denotes amino acid residues containing side-chain functional groups which are ionized under physiological conditions, including Glu, Asp, Dap, Dab, Cit, Orn, Lys and Arg. Also included are side-chain constrained analogues of Asp and Glu, such as 1- aminocyclo-pentane-1, 3-dicarboxyl and 1- aminocyclohexane-1, 3-dicarboxyl .

Group III denotes neutral and hydrophobic amino acid residues, including Gin, Asn, Ser, Thr, Leu and Nle.

Specifically excluded from the definition of 1 are the peptides H-Ala-Glu-Gln-Tyr-Val-OH and Ac-Ala-Glu- Gln-Tyr-Val-NH₂.

The two residues Saa in 2 are amino acid residues containing thiol functions in the side chain, these thiol functions being linked intramolecularly through a disulphide bond. Thus the two residues Saa may independently correspond to Cys, homocysteine or penicillamine .

Specifically excluded from the definition of 2 are the peptides disulphide- cyclo- [H-Cys-Glu-Gln-Tyr-Cys-OH] and disulphide- cyclo- [Ac-Cys-Glu-Gln-Tyr-Cys-NH₂] .

The residues Vaa and Zaa in 1 and 3 may independently correspond to hydrophobic amino acid residues, including Gly, Ala, βAla, Abu, Val, norvaline, Pro, aminoalkanoyl (4-8 C atoms) and o- , - or p- (aminomethyl) benzoyl . Furthermore, one or both of Vaa and Zaa may correspond to Asp, Glu, Dap, Dab, Orn or Lys. In the case of general structures 3b and 3c the terminal residues participating in a "side chain-to- tail" or "head-to-side chain" amide bond (Vaa and Zaa, respectively) correspond exclusively to amino acids residues containing side-chain amine (3b) or carboxyl (3c) groups .

The substituent R¹ in 1-3 may correspond to the following: H-, acetyl, R⁶-Gly-, R⁶-Gly-Gly- , R⁶-Asn-Gly- Gly-, R⁶-Dab-Gly-Gly-, Glp-Gly-Gly- , succinyl-Gly-Gly- or amidosuccinyl-Gly-Gly-, where R⁶ may be H- or acetyl. The substituent R² in 1-3 may correspond to -OH, -NH₂, -Ser-R⁷, -Ser-Asp-R⁷, -Ala-Asp-R⁷, -Ser-Abu-R⁷, -Ser-Lys-R⁷, -Ser-Dab-R⁷ or -Se -Ile-R⁷ , where R⁷ may be - OH or -NH₂.

The value of n in 3a may be 1 or 2.

In structure 4 , R³ may be -H or methyl . R⁴ may be - H, methyl, -NH₂, -NH-Ac or -NH-R¹, whereas R⁵ may be -H, methyl -COOH, -COOMe, -CONH₂ or -CO-R².

The moiety A in 4 may correspond to a saturated or unsaturated carbon chain of length C^Cg, optionally interrupted by a phenyl group or a heteroatom (N, O or S) . Both the chain and the optional phenyl group may bear (Ci-C -alkyl, amino, hydroxyl, carboxyl or carboxamide substituents.

Preferred compounds according to the above definitions are as follows: Linear peptides :

H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Asp-OH

Succinyl-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Asp-OH

H-Glp-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Asp-OH

H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ala-Asp-OH

H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Lys-OH H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Abu-OH H-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Dab-OH H-Abu-Glu-Gln-Tyr-Abu-Ser-Dab-OH H-Abu-Glu-Gln-Tyr-Abu-OH H-Nle-Glu-Gln-Tyr-Nle-OH Disulphide -cyclic peptides : disulphide - cycl o- [H-Cys -Asp -Gln- -Phe-Cys -OH] disulphide-cyclo- [H-Cys -Glu -Gin--Tyr-Cys -Ser-OH] disulphide- cycl o- [H-Cys- -Glu -Gln--Tyr-Cys -Dab-OH] disulphide-cyclo- [H-Cys- •Orn -Gln-^•Tyr-Cys -OH] disulphide- cyclo- [H-Cys- ^■Nle -Gln-^■Tyr-Cys -OH] disulphide-cyclo- [H-Cys- •Glu -Nle- Tyr-Cys -OH] disulphide- cyclo- [H-Cys- Glu -Gln- Tyr (Me) -Cys-OH] disulphide-cyclo- [H-Cys- Aph-^•Asp- Leu-Cys -OH] disulphide-cyclo- [H-Cys- Tyr-^■Glu- Gln-Cys -OH] disulphide-cyclo- [H-Cys- Cit- Tyr- Thr-Cys -OH] disulphide- cyclo [H-Cys- Glu- Gln- Hat (Me) -Cys-OH] Lac 'tarn- cyclic peptides : cyclo- [Abu-Glu-Gln-Tyr-Abu] cyclo- [βAla-Glu-Gln-Tyr-βAla] cyclo- [ (3-Amb) -Glu-Gln-Tyr- (3-Amb) ] cyclo- { [ (3-Amb) -Glu-Gln-Tyr- (3-Amb) ]₂} cyclo- [Abu-Glu-Gln-Tyr-Asp] cyclo- [ (5-Ava) -Glu-Gln-Tyr-Gly] cyclo- [Dap-Glu-Gln-Tyr-Asp] cyclo- [Gly-Glu-Gln-Tyr-Gly] cyclo- [ (Gly-Glu-Gln-Tyr-Gly) ₂]

Results with compound examples from the present invention and with the best previously disclosed peptide are summarised in the following table: Peptide compound IC₅₀ (μM)^a

H-Asn-Gly-Gly-Cys (Acm) -Glu-Gln-Tyr-Cys (Acm) -Ser-Asp-OH 260

H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Asp-OH 130

H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Lys-OH 3

H-Abu-Glu-Gln-Tyr-Abu-OH 11 disulphide- cyclo- [H-Cys-Glu-Gln-Tyr-Cys-Ser-OH] 9 disulphide- cyclo- [H-Cys-Orn-Gln-Tyr-Cys-OH] 12 disulphide- cyclo- [H-Cys-Aph-Asp-Leu-Cys-OH] 14 cyclo- [Gly-Glu-Gln-Tyr-Gly] 83 cyclo- { [ (3-Amb) -Glu-Gln-Tyr-Gly) ] ₂} 15 cyclo- [Abu-Glu-Gln-Tyr-Abu] 19

The inhibition of FVII function by a number of synthetic peptides was determined in a two-stage chromogenic assay as described (Kumar et al . , J. Biol . Chem . , 1991, 266:915-21). This consisted in incubation of peptide dilutions with mixtures of TF, FVIIa and FX, followed by kinetic measurement of FXa amidolysis of a chromogenic substrate. The concentrations of peptides required to inhibit 50 (IC₅₀) of FX activation was determined.

It can be seen from the results above that the peptides of the present invention are up to several tenfold more potent inhibitors than the previously disclosed peptide H-Asn-Gly-Gly-Cys (Acm) -Glu-Gln-Tyr- Cys (Acm) -Ser-Asp-OH, despite the fact that they are structurally simpler by virtue of having lower molecular mass and, in some cases, by virtue of possessing fewer chiral atoms. Furthermore, those cyclic peptides of the present invention not containing disulphide bonds can be expected to possess enhanced physiological stability due to the fact that they cannot be processed by amino- and carboxypeptidases .

Salts of the peptides of the invention include physiologically acceptable salts such as acid addition salts, for example the hydrochlorides . The pharmaceutical compositions comprising the peptides of the invention and/or salts thereof may be administered together with any physiologically acceptable excipient known to those skilled in the art. Examples of excipients include water and oil .

The compositions according to the invention may be presented, for example in a form suitable for oral, nasal, parenteral or rectal administration.

As used herein, the term "pharmaceutical administration" includes veterinary applications of the invention.

The compounds according to the invention may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, powders, capsules or sustained release forms. Conventional pharmaceutical excipients as the usual methods of production may be employed for the preparation of^" these forms. Tablets may be produced, for example, by mixing the active ingredient or ingredients with known excipients, such as for example with diluents, such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatine, lubricants such as magnesium stearate or talcum, and/or agents for obtaining sustained release, such as carboxypolymethylene, carboxymethylcellulose, cellulose acetate phthalate, or polyvinylacetate .

The tablets may if desired consist of several layers. Coated tablets may be produced by coating cores, obtained in a similar manner to the tablets, with agents commonly used for tablet coatings, for example, polyvinyl pyrrolidone or shellac, gum arabic, talcum, titanium dioxide or sugar. In order to obtain sustained release or to avoid incompatibilities, the core may consist of several layers in order to obtain sustained release, in which case the excipients mentioned above for tablets may be used. Organ specific carrier systems may also be used. Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are then filled into injection vials or ampoules.

Nasal sprays may be formulated similarly in aqueous solution and packed into spray containers either with an aerosol propellant or provided with means for manual compression. Capsules containing one or several active ingredients may be produced, for example, by mixing the active ingredients with inert carriers, such as lactose or sorbitol, and filling the mixture into gelatine capsules .

Suitable suppositories may, for example, be produced by mixing the active ingredient or active ingredient combinations with the conventional carriers envisaged for this purpose, such as natural fats or polyethylene glycol or derivatives thereof.

Dosage units containing the compounds of this invention preferably contain 0.1-1.0 mg, for example 1-5 mg of the peptide of formula 1-4 or salt thereof.

Blood clotting disorders in which the compounds of the invention may be used include thrombosis (particularly vascular thrombosis or deep vein thrombosis), acute myocardial infarction, restenosis, angina, reclosure, cerebrovasular disease, peripheral arterial occlusive disease, hypercoagulability and pulmonary embolism. The peptides according to the invention can also be used to prevent occurrence of blood clotting problems caused by, for example, grafting surgery, vessel wall patency restoration, etc. Blood clotting disorders may be triggered by sepsis due to production of tissue necrosis factor-α or interleukin-1.

In another aspect, the present invention provides a process for the preparation of peptides of the general structures 1-4 as defined above. The peptides of the present invention may be synthesised in any convenient way. Generally, the reactive groups present (e.g. amino, thiol, carboxyl, etc.) will be protected during overall synthesis. The final step in the synthesis will normally be deprotection of a protected derivative of the peptides of the invention. In the case of disulphide-cyclic peptides of general structure 2, the final step in the synthesis will normally be cyclisation following deprotection of a protected derivative of the peptides of the invention.

In building up the peptide chains, one can in principle start either at the carboxyl- or amino- terminus of the sequence of interest, although only the carboxyl-terminal starting procedure is in common use. Thus one can start at the carboxyl terminus by reaction of a suitably protected derivative of the terminal amino acid. This derivative will have' a free amino group and can be reacted with the free or activated carboxyl group of a protected amino acid derivative corresponding to the penultimate residue of the desired sequence. After coupling, the resulting dipeptide intermediate may be purified, for example by chromatography, and then selectively deprotected at the terminal amino group to permit addition of a further amino acid residue. This procedure is continued until the required amino acid sequence is assembled.

Carboxylic acid activating substituents which may, for example, be employed include symmetrical anhydrides or mixed anhydrides, or activated esters such as for example the p-nitrophenyl ester, 2 , 4 , 5-trichlorophenyl ester, _7-hydroxysucccinimidyl ester or N- hydroxybenzotriazolyl ester. Amino components may be acylated directly by amino acid derivatives possessing a free carboxyl group which the aid of coupling reagents such as 2- (lH-benzotriazole-1-yl) -1, 1, 3, 3- tetramethyluronium hexafluorophosphate and other reactive species derived from 1-hydroxybenzotriazole, as well as carbodiimides , 2-ethoxy-l-ethoxycarbonyl-l, 2 , - dihydroquinoline, etc. Often catalysts which possess racemisation-suppressing properties are added to the acylation mixtures, e. g. 1-hydroxybenzotriazole . In general it is convenient to effect the coupling reactions at low temperature up to ambient temperature, conveniently in a suitable solvent system such as tetrahydrofuran, dioxan, dimethylformamide, dichloromethane or a mixture of such solvents.

It may be convenient to carry out the syntheses on a solid support. Chloromethylated polystyrene (cross- linked with 1 % divinylbenzene) is one useful type of support; in this case the synthesis will start at the carboxyl terminus, for example by coupling an N- protected derivative of the terminal amino acid residue to the support. Preferred supports are p-alkoxybenzyl alcohol resins (Wang, J. Am. Chem . Soc . , 1973, 95:1328- 33) and trityl resins (Barlos et al . , Tetrahedron Lett . , 1989, 30:3943-6) for the synthesis of peptides containing free carboxyl termini. Additionally, p- nitrobenzophenone oxime resin (Kaiser et al . , Science, 1989, 243:187-92) is preferred for the synthesis of lactam-cyclic peptides.

A wide choice of protecting groups for amino acids are known (Bodanszky, Principles of peptide synthesis, Berlin :Springer-Verlag, 1984) . Thus, for example amino protecting groups which may be employed include groups such as benzyloxycarbonyl , t-butoxycarbonyl and 9- fluorenylmethoxycarbonyl . It will be appreciated that when the peptide is built up from the carboxyl terminus, an amine-protecting group will be present on the amino acid group of each new residue added and will need to be removed selectively prior to the next coupling step. Particularly useful groups for such temporary amine protection are the Fmoc group which can be removed selectively by treatment with piperidine in an organic solvent and the t-butoxycarbonyl group which may be removed with the aid of acids such as trifluoroacetic acid. Carboxyl protecting groups which may, for example, be employed include readily cleaved ester groups such as benzyl and t-butyl, as well as the linkers on solid supports, for example p-alkoxybenzyl alcohol linked to polystyrene. Thiol protecting groups include p- methoxybenzyl , trityl, acetamidomethyl and 3-nitro-2- pyrdylsulphenyl . When desired, the amide groups of e. g. asparagine and glutamine side chains can be protected with a trityl group. It will be appreciated that a wide range of other such protecting groups exist, and the use of all such groups in hereinbefore described processes fall within the scope of the present invention. A wide range of procedures exist for removing peptide protecting groups. These must, however, be consistent with the synthetic strategy employed. The side-chain protecting groups must be stable to the conditions used to remove the temporary amino protecting group prior to the next coupling step.

Disulphide-cyclic peptides (2) of the present invention may be obtained after assembly of linear S- protected precursors through cyclisation. In general, the cyclisation may be effected using oxidation, e . g. with oxygen at high dilution under basic conditions or under acidic conditions in trifluoroacetic acid with dimethylsulphoxide . If the thiol groups of the linear precursor are protected by trityl or acetamidomethyl groups, deprotection and oxidation ca be effected simultaneously using e . g. iodine or thallium (III) trifluoroacetate. Alternatively, the two thiol- containing residues of the precursors to the disulphide- cyclic peptides are protected separately, one with an acid- labile group such as trityl and one with an acid- stable disulphide- forming group such as -sulphonate or 3- nitro-2-pyridylsulphenyl (Npys) . If the peptide is assembled using acid-labile protecting groups and, where a solid support is used, an acid-labile ester linkage, conventional acidolysis (King et al . , Int . J. Peptide Protein Res . , 1990, 36:255-6) will provide the peptide in linear form which is fully deprotected apart from the sulphonate or Npys group. Under neutral or basic aqueous conditions the peptide then undergoes intramolecular disulphide exchange to yield the desired disulphide- cyclic peptide.

Cyclic peptides of the present invention other than those containing disulphide bonds (i.e. peptides of general structures 3 and 4) may be synthesised through known procedures for cyclisation of a precursor in which all reactive groups are protected with the exception of the amino and carboxyl groups to be condensed. The fully protected peptides can be obtained by conventional solution synthesis or preferably by solid-phase synthesis on super acid-labile linkers (Mergler et al . , Tetrahedron Lett . , 1988, 29:4005-4008; Barlos et al . , ibid. , 1989, 30:3943-6). In the fully protected peptide the amino and carboxyl functions taking part in the prospective lacta bond are protected in such a way as to be compatible with both temporary amino protection as well as semi-permanent side-chain protection. For example, if temporary amino protection is through Fmoc and semi-permanent side-chain protection is through t- butyl-type groups, then the amino and carboxyl groups participating in the prospective lactam bond may be protected through allyl carbamates and esters, respectively, which can be removed selectively with palladium (O) catalysts. The condensation reaction to form the lactam bond can be achieved through the wide range of coupling procedures described above for peptide bond formation. In general such condensations will be carried out in a suitable organic solvent at high peptide dilution in order to prevent polymerisation. In cases where the peptide is anchored to a support through a group other than the carboxyl group to be condensed, such cyclisation can be performed with the protected linear peptide precursor still attached to a solid synthesis support. In a preferred method of synthesis, linear precursors to lactam-cyclic peptides are assembled as described (Osapay et al., in: Techniques in Protein Chemistry II, edited by J.J. Villafranca, New YorkrAcademic Press, Inc. 1991, p. 221-31) on p- nitrobenzophenone oxime resin using the t-butoxycarbonyl group for temporary amino protection and using benzyl - type protecting groups for reactive amino acid side chain functions. The side-chain protected, resin-bound and amino-terminally deprotected precursor is then allowed to undergo intramolecular attack of the terminal amino group on the oxime-bound terminal carbonyl group, resulting in simultaneous cyclisation and resin detachment. The free cyclic peptide is then obtained after removal of the benzyl -type protecting groups, typically be treatment with strong acid such as trifluoromethanesulphonic acid or similar. A special case presents in the case of peptides with the general structure 4 and the substituents R⁴ and R⁵ containing amino and carboxyl groups, respectively. Thus if for example R³, R⁴ , R⁵ and A in the above precursor are -H, amino, carboxyl and butylene, respectively, then a suitable starting material would be Boc-NH-CH (COOBn) - (CH₂) ₄-CH(NH-Z) -COOPha, where Pha denotes phenacyl. This starting material can be selectively Boc-deprotected, followed by conventional solution synthesis to afford Boc-Waa-Xaa-Yaa-NH-CH (COOBn) - (CH₂) ₄-CH (NH-Z) -COOPha. After selective removal of the Boc group with trifluoroacetic acid and the phenacyl ester group with zinc in acetic acid, the resulting free amino and carboxyl groups are condensed to afford the cyclic protected peptide, from which the Z and Bn groups, together with any benzyl -type side chain protecting groups in Waa, Xaa and Yaa, are removed through hydrogenolysis . Abbreviations

Amino acid and peptide nomenclature follows the IUPAC- IUB recommendations {Eur. J. Biochem . , 1984, 138:9-37). Additional abbreviations are as follows:

2BrZ 2-bromobenzyloxycarbonyl

Abu 2-aminobutyric acid

ACH -cyano-4-hydroxycinnamic acid

Acm acetamidomethyl

3-Amb 3- (aminomethyl) benzoic acid

Aph p-amino-phenylalanine

5-Ava 5-aminovaleric acid

Bn benzyl

Boc t-butyoxycarbonyl

Cit citrulline

Dab 2, 4-diaminobutyric acid

Dap 2, 3-diaminopropionic acid

DIC N, _/' -di-isopropylcarbodiimide

DMF N, N' -dimethylformamide

DMSO dimethylsulphoxide

Fmoc 9-fluorenylmethoxycarbonyl

FVII human coagulation factor VII, suffix "a¹ denotes the activated form FX human coagulation factor X, suffix "a" denotes activated form Glp pyroglutamic acid

Hat 6-hydroxy-2-aminotetralin-2-carboxylic acid

Hat (Me) 6-methoxy-2-aminotetralin-2-carboxylic acid HOBt 1-hydroxybenzotriazole MALDI-TOF MS matrix-assisted laser desorption ionisation - time of flight mass spectrometry Nle norleucine

Orn ornithine

PyBOP benzotriazole-1-yl-oxy-tris-pyrrolidino- phsophonium hexafluorophosphate RP-HPLC reversed-phase high performance liquid chromatography TBTU 2- (lH-benzotriazole-1-yl) -1,1,3,3- tetramethyluronium tetrafluoroborate TF human tissue factor

Tic (OH) 4 ' -hydroxytetrahydroisoquinolinecarboxylic acid Trt trityl, triphenylmethyl

Tyr (Me) O-methyl tyrosine Z benzyloxycarbonyl

EXAMPLES

Example 1

Inhibition of FVIIa/TF-catalysed FX activation

This was determined using a two-stage chromogenic biochemical in vi tro assay, essentially as described

(Kumar et al . , J. Biol . Chem. , 1991, 266:915-21). In short: peptides were pre-incubated with lipidated TF (5 pM; from American Diagnostica Inc., Greenwich, CT, USA) and calcium (5 mM) for 10 min prior to addition of FVIIa

(5 pM; from Novo Nordisk AS, Gentofte, Denmark) and FX

(20 nM; from Enzyme Research Laboratory, South Bend, IN, USA) . Reactions were terminated by addition of EDTA (50 mM) and formation of FXa was monitored using the chromogenic FXa substrate S2765 (0.4 mM; from Chromogenix, Mδlndal, Sweden) and measuring the absorbance increase at 405 nm in a microtitre plate reader. The concentrations for half-maximal inhibition

(IC₅₀) were determined from dose-inhibition curves. IC₅₀ values were based on net peptide content (determined by amino acid analysis) . Suitable positive and negative

(unrelated peptides) control substances were used. Example 2 H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Asp-OH

The synthesis was carried out on a 0.1 mmol scale with Fmoc-Asp (OBu¹) - [p-alkoxybenzyl alcohol resin] (0.6 mmol/g) using an Applied Biosystems model 433A peptide synthesiser and standard Fmoc-chemistry program cycles. The final H-Asn (Trt) -Gly-Gly-Abu-Glu (OBu¹) -Gin (Trt) - Tyr (Bu') -Abu-Ser (Bu*¹) -Asp (OBu¹) -peptidyl resin was washed with CH₂C1₂ and Et₂0 and was dried. Cleavage/deprotection of this material was carried out using CF₃COOH (10 mL) containing PhSMe (0.5 mL) , H₂0 (0.5 mL) , 1,2- ethanedithiol (0.25 mL) and PhOH (0.75 g) during 3 h at room temperature. The crude peptide was obtained by precipitation with Et₂0, centrifugation, washing with Et₂0 and drying. It was purified by prep. RP-HPLC (Vydac 218TP1022 column, 10 mL/min, 0 to 8 % MeCN in 0.1 % aq CF₃COOH over 60 min) to afford the pure title compound (30.8 mg) after lyophilisation. Anal. RP-HPLC: t_R = 18.5 min, purity 97 % (Vydac 218TP54, 1 mL/min, 4 to 16 % MeCN in 0.1 % aq CF₃COOH over 20 min, λ = 215 nm) . ^XH- MR (d₆-DMSO, 200 MHz): inter alii δ (ppm) = 9.1 (br. s, 1 H, Gly² NH) , 8.7 (t, 1 H, Gly³ NH, J = 5.5 Hz) , 8.2 (t, 1 H, Ser⁹ NH, J = 5.7 Hz), 8.1 _ 7.8 (2 d, 2 H, Asn¹ NH₂, J = 7.63 Hz), 8.1-7.9 (m, 5 H, Tyr⁷ NH, Asp¹⁰ NH, Abu⁴' ⁸ NH, Gin⁶ NH) , 7.65 & 7.25 (2 s, 2 H, Gin⁶ δNH₂) 7.2 & 6.75 (2 s, 2 H, Asn¹ γNH₂), 7.0 & 6.97 (d, 2 H, Tyr⁷ H-2,6 J = 8.5 Hz), 6.61 & 6.58 (d, 2 H, Tyr⁷ H-3,5 J^" = 8.4 Hz), 0.9 (m, 6 H, Abu⁴' ⁸ γCH₃) . ¹³C-NMR (d₆-DMSO, 50 MHz) : δ (ppm) = 174.4 (Asn¹ CONH₂) , 174.3 (Gin⁶ CONH₂) , backbone CONH: 172.7, 172.2, 172.0, 171.7, 171.33, 171.3, 171.2, 171.1, COOH: 169.9, 169.1, 168.9, 168.8; 155.9 (Tyr⁷ C-4), 130.18 (Tyr⁷ C-2,6), 127.8 (Tyr⁷ C-l) , 115.0 (Tyr⁷ C- 3,5), 62.0 (Ser⁹ CH₂) , CH : 55.1, 54.3, 53.9, 52.2, 49.4, 48.7, 42.4, 42.0, CH₂ : 36.7, 35.7, 31.6, 30.4, 28.4, 27.1, 25.6, 25.5, CH₃ : 10.3, 10.2. MALDI-TOF MS (ACH matrix): [M + H] ⁺ = 1039.8; C₄₂H₆₂N₁₂0₁₉ = 1039.0. Amino acid analysis: Asx 1.97 (2), Ser 0.97 (1), Glx 2.01 (2), Tyr 1.01 (1), Gly 2.04 (2), Abu 1.99 (2).

Example 3 H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Lys-OH

The synthesis of this peptide was carried out using similar methods to those described in Example 2, except that Fmoc-Lys (Boc) -resin was the starting material. The title compound was purified to >97% as assessed by analytical RP-HPLC (215 nm) . Amino acid analysis: Asx 0.99 (1), Ser 1.08 (1), Glx 1.99 (2), Tyr 0.98 (1), Gly 1.95 (2), Lys 1.01 (1), Abu 2.00 (2).

Example 4 H-Abu-Glu-Gln-Tyr-Abu-OH

Fmoc-Abu-OH (1.82 g, 5.6 mmol) was dissolved in 8 : 2 CH₂C1₂ / DMF (25 mL) and DIC (^'0.44 mL, 2.8 mmol) was added. The mixture was stirred at 0 °C for 30 min to permit formation of (Fmoc-Abu- ) ₂0. CH₂C1₂ was removed by rotary evaporation and the residue was diluted with DMF (10 mL) . This solution was added to pre-swollen (in DMF) NovaSyn^® TGA resin (2 g, 0.28 mmol/g; from Novabiochem AG, Laufelfingen, Switzerland) and 4- dimethylaminopyridine (7 mg, cat.) was added. The entire mixture was stirred for 1.5 h at room temperature. The resulting Fmoc-Abu resin was collected by filtration, was washed with DMF and CH₂C1₂ and was dried. The resin loading was determined spectrophotometrically after piperidine treatment of weighed resin aliquots (using e = 4.95 x 10³ M^"1. cm^"1 at 290 nm for the dibenzolfulvene- piperidine adduct formed) and a value of 0.274 mmol/g was obtained. The resin was then treated with benzoyl chloride and Pr^NEt (10 eq each) in DMF for 2 h in order to cap unreacted hydroxyl groups before being washed and dried again.

An aliquot (912 mg, 0.25 mmol) of this resin was elongated using an Applied Biosystems model 433A peptide synthesiser. Standard instrument cycles (single coupling with capping) and chemistry ("SlowMoc$ 0.25 mmol MonPrevPK) were used. The H-Abu-Glu (OBu¹¹) -Gin (Trt) - Tyr (Bu¹) -Abu-peptidyl resin was washed successively with CH₂C1₂ and Et₂0 and was dried (1.054 g) . An aliquot (0.5 g, 0.117 mmol) of this material was resuspended in CF₃COOH (10 mL) containing PhSMe (0.5 mL) , H₂0 (0.5 mL) , 1,2-ethanedithiol (0.25 mL) and PhOH (0.75 g) and the mixture was shaken at room temperature for 3 h. Resin residue was then filtered off and washed with neat CF₃COOH (10 L) . The combined filtrate and washings were added to stirred Et₂0 (250 mL) . The precipitate was filtered off, washed with more Et₂0, redissolved in AcOH and lyophilised to afford the crude title compound (62.1 mg) . This material was redissolved in 0.1 % aq CF₃COOH (250 mL) , filtered and pumped (at 10 mL/min) onto a prep. RP-HPLC column (Vydac 218TP1022, 2.5 x 25 cm). The column was then eluted at 10 mL/min using a gradient from 0 to 12 % MeCN in 0.1 % aq CF₃COOH over 60 min, followed by isocratic elution. The eluant was monitored (260 nm) and appropriate peak fractions were collected, pooled and lyophilised to afford pure title compound (44.7 mg) . Anal. RP-HPLC: t_R = 18.9 min, purity > 98 % (Vydac 218TP54, 1 mL/min, 0 to 15 % MeCN in 0.1 % aq CF₃COOH over 20 min, λ = 215 nm) . ^XH-NMR (300 MHz, d₆- DMSO) : δ (ppm) = 8.52 (d, 1 H, CONH) , 8.13 (d, 1 H, CONH) , 8.08 (d, 1 H, CONH), 7.92 (d, 1 H, CONH), 7.16 & 6.75 (2 s, 2 H, Gin CONH₂) , 7.01 & 6.60 (2 d, 4 H, Ar-H) , 4.4-4.5 & 4.25-4.4 & 4.08-4.22 (3 m, 1 H, 1 H and 2 H, resp., CH) , 3.72 (t, 1 H, Abu αCH) , 2.85-2.95 & 2.65- 2.75 (2 m, 2 H, Tyr βCH₂) , 2.18-2.38 (m, 2 H, γCH₂) , 2.0- 2.15 (m, 2 H, γCH₂) , 1.58-1.95 (m, 8 H, βCH₂) , 0.83-0.89 (m, 6 H, Abu γCH₃) . MALDI-TOF MS (ACH matrix) : [M + H] ⁺ = 609.9, [M + Na] ⁺ = 631.8; C₂₇H₄₀N₆O₁₀ = 608.6. Amino acid analysis: Glx 1.99 (2), Tyr 1.01 (1), Abu 2.00 (2). Example 5

Disulphide- cyclo- [H-Cys-Aph-Asp- eu-Cys-OH]

The chain assembly was performed using Fmoc- Cys (Trt) -Tentagel S Trityl resin (1.39 g, 0.25 mmol; from Rapp Polymere GmbH, Tubingen, Germany) . Fmoc- deprotection was carried out with 20 % piperidine in DMF during 20 min. Successive acylations were carried out for 1 h using four-fold molar excesses of the following amino acid derivatives in DMF (15 mL) : Fmoc-Leu-OH, Fmoc-Asp (OBu') -OH, Fmoc-Aph (Boc) -OH and Boc-Cys (S0₃ ^~) -O^" .2 Na⁺, together with PyBOP, HOBt and Pr^NEt (in the molar ratio 1 : 1 : 1: 1.5) . After each acylation and deprotection cycle the peptidyl resin was washed exhaustively with DMF and completeness of reactions was assessed using the Kaiser ninhydrin test. The final material was washed successively with DMF, CH₂C1₂ and Et₂0 and was dried in vacuo . It was then resuspended in CF₃COOH (15 mL) containing PhOH (1.12 g) , H₂0 (0.75 L) , PhSMe (0.75 mL) and 1, 2-ethanedithiol (0.325 mL) and the mixture was agitated for 2 h. Resin residue was then filtered off and washed with neat CF₃COOH. The combined filtrate and washings were rotary evaporated and the crude peptide H-Cys (S0₃H) -Aph-Asp-Leu-Cys-OH (213.5 mg) was obtained by precipitation with Et₂0, centrifugation, washing with more Et₂0 and drying. Some of this material (200 mg) was redissolved in 0.1 % aq CF₃COOH (6 mL) and purified in three separate runs by prep. RP-HPLC (Vydac 218TP1022 column, 10 mL/min, gradient from 2 to 8 % MeCN in 0.1 % aq CF₃COOH over 60 min, λ = 230 nm) . Appropriate peak fractions were collected, pooled and lyophilised to afford the pure linear peptide (104.5 mg) .

An aliquot of this material (36.8 mg) was dissolved in phosphate buffer (50 mM, pH 6.0; 37 mL) and the solution was stirred magnetically. The cyclisation reaction was monitored by anal. RP-HPLC_? which indicated complete reaction after 45 min. The mixture was then acidified by addition of aq HC1 (1 M; 2 mL) and was concentrated by rotary evaporation. The residue was chromatographed (two runs) by prep. RP-HPLC using the same conditions as above for the linear precursor. Appropriate peak fractions were collected, pooled and lyophilised to afford the pure cyclic title compound (19.4 mg) . Anal. RP-HPLC: t_R = 14.8 min, > 98 % purity (Vydac 218TP54 column, 1 mL/min, 4 to 16 % MeCN in 0.1 ^; aq CF₃COOH over 20 min, λ = 215 nm) . MALDI-TOF MS (ACH matrix): [M + H] ⁺ = 613.6; C₂₅H₃₆N₆0₈S₂ = 612.7. Amino acid analysis: Asx 1.02 (1), Cys (as cysteic acid) 1.98 (2), Leu 1.00 (1), Aph n.d. (1).

Example 6

Disulphide-cyclo- [H-Cys-Glu-Gln-Hat (Me) -Cys-OH]

Fmoc-Cys (Trt) - [p-alkoxybenzyl alcohol resin] (0.18 g, 0.55 mmol/g) was deprotected with 50 % piperidine in DMF for 15 min and was then washed with DMF. Fmoc-D/L- Hat (Me) -OH (synthesised according to Rastogi et al . , Indian J. Chem . , 1971, 9:1175-82, Cardinaux & Pless, in: Peptides 1984 , edited by U. Ragnarssson, Stockholm: Almqvist _ Wiksell International, 1984, p. 321-4; 133 mg, 0.3 mmol), TBTU (96 mg, 3 mmol), HOBt (45 mg, 0.3 mmol) and Prⁱ ₂NEt (73 μL, 0.45 mmol) were pre-activated for 5 min (in 3 mL DMF) , added to the drained resin and allowed to react with agitation of the mixture for 5 h. The resin was then again washed and drained. Further chain elongation was performed similarly using 5 eq each of Fmoc-Gln(Trt) -OH, Fmoc-Glu (OBu') -OH and Fmoc-Cys (Trt ) - OH. After the final Fmoc-deprotection, the resin was washed successively with DMF, CH₂C1₂ and Et₂0 and was dried in vacuo (202 mg) . This material was resuspended in CF₃COOH (10 mL) containing PhOH (0.75 g) , H₂0 (0.25 mL) , PhSMe (0.5 mL) and 1 , 2-ethanedithiol (0.25 mL) and the mixture was shaken for 1.5 h. Resin- residue was filtered and washed with neat CF₃COOH. The combined filtrate and washings were rotary evaporated. The residue was triturated with Et₂0 and centrifuged. The pellet was further washed three times with Et₂0 and was dried to afford the crude linear peptide H-Cys-Glu-Gln- D/L-Hat (Me) -Cys-OH (75 mg, quant.). An aliquot of this material (75 mg) was redissolved in 0.1 % aq CF₃COOH (4 mL) and chromatographed by prep. RP-HPLC (Vydac 218TP1022 column, 9 mL/min, 12 - 25 % MeCN in 0.1 % aq CF₃COOH over 50 min, λ = 230 nm) . Appropriate peak fractions were collected and pooled to afford a solution of the pure diastereomeric linear peptide. Anal. RP- HPLC: t_R = 19.8 min (Vydac 218TP54, 1 mL/min, 10 to 20 % MeCN in 0.1 % aq CF₃C00H over 20 min) . MALDI-TOF MS (ACH matrix): [M + H] ⁺ = 685.9; C₂₈H₄₀N₆O₁₀S₂ = 684.8. The pool was made 0.1 M in NH₄HC0₄ by addition of 1 M aq NH₄HC0₄ soln. This mixture was stirred magnetically with access to air. Anal. RP-HPLC indicated complete oxidation after 36 h, after which time the solution was dried by vacuum centrifugation. The residue was^" redissolved (5 mL of 10 % MeCN, 0.1 % CF₃COOH in H₂0) and chromatographed by prep. RP-HPLC (conditions as above except 7.5 to 15.5 % MeCN gradient over 100 min) . Appropriate peak fractions were collected, pooled and lyophilised to afford the pure cyclic peptide diastereomers (9.27 mg, isomer 1; 2.70 mg, isomer 2). Anal. RP-HPLC: t_R = 20.66 min, purity 100 % (isomer 1) and 22.02 min, purity 98.7 % (isomer 2) (Vydac 218TP54, 1 mL/min, 4 to 16 % MeCN in 0.1 % aq CF₃COOH over 20 min, λ = 215 nm) . MALDI-TOF MS (ACH matrix): [M + H] ⁺ = 683.9 (both isomers) ; C₂₈H₃eN₆O₁₀S₂ = 682.8. Amino acid analysis (found isomer 1, found isomer 2 (calculated)): Glx 2.09, 2.04 (2); Cys (as cysteic acid) 2.10, 2.04 (2), Hat (Me) 0.81, 0.92 (1).

Example 7

Disulphide-cyclo- [H-Cys-Orn-Gln-Tyr-Cys-OH]

This peptide was synthesised using chain assembly (using Fmoc-Orn (Boc) -OH and Fmoc-Tyr (Bu^c) -OH in the appropriate acylation cycles) , cleavage, deprotection, cyclisation (of the fully deprotected linear precursor at high dilution in basic aqueous solution) and purification methods analogous to those described in Example 6. The following analytical details for the title compound were recorded: Anal. RP-HPLC: t_R = 15.6 min, 99% purity at 215 nm (Vydac 218TP54, 1 mL/min, 0 to 12% MeCN in 0.1% aq CF₃COOH over 20 min) . Amino acid analysis: Glx 1.02 (1), Tyr 0.99 (1), Cys (as cysteic acid) 1.96 (2), Orn 1.03 (1). MALDI-TOF MS (ACH matrix): [M + H] ⁺ = 628.4, C₂₅H₃₇N₇0₈S₂ = 627.7.

Example 8

Disulphide-cyclo- [H-Cys-Glu-Gln-Tyr-Cys-Ser-OH]

This peptide was assembled on Fmoc-Ser (Bu¹) -Sasrin resin (from Bachem AG, Bubendorf , Switzerland) . Cleavage, deprotection, cyclisation (of the fully deprotected linear precursor at high dilution in basic aqueous solution) and purification methods analogous to those described in Example 6 were then applied. Analytical details for the title compound are as follows: Anal. RP-HPLC: t_R = 16.4 min, 99% purity at 215 nm (Vydac 218TP54, a mL/min, 0 to 12% MeCN in 0.1% aq CF₃COOH over 20 min). Amino acid analysis: Ser 1.03 (1) , Glx 2.03 (2), Tyr 1.02 (1), Cys (as cysteic acid) 1.91 (2). MALDI-TOF MS (ACH matrix): [M + H] ⁺ = 733.1, C₂₈H₃₉N₇0₁₂S₂ = 729.8.

Example 9

Cyclo- [Abu-Glu-Gln-Tyr-Abu]

Coupling first residue to oxime resin: p- Nitrobenzophenone oxime on polystyrene - 1 % divinylbenzene resin (1.1 mmol/g, 1.5 g; from Novabiochem AG, Laufelfingen, Switzerland; cf . Degrado & Kaiser, J. Org . Che . , 1980, 45:1295) was reacted with Boc-Abu-OH (91 mg, 0.45 mmol), HOBt (79 mg, 0.45 mmol) and DIC (71μL, 0.45 mmol) in 10 % DMF / CH₂C1₂ (15 mL) for 24 h in a bubbler reactor. The resin was then drained, washed with CH₂C1₂ and Et₂0 and was dried. A weighed aliquot was Boc-deprotected, neutralised, washed and dried before being submitted to picric acid titration (Gisin, Anal. Chem. Acta, 1972, 58:248), which provided an Abu/resin substitution value of 0.20 mmol/g. Unreacted oxime functions on the Boc-Abu- derivatised resin were then capped by reaction for 24 h with pivaloyl chloride (3.75 mL) and Pr^NEt (5.1 mL) in CH₂C1₂ (1 mL) . The resulting resin was again washed and dried.

Peptide chain assembly: The capped Boc-Abu-resin was then deprotected by treatment with 25 % CF₃COOH / CH₂C1₂ during 30 min. After draining, the resin was washed with CH₂C1₂ and was then reacted with Boc- Tyr(2BrZ)-OH (5 eq) , HOBt (5 eq) , PyBOP (5 eq) and Pr^NEt (20 eq) in DMF for 2 h. Further peptide chain extension was then achieved similarly by applying alternating deprotection and coupling steps. The amino acid derivatives Boc-Gln-OH, Boc-Glu (OBn) -OH and Boc- Abu-OH were used successively. The Boc-Abu-Glu (OBn) -Gln- Tyr (2BrZ) -Abu-resin was finally obtained after washing with CH₂C1₂, Et₂0 and drying.

Cyclisation : The protected peptidyl resin ( ca . 0.088 mmol) was Boc-deprotected as above. After draining and washing the resin was suspended in DMF (5 mL) , containing Pr^NEt (75 μL) and AcOH (50 μL) and was agitated during 24 h. Resin residue was the filtered and washed with CH₂C1₂. The combined filtrate and washings were rotary evaporated to a small volume. Water was then added and the precipitated protected cyclic peptide material was filtered, washed thoroughly with water, redissolved in AcOH (5 mL) and lyophilised. Crude material (15.5 mg, ca . 20 % cyclisation yield) contained two main products: RP-HPLC: t_R = 10.3 & 16.3 min, 4 : 1 ratio at 1 = 215 nm (Vydac 218TP54, 30 to 70 % MeCN in 0.1 % aq CF₃COOH over 20 min at 1 mL/min), corresponding to the cyclic monomer cyclo- [Abu-Glu (OBn) -Gln-Tyr (2BrZ) - Abu] and the cyclic dimer cyclo- [Abu-Glu (OBn) -Gln- Tyr (2BrZ) -Abu] ₂, respectively, as evidenced by mass spectrometric analysis.

Deprotection and purifica tion : The crude product from the cyclisation reaction was treated at 0 °C during 2 h with a mixture of CF₃S0₃SiMe₃ (0.98 mL) , PhSMe (0.63 mL) , m-cresol (0.12 mL) and CF₃C00H (3.75 mL) . Excess Et₂0 was then added and the precipitated peptide material was filtered and washed with Et₂0. It was then redissolved in AcOH (10 mL) and was lyophilised. The residue was redissolved in 7 : 3 H₂0 / MeCN containing 0.1 % CF₃COOH (5 mL) and was chromatographed on a Vydac 218TP1022 column at 10 mL/min using a gradient from 0 to 20 % (over 60 min) and then to 30 % MeCN (over 30 min) in 0.1 % aq CF₃COOH. The eluant ^' was monitored at 260 nm. Fractions containing pure title compound (judged by anal. RP-HPLC) were pooled and lyophilised (2.5 mg yield). Anal. RP-HPLC: t_R = 18.2 min, 98 % purity at 215 nm (Vydac 218TP54, 1 mL/min, 5 to 25 % MeCN in 0.1 % aq CF₃COOH over 20 min). Amino acid analysis: Glx 2.03 (2), Tyr 1.01 (1), Abu 1.96 (2). MALDI-TOF MS (ACH matrix): [M - H]^~ = 589.5, C₂₇H₃₈N₆0₉ = 590.6.

Example 10

Cyclo- [Gly-Glu-Gln-Tyr-Gly] and cyclo- [Gly-Glu-Gln-Tyr- Gly] 2

Coupling first residue to oxime resin: p- Nitrobenzophenone oxime on polystyrene - 1 % divinylbenzene resin (same as in above example; 1.5 g, 1.65 mmol) was reacted with Boc-Gly-OH (79 mg, 0.45 mmol), HOBt (79 mg, 0.45 mmol) and DIC -(71 μL, 0.45 mmol) in 10 % DMF/CH₂C1₂ (15 mL) for 24 h in a bubbler reactor. The resin was then drained, washed with CH₂C1₂ and Et₂0 and was dried. A weighed aliquot was Boc- deprotected, neutralised, washed and dried before being submitted to picric acid titration, which provided Gly/resin substitution value of 0.156 mmol/g. Unreacted oxime functions on the Boc-Gly-derivatised resin were then capped by reaction for 24 h with pivaloyl chloride (3.75 mL) and Pr^NEt (5.1 mL) in CH₂C1₂ (1 mL) . The resulting resin was again washed and dried.

Peptide chain assembly: The capped Boc-Abu-resin was then deprotected by treatment with 25 % CF₃C00H/ CH₂C1₂ during 30 min. After draining, the resin was washed with CH₂C1₂ and was then reacted with Boc- Tyr(2BrZ)-0H (5 eq) , HOBt (5 eq) , PyBOP (5 eq) and Pr^NEt (20 eq) in DMF for 2 h. Further peptide chain extension was then achieved similarly by applying alternating deprotection and coupling steps. The amino acid derivatives Boc-Gln-OH, Boc-Glu (OBn) -OH and Boc- Gly-OH were used successively. The Boc-Gly-Glu (OBn) -Gln- Tyr (2BrZ) -Gly-resin was finally obtained after washing with CH₂C1_2/ Et₂0 and drying.

Cyclisation : The protected peptidyl resin ( ca . 0.115 mmol) was Boc-deprotected as above. After draining and washing the resin was suspended in DMF (7.5 mL) , containing Pr^NEt (90 μL) and AcOH (60 μL) and was agitated during 22 h. Resin residue was the filtered and washed with CH₂C1₂. The combined filtrate and washings were rotary evaporated to a small volume. Water was then added and the precipitated protected cyclic peptide material was filtered, washed thoroughly with water, redissolved in AcOH (5 mL) and lyophilised. Crude material (60.8 mg, ca . 63 % cyclisation yield) contained two main products: RP-HPLC: t_R = 12.5 & 21.1 min, 3 : 2 ratio at 1 = 215 nm (Vydac 218TP54, 30 to 70 % MeCN in 0.1 % aq CF₃COOH over 20 min at 1 mL/min) , corresponding to the cyclic monomer cyclo- [Gly-Glu (OBn) -Gln-Tyr (2BrZ) - Gly] and the cyclic dimer cyclo- [Gly-Glu (OBn) -Gln- Tyr (2BrZ) -Gly] ₂, respectively, as evidenced by mass spectrometric analysis. This material was redissolved ( ca . 35 % MeCN, 0.1 % CF₃COOH in H₂0; ca . 400 mL) and pumped onto a prep. RP-HPLC column (Vydac 218TP1022, 2.5 x 25 cm) ) , which was then eluted at 10 mL/min with a gradient from 0 to 30 % (30 min) to 45 % (60 min) MeCN in 0.1 % aq CF₃COOH. Appropriate peak fractions corresponding to the cyclic monomer and dimer were collected, pooled and lyophilised.

Deprotection and purification: The purified products from the cyclisation reaction were treated at 0 °C during 2 h with a mixture of CF₃S0₃SiMe₃ (1 M) , PhSMe (1 M) and m-cresol (50 eq) in CF₃C00H (1 mL/O.01 mmol peptide) . Excess Et₂0 was then added and the precipitated peptide materials were filtered and washed with Et₂0. They were then redissolved in AcOH (10 mL) and were lyophilised. The residues were redissolved in 0.1 % aq CF₃COOH (5 mL) and were chromatographed on a Vydac 218TP1022 column at 10 mL/min using a gradient from 0 to 10 % (monomer) or 15 % MeCN in 0.1 % aq CF₃COOH (dimer) over 60 min, followed by isocratic elution. Appropriate peak fractions were collected, pooled and lyophilised to afford the pure title compounds (8.4 mg cyclic monomer and 9.9 mg cyclic dimer). Anal. RP-HPLC: t_R = 10.9 min (monomer); 20.7 min (dimer), > 97 % purity for both peptides at 215 nm (Vydac 218TP54, 1 mL/min, 4 to 16 % MeCN in 0.1 % aq CF₃COOH over 20 min) . Amino acid analysis: Glx 2.01 (2), Tyr 1.01 (1), Gly 1.98 (2) for monomer; Glx 4.00 (4), 2.02 (2) Tyr, Gly 3.98 (4) for dimer. MALDI-TOF MS (ACH matrix): [M - H] ^~ = 533.6, C₂₃H₃₀N₆O₉ = 534.5 for monomer; [M - H] ^" = 1067.4, [M + H] ⁺ = 1070.3, C₄₆H₆₀N₁₂O₁₈ = 1069.0 for dimer.

Example 11

Cyclo- [ (3-Amb) -Glu-Gln-Tyr-Gly] and cyclo- [ (3-Amb) -Glu-

Gln-Tyr-Gly] ₂

These peptides were prepared starting from the same Boc-Gly-Oxime resin as in Example 10. Peptide chain assembly, cyclisation, deprotection and purification were carried out analogously to the method described in Example 10, including obtention and separation of the monomeric and dimeric cyclic products. A custom- synthesised N-Fmoc protected derivative of 3- (aminomethyl) benzoic acid was used for the introduction of the 3-Amb residue. Analytical details for the title compounds are as follows: Anal. RP-HPLC: t_R = 18.3 min (monomer); 13.5 min (dimer), >96% purity for both peptides at 215 nm (Vydac 218TP54, 1 mL/min, 4 to 16% MeCN (monomer) or 10 to 30% MeCN (dimer) in 0.1% aq CF₃COOH over 20 min). Amino acid analysis: Glx 2.00 (2), Tyr 0.99 (1), Gly 1.01 (1), 3-Amb 1.00 (1) for monomer; Glx 4.00 (4), Tyr 1.96 (2), Gly 2.01 (2), 3-Amb 2.04 (2) for dimer. MALDI-TOF MS (ACH matrix): [M + H] ⁺ = 612.6, C₂₉H₃₄N₆0₉ = 610.06 for monomer; [M - H] ^" = 1220.2, C₅₆H₆₈N₁₂0₁₈ = 1221.2 for dimer.

Claims

CLAIMS :

1) A compound which is capable of interacting with the internal receptor in the catalytic domain of FIXa or

FX defined by the residues He²⁹⁰, Ala²⁹¹, Asp²⁹², Tyr²⁹³, Thr²⁹⁴, Glu³⁷⁴, and Phe³⁷⁸ in FIXa and Leu³⁰⁰, Pro³⁰¹' Glu³⁰², Trp³⁰⁵, Ala³⁰⁶, Lys³⁸⁵ and Phe³⁸⁹ in FXa or the ligand defined by residues Cys⁹⁵ to Cys⁹⁹ in FIXa or Cys⁹⁶ to Cys¹⁰⁰ in FXa to prevent the formation of a functional FVIIIa/FIXa complex or FXa/FV complex respectively.

2) A compound as claimed in claim 1 wherein said compound is a peptide.

3) A compound as claimed in claim 2 wherein said peptide is of formula

R^Vaa-Waa-Xaa-Yaa-Zaa-R² (1)

R^Saa-Waa-Xaa-Yaa-Saa-R² (2)

R^Vaa-Waa-Xaa-Yaa-Zaa- (3b)

pVaa-Waa-Xaa-Yaa-Zaa-R² (3c)

O

in which one of the three residues Waa, Xaa and Yaa is an amino acid residue from group I, the remaining two residues are from groups II and III, respectively, or they are both from group III wherein

Group I denotes aromatic amino acid residues,

Group II denotes amino acid residues containing side-chain functional groups which are ionized under physiological conditions, and

Group III denotes neutral and hydrophobic amino acid residues; the two residues Saa in 2 are amino acid residues containing thiol functions in the side chain, these thiol functions being linked intramolecularly through a disulphide bond; the residues Vaa and Zaa in 1 and 3 may independently correspond to hydrophobic amino acid residues, and in the case of general structures 3b and 3c the terminal residues participating in a "side chain- to-tail" or "head-to-side chain" amide bond (Vaa and Zaa, respectively) correspond exclusively to amino acids residues containing side-chain amine (3b) or carboxyl (3c) groups;

R¹ in 1-3 may correspond to the following: H- , acetyl, R⁶-Gly-, R⁶-Gly-Gly-, R⁶-Asn-Gly-Gly- , R⁶-Dab-Gly- Gly- , Glp-Gly-Gly- , succinyl-Gly-Gly- or amidosuccinyl- Gly-Gly-, where R⁶ may be H- or acetyl;

R² in 1-3 may correspond to -OH, -NH₂, -Ser-R⁷, Ser-Asp-R⁷, -Ala-Asp-R⁷, -Ser-Abu-R⁷,

-Ser-Lys-R⁷, -Ser-Dab-R⁷ or -Ser-Ile-R⁷, where R⁷ may be - OH or -NH₂; n in 3a may be 1 or 2;

R³ in 4 may be -H or methyl ;

R⁴ may be -H, methyl, -NH₂, -NH-Ac or -NH-R¹;

R⁵ may be -H, methyl -COOH, -COOMe, -CONH₂ or -CO- R² ; and moiety A in 4 may correspond to a saturated or unsaturated carbon chain of length C_!-C₆, optionally interrupted by a phenyl group or a heteroatom (N, O or S) . 4) A compound as claimed in claim 3 wherein said group

I amino acid residues are selected from Tyr, Aph and Phe, Tic (OH) and Hat.

5) A compound as claimed in claim 3 wherein said group

II amino acid residues are selected from Glu, Asp, Dap, Dab, Cit, Orn, Lys, Arg, 1-aminocyclo-pentane-l, 3- dicarboxyl and 1-aminocyclohexane-l, 3-dicarboxyl .

6) A compound as claimed in claim 3 wherein said group

III amino acid residues are selected from Gin, Asn, Ser, Thr, Leu and Nle.

7) A compound as claimed in claim 3 wherein residues Vaa and Zaa are independently selected from Gly, Ala, βAla, Abu, Val, norvaline, Pro, aminoalkanoyl (4-8 C atoms) and o- , m- or p- (aminomethyl) benzoyl, Asp, Glu, Dap, Dab, Orn or Lys .

8) A compound as claimed in claim 3 wherein in said moiety A both the chain and the optional phenyl group may bear (C-C,,) -alkyl, amino, hydroxyl, carboxyl or carboxamide substituents.

9) A compound as claimed in claim 3 wherein Saa represents Cys, homocysteine or penicillamine .

10) A compound as claimed in claim 3 of formula H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Asp-OH Succinyl-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Asp-OH H-Glp-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Asp-OH H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ala-Asp-OH H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Lys-OH H-Asn-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Abu-OH H-Gly-Gly-Abu-Glu-Gln-Tyr-Abu-Ser-Dab-OH H-Abu-Glu-Gln-Tyr-Abu-Ser-Dab-OH H-Abu-Glu-Gln-Tyr-Abu-OH H-Nle-Glu-Gln-Tyr-Nle -OH disulphide- cyclo- [H-Cys- ^•Glu- Gln- Tyr-Cys -Ser-OH] disulphide- cyclo- [H-Cys-^•Glu- Gln- Tyr-Cys -Dab-OH] disulphide- cyclo- [H-Cys- Orn- Gln- Tyr-Cys -OH] disulphide- cyclo- [H-Cys-^•Nle- Gln- Tyr-Cys -OH] disulphide- cyclo- [H-Cys- Glu- Nle- Tyr-Cys -OH] disulphide- cyclo- [H-Cys- Glu- Gln- Tyr (Me) -Cys-OH] disulphide- cyclo- [H-Cys- Aph- Asp- Leu-Cys -OH] disulphide- cyclo- [H-Cys- Tyr- Glu- Gin-Cys^■ -OH] disulphide- cyclo- [H-Cys- Cit- Tyr- Thr-Cys^■ -OH] disulphide- cyclo- [H-Cys- Glu- Gln- Hat (Me) ^■ -Cys-OH] cyclo- [Abu-Glu-Gln-Tyr-Abu] cyclo- [ βAla-Glu-Gln-Tyr- βAla] cyclo- [ ( 3 -Amb) -Glu-Gln-Tyr- ( 3 -Amb) ] cyclo- { [ ( 3 -Amb) -Glu-Gln-Tyr- ( 3 -Amb) ] ₂ } cyclo- [Abu-Glu-Gln-Tyr-Asp] cyclo- [ ( 5 -Ava) -Glu-Gln-Tyr-Gly] cyclo- [Dap-Glu-Gln-Tyr-Asp] cyclo- [Gly-Glu-Gln-Tyr-Gly] cyclo- [ (Gly-Glu-Gln-Tyr-Gly) ₂]

11) A pharmaceutical composition containing one or more compounds as claimed in any one of claims 1 to 10.

12) Use of a compound as claimed in any one of claims 1 to 10 in the prevention of the formation of a functional FVIIIa/FIXa complex or FXa/FV complex respectively.

13) The use of a compound as claimed in any one of claims 1 to 10 in the manufacture of a medicament for the prevention of the formation of a functional FVIIIa/FIXa complex or FXa/FV complex respectively.

14) A method of treatment of the human or animal body to combat or prevent blood clotting disorders said method comprising administering to said body one or more compounds as claimed in any one of claims 1 to 10 to prevent the formation of a functional FVIIIa/FIXa complex or FXa/FV complex respectively.