CA2215702A1 - Thrombin inhibitors based on the amino acid sequence of hirudin - Google Patents

Abstract

A thrombin inhibitor comprising a first bulky hydrophobic portion interacting with the catalytic side of thrombin responsible for proteolysis and a second portion maintaining the hydrophobic and acidic character of amino acids 55 to 60 of native hirudin at the C-terminal non-catalytic region of N-acetyl-hirudin45-65. Between the first and second portions is a divalent linker moiety. The compound is useful in the treatment of thrombotic disorders.

Description

CA 0221~702 1997-09-17 THROMBIN INHIBITORS BASED ON
THE AMINO ACID SEQUENCE OF HIRUDIN

FT~T~n OF THE INVE~TION

The present invention relates to peptide derivatives useful as thrombin inhibitors and in particular to a peptide derivative based on the se~uence of the segment of hirudin cont~; n; ng amino acids 45 to 65.

BACKGROUND ~F THE INVENTION

Thrombin is an important serine proteinase component o~
lS the blood coagulation cascade. Besides initiating blood clotting by cleaving fibrinogen, thrombin activates other hemocoagulant ~actors including ~actors V, VIII and XIII
and the anticoagulant enzyme protein C. Thrombin is also a potent platelet activator that impairs thrombolysis mediated by tissue plasminogen activator in vivo. Thus, thrombin's positive feedback regulation serves to amplify hemostatic events but causes life-threatening thrombi in response to aberrations with vascular and cerebrovascular arteries.

Given the diverse functions of this enzyme, its inhibition by potent and specific compounds could provide an invaluable addition to the treatment of disorders related to thrombosis. These include: coronary artery disease, cerebrovascular disease, peripheral arterial occlusive disease, deep vein thrombosis and pulmonary embolism.
c ~ The most potent inhibitor of thrombin known is hirudin, a family o~ iso-proteins isolated ~rom the glandular secretions of the leech Hirudo Medicinal is . The anticoagulant properties of hirudin have been known ~or a long time. However, it has so far been of little therapeutic value since the formulation of this protein in CA 0221~702 1997-09-17 a readily efficient and administrable form seems to be difficult as both enteral and cutaneous absorptions are very low so it has not been possible to produce adequate levels o~ the protein in the bloodstream.
s Furthermore, clinical use of hirudin isolated from leech extracts is unlikely because of its limited quantity, expense and allergic reactions whlch may commonly occur upon administration of foreign proteins having the size of hirudin.

In his publication entitled "Pharmacology o~ selective thrombin inhibitors", (1988), Nouv. Rev. Fr. Hematol., 30, pages 161-165, Markwardt provided further clinical information on hirudin based on results o~ pharmacological studies performed for both natural and synthetic thrombin inhibitors.

The author makes general observations concerning hirudin, mentioning that the peptide, which contains a highly acidic C-terminal portion, is highly specific for ~-thrombin. He then~concludes that the C-t~rm;n~l portion of hirudin is likely to bind to the anionic b;n~;ng site region of the enzyme whereas the compact N-terminal portion appears to bind to the active site region of the enzyme.

It has been found that native desulfo hirudin45~65 inhibits fibrinogen clotting by both bovine and human ~-thrombin in a dose dependent manner. The IC50 value of 940+200 nM for bovine ~-thrombin is in good agreement with the reported value o~ plasma fibrin clot ~ormation for the same fragment and three times lower than hirudin55~65, which had been assigned as the m;n;mllm core required for anticoagulant activity. It has also been ~m~trated that the same peptides were consistently more potent against human ~-thrombin than bovine ~-thrombin.

CA 0221~702 1997-09-17 WO 96129347 PCT~CA96~0016 Various prior art documents have also ~m~n~trated that the active fragment of the amino acid sequence of hirudin appears to be the amino acid sequence including amino acids 45 to 65. Hence, e~forts have been made to enhance S the inhibitory activities of the peptide by substituting some of the amino acids present in this sequence.

Krstenansky et al., in "Antithrom.bin properties of C-terminus of hirudin using synthetic unsul~ated M~-acetyl-hirudin", (1987), Febs Letters, Vol. 211, No. 1, pages 10-16, describe the synthesis of the C-terminal fragment unsulfated Na-acetyl-hirudin45~65. The authors refer to previous work (Chang, J.-V., FEBS Letters, 164, 307 (1983)) and mention that this fragment could potentially contain two specific binding dom~; n.~, one binding to the catalytic site of throm.bin and the other binding to another recognition site on thrombin. This was concluded not to be the case by either authors.

Still, the authors demonstrated that the 45-65 sequence o~
hirudin has the ability to inhibit clotting activity as well as the release of fibrinopeptide A by thrombin. They also suggested that the same sequence of hirudin45~65 may not be directly involved in the binding with the catalytic site of thro-mbin since the amidolytic properties of thrombin toward synthetic substrates is not perturbed.

In the Krstenansky et al. article entitled "Anticoagulant peptides: nature of the interaction of the C-terminal region of hirudin with a noncatalytic site on thro-m-bin (1987), J. Med. Chem., 30, pages 1688-1691, the authors report that the m; n; mllm active sequence at the noncatalytic binding site of thro-m-bin is hirudin56~64.
Based on this assumption, the authors report the synthesis 35 o~ several C-t~rm;n~l hirudin54~65 analogs and their ability to inhibit thrombin-induced fibrin clot formation for the purpose of establishing the nature of the interaction CA 0221~702 1997-09-17 between hirudin56~64 and a noncatalytic binding site of thrombin.

In their conclusion, the authors mention that the C-terminal region of hirudin may bind to a region of fibrinogen b;n~;ng on thrombin that is not the region proposed so far in the literature.

In the articles by Dodt et al. (Interaction of site speci~ic hirudin variants with ~-thrombin, (1988), Febs Letters, Vol. 229, No. 1, pages 87-90), Degryse et al.
(Point mutations modifying the thrombin inhibition kinetics and antithrombotic activity in vivo of recombinant hirudin, (1989), Protein Engineering, Vol. 2, lS No. 6, pages 459-465) and Braun et al. (Use of site-directed mutagenesis to investigate the basis for the specificity of hirudin, (1988), Biochemistry, 27, pages 6517-6522), the authors report the results of site-directed mutagenesis performed on the hirudin gene. The inhibition of thrombin by mutant hirudin peptides is studied.

In these publications, the authors studied mutations effected on the whole protein and did not restrict themselves to the 45-65 segment of hirudin. Furthermore, the modifications performed on the 45-65 segment were restricted to a single modification, usually at position 47, to illustrate that this residue does not interact with the active site, although these publications also show mutations at positions 51, 57, 58, 61 and 62.

In a similar fashion, the article by Dodt et al. entitled "Distinct binding sites of Ala48-Hirudin1~47 and Ala48-Hirudin48~65 on ~-thrombin", (1990), The Journal of Biological Chemistry, Vol. 265, No. 2, pp. 713-718, describes experiments aimed at conducting site-directed mutagenesis of hirudin at position 48 in the sequence. The work done by Dodt et al. in this case seems to have been CA 0221~702 1997-09-17 W O 96/29347 P ~CA96~W164 s restricted to the substitution of alanine for proline at this position in order to facilitate the re~uired proteolysis necessary ~or their experiment.

Finally, Maraganore et al., in "Anticoagulant activity o~
synthetic hirudin peptides", (1989), The Journal of Biological Chemistry, Vol. 264, No. 15, pages 8692-8698, Dennis et al. in "Use of ~ragments of hirudin to investigate throm.bin-hirudin interaction", (1990), Eur. L~ .
Biochem. 188, pages 61-66 and Chang et al. in "The structural elements of hirudin which bind to the ~ibrinogen recognition site of thrombin are exclusively located within its acidic C-terminal tail", (1990), Febs., Vol. 261, No. 2, pages 287-290, describe the synthesis and anticoagulant properties o~ a num.ber of peptides whose se~uences are based on the se~uence o~ various fragments of native hirudin.

Compounds having anticoagulating properties are valuable therapeutics which may be used in vivo in the treatment o~
various pathologic states. Among the most important conditions in which an anticoagulant treatment may be useful, there may be mentioned myocardial in~arction, pulmonary em.bolism and cerebral vascular diseases, deep vein throrbosis and other indications of thrombotic disorders.

Currently available anticoagulants are in many respects unsatisfactor~, For ex&mple, heparin has been eLmployed to inhibit the activity of throm.bin and therefore in the treatment of conditions such as venous throm.bosis and throm.bo embolism. However, heparin exhibits a wide array of undesirable side effects that demonstrate the need for anticoagulants presenting more favorable toxicity levels.
The design of low molecular weight and speci~ic inhibitors o~ thrombin that utilize accessory b; n~; ng loci remote from or in conjunction with the catalytic center, similar CA 0221~702 1997-09-17 to the way flbrlnogen or hirudln binds to thrombln, constltutes a challenge ln proteln chemlstry. Concelvably, such a multifunctlonal lnhibitor lntegrates two or more recognltlve elements, separated by a sultable spacer, that favor multiple slmultaneous lnteractions and which could manifest enhanced potency and speciflclty. Incorporatlon o~ "foreign" chemlcal elements embodled in a structure of low molecular welght could confer reslstance agalnst proteolysls and favourable bloavallablllty. Also, because they are smaller than hirudin, these compounds are less llkely to stimulate an undesirable lmmune response ln patlents treated with them.

PCT applicatlon WO 91/02750 lndlcates that certaln thrombln inhlbltors possess CSDMs that may be cleaved slowly or not cleaved at all. However, all they dlsclose are modlfled bonds between the Arg and Gly or Pro such as Arg[psi CH2-NH]-Gly; ~-HomoArg-Gly; ~-HomoArg-Pro; ~-HomoArg-Val; or Arg-[~CO-CH23-CH2-(CONH)-Gly. There ls no indicatlon that the Gly or Pro amlno acld may be completely ellmlnated and followed by a synthetlc llnker that ls completely reslstant to thrombln cleavage.

SUMMARY OF THE INVENTION

The compound of the present lnventlon ls a peptlde deriva-tlve (I) of the formula (D)-Phe-Pro-Arg-(CH2)4(CO)-[NH-(CH2)4CO]2-DFEPIPL whlch mlmics the carboxyl domaln of hlrudln comprlsing resldues 45-65. The letters D, F, E, P, I and L identlfy amino aclds ln accordance wlth the known single letter code.

In another aspect of the inventlon, there ls provlded pharmaceutlcal compositlon for the treatment of thrombotlc dlsorders comprlslng an effectlve amount of the peptlde W ~96129347 PCT~CA96/00164 6a derivatlve (I) (D)-Phe-Pro-Arg-(CH2)4(CO)-[NH(CH2)4CO]2-DFEPIPL and pharmaceutlcally acceptable salts thereof.

In another aspect of the invention, there is provided a method for the treatment or prophylaxls of vascular CA 0221~702 1997-09-17 diseases related to thrombosis which comprises administering to a patient an effective amount of a composition comprising an effective amount of the peptide derivative (I) (D)-Phe-Pro-Arg-(CH2)4(CO)-[NH(CH2)4CO]2-DFEPIPL .

In further aspects the compound of the invention may also be employed in compositions and methods for in vivo diagnostic imaging, for storing extracorporeal blood in vitro and coating invasive devices, and for ex vivo treatment of blood.

DFT~TTFn DESCRIPTION QF THE INVENTION
The present invention relates to a peptide derivative useful as a thrombin inhibitor. It has been found that the native fragment of hirudin comprising residues 45-65 can interact concurrently with two independent and remote sites on thrombin, one site being the putative anion exosite while the other is the catalytic site responsible for proteolysis. This b; n~; ng mode simulates but is different from the mechanism of the native hirudin molecule which has now been shown to interact with thrombin's active site through its N-terminal three residues. Thus, it appears that residues 45, 46 and 47 do not serve a b; n~; ng role in the native protein but, in the absence of the N-terminal core, are spatially correctly predisposed to interact, albeit weakly.
Based on this observation, we have synthesized a peptide derivative that bears modifications in both inhibitory components of the molecule and which exhibit anti-thrombin activity beyond the level of either portions alone.
Furthermore, chemical modification of the newly formed scissile bond affords a more active compound that has the advantage of being proteolytically stable to thrombin.
The peptide derivative is useful as an anticoagulant and CA 0221~702 1997-09-17 W~ 96~29347 PCT~CA96~00164 as an inhibitor of platelet aggregation, thereby decreasing the risk factor in indications of arterial thrombosis and other related cardiovascular disorders. The compound of the invention may also be used in the treatment of tumor metastasis, such as in the case of carcinomas.

The term "residue", when applied to an ~-amino acid, means a radical derived from the corresponding a-a-mino acid by removing the hydroxyl of the carboxyl group and one hydrogen ~rom the a-amino group.

The abbreviations used herein for designating individual residues are based on the recomm~n~tions of the IUPAC-IUB
Co-m-mission on Biochemical Momenclature [Biochemistry, 11, 1726-1732, (1972)]. The term "amino acid~ used herein includes naturally-occurring amino acids as well as non natural analogs as those 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.

Also within the scope of the present invention is a method 2s for the treatment or prophylaxis of vascular diseases related to thrombosis. The method comprises a~m;n;stering to a patient an effective amount of a composition comprising the peptide derivative in admixture with a pharmaceutically acceptable carrier.
The invention also relates to a method for decreasing reperfusion time or increasing reocclusion time in a patient treated with a thrombolytic agent. The method comprises ~m;n;stering to a patient an effective amount of a composition comprising a peptide derivative according to the invention and a thrombolytic agent in admixture with a pharmaceutically acceptable carrier.

CA 0221~702 1997-09-17 According to another aspect of the present invention, a peptide derivative according to the invention may be used in the treatment of tumor metastases. The efficacy of the derivative for the treatment of tumor metastases is manifested by the inhibition of metastatic growth. This is based upon the presence of a procoagulant enzyme in certain cancer cells. This enzyme activates the conversion of Factor X and Factor Xa in the coagulation cascade, resulting in fibrin deposition which, in turn, serves as a substrate for tumor growth. By inhibiting fibrin deposition through inhibition of thrombin, the oleculs of the present invention serve as effective anti-metastatic tumor agents. Examples of metastatic tumors which may be treated by the trombin inhibitors of this invention include, but are not limited to, carcinoma of the brain, carcinoma of the liver, carcinoma of the lung, osteocarcinoma and neoplastic plasma cell carcinoma.

According to another aspect, a thrombin inhibitor 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 thrombin inhibitor-cont~;n;ng compositions to the surface of the devices.

According to a further embodiment of the present invention, the thrombin inhibitor may be used for diagnostic thrombus imaging in a patient. In this embodiment, the thrombin inhibitor is labelled with a radioisotope. The choice of radioisotope is based upon a number of well-known factors, for example, toxicity, biological half-life, and detectability. Prefered isotopes CA 0221~702 1997-09-17 WO 961293~7 PCT~CA96/00164 include, but are not limited to, 125I 123I and 111In Techni~ues for labelling the thrombin inhibitor are well known in the art. Most preferably, the radioisotope is 123I
and the labelling is achieved using 123I-Bolton-Hunter Reagent. The labelled throbin inhibitor is administered to a patient and allowed to bind to the throbin 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 compouter imaging system. This techni~ue also yeilds images of platelet-bound thrombin and meizothrombin.

In another aspect the above-described thrombin inhibitor, or compositions thereof, may be used as anticoagulants for ex vivo treatment of blood or extracorporeal (in vitro) blood. As used herein, the term "ex vivo" treatment includes blood removed in line from a patient, subjected to extracorporeal treatment, and then returned to the patient such as dialysis procedures, blood ~iltration, or blood bypass during surgery. As used herein, the term llextracorporeal blood" refers to blood products that are stored extracroporeally ~or eventual a~m; n; stration to a patient and blood collected from a patient to be used for various assays. Such products include whole blood, plasma, or any blood ~raction in which inhibition of coagulation is desired.

The peptide derivative 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 such as that described by Stewart et al. in "Solid phase peptide synthesis", Freeman ~ Co., San Francisco, 1969 on a suitable peptide synthesizer. The non-amino acid regions of the peptide derivative re~uires chemical synthesis prior to linking this moiety with the other amino acids to yield the desired peptide through conventional solid phase synthesis. It will be CA 0221~702 1997-09-17 appreciated by those skilled in the art that a skilled organic chemist can readily prepare the synthetic regions o~ the peptide derivative.

Svnthesis of the ~e~tide derivative Peptide derivative may be synthesized on an Applied Biosystems 43OA peptide synthesizer with BOC-GlnPAM resin (Applied Biosystems; 0.64 mmol/gram) as the solid phase support. Amino acid coupling is mediated by dicyclohexylcarbodiimide/N-hydroxybenzoltriazole and deprotection 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), Arg(Tos). A fully protected peptide resin may then be treated with li~uid hydrogen fluoride containing anisole and dimethyl sulfide (10% by volume) at -5~C for 60 minutes. Excess HF may then be removed under a stream of nitrogen and the residual solid extracted with ether and filtered. The resin may then be extracted with glacial acetic acid and water followed by lyophilization.

Pll~ification and analvsis of the pe~tide derivative The resulting lyophilized crude peptide may be purified to homogeneity by using generally accepted peptide puri~ication techniques. One suitable technique is reverse phase chromatography on a Vydac octadecyl silica glass column (15 ~, 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, lL 60% Acetonitrite/H20 contA;ning 0.1%
TFA. The fractions may be 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 may be 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 HCl (200 ul) containing 1% phenol is added to the vial and CA 0221~702 1997-09-17 WO 96129347 PCT~CA96~00~-5i4 alternatively purged (with dry nitrogen) and evacuated after three purges. Finally, the vial contA;n;ng the sample is heated at 150~C for 1 hour under vacuum. Mass - spectral analyses may be carried out on a SCIEX~ API III
S spectrometer equipped with an ionspray inlet source.

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

Pharm~ceutical comnos; tion~.
The peptide derivative of the present invention may be lS obtained in the form of therapeutically acceptable salts.
Since the peptide derivative has residues that function both as acids and/or bases, then salts of organic acids (e.g. acetic, trifluoracetic, lactic, succinic or malic) or bases (e.g. sodium, potassium or calcium) could be derived. These salts of the peptide derivative are fully biologically active. Therapeutically acceptable salts may be converted from one salt form to another by employing a suitable ion exchange resin in a manner described by R.A.
Boissonas et al., Helv. Chim. Acta. 43, 1849 (1960).
The peptide derivative or therapeutically acceptable salts thereof, may be employed alone or in combinations for the treatment of prophylaxis of vascular diseases due to thromboses. It is administered systemically to warm blooded ~n; m~ls, e.g. humans, horses or dogs, with pharmaceutically acceptable carriers, the proportion of composition of which depends on solubility and chosen route of A~m;n;stration. The peptide derivative of the present invention are administered either intravenously, subcutaneously or by intramuscular injection in combination with p~A~mAceutically acceptable carriers.
Examples of suitable carriers are found in standard pharmaceutical texts, e.g. "Remington's Pharmaceutical CA 0221~702 1997-09-17 Sciences", 16th edition, Mack Publishing Company, Easton, Penn., 1980.

The dosage of the peptide derivative will vary depending on the form of a~m;n;,ctration and possibly on the particular salt. In the case o~ an injection, the therapeutically effective dose of the peptide derivative is in a dosage range of approximately 0.05 mg/kg to 10 mg/kg body weight. In addition to the active ingredient, the compositions usually also contain suitable buffers, for example phosphate buffer, to maintain an appropriate pH and sodium chloride, glucose or mannitol to make the solution isotonic.

The peptide derivative of the present invention may be administered alone or in combination with other p~rm~ceuticalS. For example, the peptide derivative may be administered in combination with a fibrinolytic such as tissue pl~cm;nogen activator, streptokinase or urokinase to prevent reocclusion of coronary arteries.
Alternatively, the peptide derivative could be administered with heparin or low molecular weight heparin, a combination which could advantageously lower the dosage of heparin or low molecular weight heparin. Other compounds which may be administered along with the peptide derivative include thromboxane and EPIIb3a.

Abbreviations used in the ~ollowing examples include BOC:
tert-butoxycarbonyl; Tos: p-toluene sulfonyl; CH2C12:
methylene chloridei TEA: triethylaminei BOP:
benzotriazolyl N-oxytrisdimethylamino phosphonium hexafluorophosphate; DMF: dimethyl formamidei EtOAc: ethyl acetate; DCC: N,N'-dicyclohexylcarbodiimide; DPPA:
diphenyl-phosphoryl azide; THF: tetrahydro~uran; HF:
hydrogen fluoride, CBZ: benzyloxycarbonyl.

CA 0221~702 1997-09-17 ~MPLE 1 Synthesis of (2S)-2-(BOC)-N-methoxy-N-methyl-5-tosylguanadinopent~nAm;de.
To a solution of N~-BOC-NG-Tosyl Arginine (428 mg, 1 mmol) in 30 ml o~ DMF, at 0~C in an ice bath, containing TEA (O.4 ml, 3 mmol) and N,O-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.
10 Selve, Te~rahedron 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 H20. The organic phase was extracted further with 5~ NaHCO3 (3 times), lN HCl (3 times) and dried over Na2SO4. The solvent was filtered over celite and concentrated in vacuo. Addition o~ a small amount o~ hexane to the concentrate, deposited a white solid (500 mg) corresponding to the title compound. Mass spectral analysis: M/Z = 472 (M+H)+.

~.~Z~MPT.~. 2 Synthesis of 6-BOC-9-tosylguanidino-1-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-1-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-l-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 guenched with aqueous NH4Cl after TLC showed disappearance o~ starting material (TLC
was performed on Kieselgel~ 60F 254, Merck, glass plates).
The phases were separated and the organic phase was washed further with lN HCl and H2O, dried (Na2SO4) and evaporated under vacuum. Chromatography on silica gel (eluting with CA 0221~702 1997-09-17 4:l EtOAc/hexane afforded a clear oil corresponding to the title compound. Mass spectral analysis M/Z = 469 (M+H)+.

F~MPLE 3 s Synthesis of 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 l00 mg of ruthenium chloride. After one hour vigorous stirring at room temperature, no starting material was observed by TLC. The mixture was diluted with l00 ml of H20 and l00 ml of ether. The phases were separated and the aqueous phase was extracted further with ether. The combined organic extracts were washed with H20, dried ((Na2SO4) and evaporated to dryness affording l.5 g of a foam corresponding to the title compound. M/Z = 485 (M+H)+.

20 EX.PMPT .F. 4 Synthesis of 6-BOC-5-oxo-9-tosylguanidinononanoic acid.
The title compound of this example was synthesized in a manner analogous to examples l to 3. Briefly the product 25 from example l 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)+.

F~MPT~ 5 Synthesis of 7-BOC-6-oxo-l0-tosylguanidinodecanoic acid.
The title compound of this example was prepared in a manner analogous to examples l to 4. In this example, the product from example l was reacted with the Grignard reagent prepared from Magnesium and 6-bromo-l-hexene.

CA 0221~702 1997-09-17 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 a~forded the title compou~d as an oil. M/Z = 513 (M+H)+.

~Z~MPT.~. 6 O O
tBoc-NH ~'D"S-C2H5 NH
~= NH
Tos~
Ethyl; 4N-t-BOC-3-oxo-7-tosylguanidine thioheptanoate (mixed anhydride method).
Formation of mixed anhydride: to a stirring solution of 1 g (2.4 mmol) of (L)-N~-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 0.40 ml (0.3 mmol) o~
isobutylchloroformate dropwise over 15 minutes. After 1 hour the mixture was diluted with 15 ml of ether and the precipitated solid was filtered. The filtrate contA;n;ng 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 e~uivalent of N-But Li in THF
dropwise over 30 minutes. After, the reqction 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 CA 0221~702 1997-09-17 was continued for 5 hours until reaction was complete by HPLC.
The reaction mixture was treated dropwise with 6 M
NH4C1 and the phases were separated. The organic phase was diluted with 50 ml of EtOAc and extracted with lN HC1 (3 X), H2O (3 X) dried with Na2SO4 and evaporated under high vacuum affording the title compound as an oil M/Z =
515 (M + H)+.

10 E~Z~MPT.F. 7 .

Coupling of the thioester from example 6 to ~-amino acid esters and deprotected amino acyl polystyrene resins.
The protected arginyl statone from example 6 (2 equivalents) was dissolved in CH2Cl2 and added to a mixture of a-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 o~ polystyrene bound peptides.

Preparation of the subunit of the synthetic spacer of formula II: -[NH-CH2-CH=CH-CH2-(CO)]3-The synthesis was modeled upon (Cox M.T., Heston D.W.and Horbury J., J. Chem. Soc. Chem. Comm., 1980, 799-800) with major modi~ications. The complete process is outlined as follows.
a) Synthesis of trans-~-hydromuconic acid dimethyl ester.
22 g (153 mmol) o~ trans-~-hydromuconic acid was dissolved in 200 ml of benzene cont~;nlng 500 mg of p-toluene sulfonic acid and 100 ml of methanol. Thesolution 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~ NaHCO3 and H2O.

CA 0221~702 1997-09-17 WO 96r29347 PCT/CA96~00~6' After drying (Na2SO4), 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-~-hydromuconic acid monomethyl ester.
5 g (27.5) mmol) o~ the product from step a) was suspended in 100 ml o~ a solution o~ 0.1 M KH2P04 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 o~ 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 fittered over celite. The filtrate was extracted with ether and the combined organic extracts were discarded.
The aqueous phase was made acidic with 3 N HCl and reextracted with ether. The combined ethereal extracts were dried (Na2SO4) 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. A~ter 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 (Na2SO4) and filtered.
The filtrate was heated in an oil bath at 80~C until no ~urther nitrogen evolution was observed. The solvent was evaporated under vacuum and the residue distilled under reduced pressure (80-85~C, 0.5 mmHg) affording 700 mg of the title compound.
d) Synthesis of 4-N-Butyloxycarbonyl-pent-3-en-oic acid.

=
CA 0221~702 1997-09-17 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 o~ 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 ~orm spacer (II) using techniques that are well known to those skilled in the art.

F.~MPT.~ 9 Preparation of P79 Ac-(D)Phe-Pro~-Arg -(COCH2)CH2-(CO)-Gln-Ser50-His-Asn-Asp-Gly-Asp55-Phe-Glu-Glu-Ile-Pro60-Glu-Glu-Tyr-Leu-GlnOH
1 g of tert-Butyloxycarbonyl-Gln phenylacetami-domethyl resin (Applied Biosystems; 0.64 mmol/g) was carried through 16 cycles of synthesis involving n~-side chain deprotection (50% TFA in CH2Cl2 and coupling using 2.5 me~ of protected amino acid/DCC and N-hydroxybenzo-triazole. Side chain protecting groups of st~n~d amino acids were as follows: Asp (cyclohexyl), Glu (benzyl), His (benzyloxymethyl), Tyr (bromobenzyl), Ser (benzyl).
The synthetic protected amino acid from example 3 was coupled to Gln49 also using DCC/N-hydroxybenzo-triazole.
For optimum results, N-BOC-(D)-Phe-Pro-OH could be added at a single unit instead of individual amino acids.
The fully protected peptide resin (500 mg) was treated with hydrogen fluoride in a te~lon vessel containing anisole and dimethyl sulfide (10~ by volume) at -5~C for 60 minutes. Excess HF was removed under a stream of N2 and the residual mass was extracted with ether and filtered. The resin was extracted three times with glacial acetic acid and water, followed by lyophilization.
The lyophilized crude peptide was purified to homogeneity by reverse phase chromatography on an CA 0221~702 1997-09-17 W ~96129347 PCT/CA96~00164 octadecyl silica (15 A, Vydac) glass column (1.5 X 30 cm), 40 psi using a linear gradient of a solvent system consisting of (a) 500 ml of 0.1% TFA/H20 and (B) 1 liter of 60% acetonitrile H2O cont~; n; ng O .1% TFA. Fractions S corresponding to 98% purity or higher were pooled and lyophilized.
Amino acid analysis indicated: Asp (3), Ser (1), Glu (6), Gly (1), Ile (l), Leu (1), Tyr (1), Phe (2), His (1), Pro (2).
lo The resulting peptide showed a pseudo molecular ion corresponding to 2548.6.

~.X~MPLE 10 Preparation of P183.
Ac-(D)-Phe-Pro-Arg-[(CO)-CH2]-CH2CH2CH2-(CO)-[NH-CH2-CH=CH-CH2-(CO)]-Asp-Phe-Glu-Pro-Ile-Pro-Leu-OH.
The ~itle peptide derivative was synthesized and purified essentially as described for P79 and its homologs with minor modifications. For example, the solid phase synthesis began with tert-butyloxycarb- onyl-Leu phenylacetamidomethyl polystyrene resin (Applied Biosystems, 0.64 mmol/g). For deprotection of the t-BOC
group following the Asp residue, 50% TFA in CH2C12 cont~;n;ng 10% ethylmethyl sulfide was used. In this way, the yield of the title peptide was optimized to greater than 60%, by HPLC (W absorption at 215 nm).

P184 and P185 were prepared in a similar manner as P183 Ac-(D)-Phe-Pro-Arg-[(CO)CH2]CH2CH2CH2(CO)-[NH-CH2-CH=CH-CH2-(CO)]2-Asp-Phe-Glu-Pro-Ile-Pro-Leu-OH.
Amino acid analysis indicated: Asp (1.00), Glu 3s (1.08), Ile (0.96), Leu (1.01), Phe (1.91), Pro (3.48).
Pseudo molecular ion: 1553.

P185.

CA 0221~702 1997-09-17 Ac-(D)-Phe-Pro-Arg-[(CO)CH2]CH2CH2CH2-(CO)-[NH-CH2-CH=CH=CH2-(CO)]3-Asp-Phe-Glu-Pro-Ile-Pro-Leu-OH.
Amino acid analysis indicated: Asp (1.00), Glu (1.06), Ile (0.93), Leu (0.98), Phe (1.88), Pro (3.6).
s Pseudo molecular ion: 1647.

Peptide derivative (I) (D)-Phe-Pro-Arg-(CH2)4(CO)-[NH(CH2)4CO]2-DFEPIPL
was prepared in a similar manner as P183, P184 and P185 except the synthetic residue aminovaleric acid was used in place of [NH-CH2-CH=CH-CH2-(CO)].
Amino acid analysis indicated: Asp (0.97), Glu (1.00), Ile (1.04), Leu (1.02), Phe (1.96), Pro (2.86).

~MPLE 11 ~;dolytic assay of thrombin activitv Thrombin-catalyzed hydrolysis of Tos-Gly-Pro-Arg-pNA was monitored at 405 nm on a Varian Cary 2000 double beam spectrophotometer using substrate concentrations of 2.5, 3.5, 5 and 10 ~M in a final volume o~ lmL. The hydrolytic reactions were performed at 25~C in 0.1 M Tris-HCl buffer, pH 7.8, cont~;n;ng 0.1 M NaCl and 0.1% PEG 6000. The reactions were initiated by addition of the substrate dissolved in 0.1 M Tris-HCl buffer, pH 7.8 to a preincubated solution of enzyme (0.4 or 0.04 nM) and variable concentrations of inhibitor dissolved in the same buffer. Initial velocities were recorded and Ki values were determined graphically by weighted linear regression of Dixon plots in the case of competitive inhibition, or by the method of Baici (Baici, 1981) for hyperbolic inhibition. Fluorogenic assays were conducted using the same conditions and instrument as above operating in the fluorescence mode in the Ratio (~ex=383 nm, ~em=455 nm). Fluorescence intensities were calibrated with 7-CA 0221~702 1997-09-17 amino-4-methyl coumarin solution of known concentration.
The specificity of the synthetic peptide derivative of the present i~vention for ~l-m~n a-thrombin may also be - determined by comparing its relative inhibitory activities towards both human ~-thrombin and bovine a-thrombin and trypsin by comparing Ki values obtained in the amidolytic assay of thrombin activity.

Hence, the inhibitory activity of the peptide derivative of the present invention towards thrombin may also be assayed by determination of prothrombin time (PT, extrinsic pathway) or activated partial thromboplastin time (APTT, intrinsic pathway) of pooled reconstituted normal ~llm~n plasma using a Coag-A-Mate 2001 instrument (General Diagnostics Inc., Morris Planes, New Jersey) or other suitable spectrophotometer.

Thus, for the determination of prothrombin time, 50 ~1 of reconstituted citrated normal human plasma (Sigma, St-Louis, MO.) is mixed with 50 ~1 o~ thromboplastin solutionat 37 ~C in a 400 ~1 cuvette. The mixture is then treated with either 200 ~1 of Tris-HCl buffer pH 7.8 (cont~;n;ng O.lM NaCl, 0.1% PEG 6000) or variable concentrations of inhibitor in the same buffer. The clotting time is recorded following recalcification with 100 ~1 of 25 mM
CaC12. The clotting time in the absence of inhibitor was between 19-22 sec.

The same procedure is adopted for the determination of activated partial thromboplastin time except that reconstituted plasma is activated for 3 minutes with cephalin (Sigma, St-Louis, MO.).

The inhibitory activity of the peptide derivative toward thrombin is reflected by its ability to inhibit thrombin-mediated platelet aggregation which is indicated by increased light transmittance which is measured on a Bio Data PAP-4 aggregometer.

CA 0221~702 1997-09-17 W 096/29347 PCTtCA96100164 F;hrino~en clottina assay Inhibition of fibrinogen clot formation was measured spectrophotometrically at 405 nm on a Varian DMS 90 at 37~C. 300 ~L of 0.1% fibrinogen (Sigma) in O.lM Tris-HCl, pH 7.8, contA;n;n~ O.lM NaCl, 0.1% PEG 600 and variable concentrations of inhibitor in the same buffer were mixed in polystyrene cuvettes and the reaction was initiated by the addition of the enzyme (human or bovine ~-thrombin 0.4 nM) in a total volume of 1 mL. The time from mixing to inflection due to clot formation was recorded for various inhibitor concentrations and IC50 values were calculated by log probit analysis. The concentrations of the inhibitors in the assays was based on the peptide content.
Various other assays may be used to determine the anticoagulant activity of the peptide derivative of the present invention. Hence, the inhibitory activity of the peptide derivative towards thrombin may also be assayed by the inhibition of activated partial thromboplastin times (APTT intrinsic pathway or prothrombin time PT extrinsic pathway). Thus, the anticoagulant activity may be determined by assaying APTT of pooled, normal human plasma with a Coag-A-Mate 2001 instrument (General Diagnostics Inc., Morris Planes, Mew Jersey).

Furthermore, the peptide derivative of the present invention may be tested for inhibition o~ thrombin-catalyzed hydrolysis o~ the tripeptidyl P-nitroanilide substrate tosyl-Gly-Pro-Arg-P-nitroanilide (Chromozym TH, Boehringer-MAnnheim, Indianapolis, In.) spectrophotometrically at 420 nm on a Cary 219 double-beam spectrophotometer. The reactions may be prepared by mixing a thrombin solution with a Tris-HCl, pH 7.4, NaCl buffer.

These assays, when per~ormed using the peptide derivative of the present invention, demonstrated that the it acts as CA 0221~702 1997-09-17 WC> 96129347 PCT~CA96/00164 a bifunctional inhibitor o~ thrombin. Indeed, it was ~mo~trated that the incorporation of the two critical regions o~ the peptide separated by a suitable spacer provided strong thrombin inhibitors. Results are shown in Table I.
.~
It is clear that the incorporation of the two b; n~; ng regions into a single molecule separated by a suitable linker substantially increases the af~inity o~ the compounds for thrombin. In fact, the combination of separate IC50 doses of the two independent regions results in the exact doubling of the clotting time whereas greater activity is obtained if the two regions are joined by a linker. It there~ore appears that dual cooperative 15 b; n~; ng of the bifunctional inhibitors of the present invention takes place when these are contacted with thrombin. The linker serves as a suitable spacer for the bridging of an auxiliary site (region (ii)) and a catalytic site (region (i)) as well as an apolar binding site adjacent to the catalytic site.

Ferric chloride iniurv-induced thrombosis model Species: Rat, male, Sprague-Dawley.
Weight: 375-450g The FeC13 induced arterial injury model assays were conducted according to 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 ( 375- 450 g) are anesthetized with Urethane ( 1500 mg\kg IP). ~n;m~l s are laid on a heating pad which is maintained at 37~C. The carotid artery is approached through a midline cervical incision. Care~ully blunt dissection is used to expose and isolate the vessel CA 0221~702 1997-09-17 from the carotid sheath. Using forceps, the artery is lifted to provide clearance to insert two small polyethylene tubing (PE-205) underneath it. The temperature probe (Physitemp MT23/3) TM is placed between the PE-205 and the artery. The vessel temperature is monitored for 60 minutes after application oi~ FeC13. J
Vessel temperature changes are recorded on a thermister (Cole-Palmer Model 08533-41). Injury is induced by application of a small disc (3 mm dia.) of WhatmanTM No.l 10 filter paper previously dipped in a 35% solution of FeCl3 on the carotid artery above the temperature probe. The site of the experiment is covered with in Aluminum foil in order to protect the FeCl3 from degradation by light.

lS The time between the Ferric Chloride application and the time at which the vessel temperature decreases abruptly (>2.4~C), is recorded as the time to occlusion (TTO) of the vessel.

Before the start o~ the experiment, one blood sample is drawn (1 ml) in a tube of 0.105M buffered citrate solution (from the eye's sinus) and the ~n;m~l is exsanguinated at the end. All the samples are kept on ice and centrifuged as soon as possible at 2000 Rpm for 10 min., 4~C. The 25 plasma is analyzed in duplicate for activated partial thromboplastin time on a haemostasis analyzer (STAG0 ST4TM
) -From a group of four ~n;m~l S, two arteries are stored at -30 80C for further analysis. The others are observed under a light microscope at 40X (LeicarM) for quantification of the occlusion ( complete, partial, no occlusion).

Platelet aa~reaation assay Human platelets were separated from donated blood and the twice-washed suspensions were prepared according to the method described by Packham et al. Rat blood was CA 0221~702 1997-09-17 WO 96129347 PCT~CAg6~0016 collected into ACD (6:1, v/v) by cardiac puncture.
Suspensions of washed platelets were prepared as described by Ardlie et al (Br. J. Haematol. 1970, 19:7, Proc. Soc.
~ Exp. Bio. Med. 1971, 136:1021). The ~inal suspending medium was a modi~ied Tyrode solution (NaCl 138 mM, KCl 2.9 mM, HEPES 20mM, NaH2P04 0.42mM, CaCl2 lM, MgCl2 2mM, 0.1~ glucose, 0.35% albumin, apyrase (lul/ml) pH7.4).
Platelet counts were adjusted to 5000,000/ul. To permit measurement of the extent of the release of the contents of the dense granules, the platelets were labeled in the first washing solution with 14C-serotonin (luCi/lOml of washing fluid), and release of 14C-serotonin was determined. Impramine (5uM final conc) was added to prevent re-uptake of released serotonin. A platelet aggregome}er was used for the assay (4 c~nn~l BioData PAP4, Hatboro, PA, USA). Percentage of aggregation was determined 3 min after the addition of the stimulating agent (human O.1 U/ml final conc). Inhibitors were preincubated 1 min at 37C before addition of stimulating agent. IC50 values were the concentration necessary to inhibit platelet aggregation or secretion to 50% of the control.

TABLE I
Compound Ki IC50 IC50 Dose to double IC50 (pM) dTT (nM)~ plasma patency time thrombin-induced clotting time (m9/k9)platelet aggregation (nM)~ (nM) Hirulog 230 1.8 12 2 8 P184 1500 10.5 52 1-2 6 _ (I) 100 1.3 3.1 1 0.7 The dose of heparin nede to cause a doubling a patency time is 200 U/kg.
- denotes not d~l~r",;"ed. Values are means for 3-5 observations.
Patency time in control (saline-treated) rats is 19~ 1 min (n=11).
* Concenlr~lion of compound required to double ll " u". , time in buffer cor,l~i" ,g fibrinogen.
30 ** Concellt,~liorb of compound required to inhibit human plasma clotting time by 50%.

,

Claims (3)

WE CLAIM

1. The peptide derivative (I):
(D)-Phe-Pro-Arg--(CH2)4(CO)-[NH(CH2)4CO]2-DFEPIPL and pharmaceutically acceptable salts thereof.

2. A composition for the treatment of thrombotic disorders comprising an ef fective amount of the peptide derivative (I) (D)-Phe-Pro-Arg-(CH2)4(CO)-[NH(CH2)4CO]2-DFEPIPL and pharmaceutically acceptable salts thereof.

3. 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 2.