AU6539190A - Anti-thrombotic peptides and pseudopeptides - Google Patents

Description

TO WHOM IT MAY CONCERN:

Be it known that we, Scott I. Klein, 436 Mill Grove Drive, Audubon, PA 19403; Bruce F. Molino, 2825 North

Ford Drive, Hatfield, PA 19440; Mark Czekaj, 11 Pine Run Drive, Holland, PA 18966; Charles J. Gardner, 644 South 4th Avenue, Royersford, PA 19468; and Jeffrey C.

Pelletier, 1308 Lakeview Drive, Lansdale, PA 19446, all citizens of the United States of America, have invented new and useful improvements in

ANTI-THROMBOTIC PEPTIDES AND PSEUDOPEPTIDES of which the following is a full and exact description as will enable those skilled in the art to which it

appertains to make and use the same.

ANTI-THROMBOTIC PEPTIDES AND PSEUDOPEPTIDES

This application is a continuation-in-part of

application Serial No. 415,006 filed on September 29, 1989, and issued on August 28, 1990, as U.S. Patent No. 4,952,562.

Background of the Invention 1. Field of the Invention

This invention relates to novel compounds having anti-thrombotic activity. More particularly, the

invention relates to novel peptides and pseudopeptides that inhibit platelet aggregation and thrombus formation in mammalian blood thereby being useful in the prevention and treatment of thrombosis associated with certain disease states, such as, myocardial infarction, stroke, peripheral arterial disease and disseminated intravascular coagulation.

2. Description of the Prior Art

Haemostasis, the biochemistry of blood coagulation, is an extremely complex and as yet not completely

understood phenomena whereby normal whole blood and body tissue spontaneously arrest bleeding from injured blood vessels. Effective haemostasis requires the combined activity of vascular, platelet and plasma factors as well as a controlling mechanism to prevent excessive clotting. Defects, deficiencies, or excesses of any of these components can lead to hemorrhagic or thrombotic

consequences. Platelet adhesion, spreading and aggregation on extracellular matrices are central events in thrombus formation. These events are mediated by a family of platelet adhesive glycoproteins, i.e., fibrinogen, fibronectin, and von Willebrand factor. Fibrinogen is a co-factor for platelet aggregation, fibronectin supports platelet attachments and spreading reactions, and von Willebrand factor is important in platelet attachment to and spreading on subendothelial matrices. The binding sites for fibrinogen, fibronectin and von Willebrand factor have been located on the platelet membrane

glycoprotein complex Ilb/IIIa.

Adhesive glycoprotein, like fibrinogen, do not bind with normal resting platelets. However, when a platelet is activated with an agonist such as thrombin or adenosine diphosphate, the platelet changes its shape, perhaps making the GPIIb/IIIa binding site accessible to

fibrinogen. The novel molecules described in this

invention may block the fibrinogen receptor, thus

inhibiting platelet aggregation and subsequent thrombus formation. Pharmaceutical agents and/or compositions possessing such an inhibiting effect may be provided for the prophylaxis and treatment of thrombogenic diseases, such as myocardial infarction, stroke, peripheral arterial disease and disseminated intravascular coagulation.

It has been observed that the presence of Arg-Gly-Asp (RGD) is necessary in fibrinogen, fibronectin and von Willebrand factor for their interaction with the cell surface receptor (Ruoslahti E., Pierschbacher, Cell 1986, 44, 517-18). Two other amino acid sequences also seem to take part in the platelet attachment function of

fibrinogen, namely, the Gly-Pro-Arg sequence, and the dodecapeptide, His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-

Asp-Val sequence, small synthetic peptides containing the RGD or dodecapeptide have been shown to bind to the platelet GPIIb/IIIa receptor and competitively inhibit binding of fibrinogen, fibronectin and von Willebrand factor as well as inhibit aggregation of activated

platelets (Plow et al. Proc. Natl. Acad. Sci. USA 1985, 82, 8057-61; Ruggeri et al. Proc. Natl. Acad. Sci. USA 1986, 5708-12; Ginsberg et al. J. Biol. Chem. 1985, 260, 3931-36; and Gartner et al. J. Biol. Chem. 1987, 260, 11,891-94).

The present invention is directed to novel peptides and pseudopeptides which inhibit platelet aggregation and subsequent thrombus formation.

SUMMARY OF THE INVENTION

In accordance with the present invention, novel peptides and pseudopeptides are provided for the

prophylaxis and/or treatment of thrombotic disease states having the general formulae: 2

I

II

III

R

IV and pharmaceutically acceptable salts thereof, wherein:

X is H,

1

- -

Y is H,

alkyl,

cycloalkyl,

aralkyl,

-R¹

-NR¹R² or - R²;

A, B and D are independently -NR²,

CH₂O, or

CH₂-NR²;

E is H,

CH₃, or K is H,

CH₃,

CH₂- OH,

CH₃CH-OH,

C

;

'

F is

or

CH₂;

G is OR²,

R¹ and R² are independently:

H,

alkyl,

cycloalkyl,

aryl, and

aralkyl;

Z is OR¹, NH₂ or NR¹R²; m is 0-9 ; and n is 0-5 ; provided that, in formula I, when X is NH₂, then: m is 3,

Y is -NH₂,

NH E is ; and

that at least one radical in A, B and D is not -NH; and provided further that, in formula III, when X is

NH₂ then:

Y is NH₂, m is 3, F is G is OH; and that one radical in A and B is not -NH.

DETAILED DESCRIPTION OF THE INVENTION

As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

"Alkyl" means, either alone or within the various substitutents, defined hereinbefore, a hydrocarbon having one to about 20 carbon atoms. "Lower alkyl" means alkyl having one to about six carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, amyl and hexyl. Preferred lower alkyl includes methyl, ethyl and propyl.

"Aryl" means a mononuclear and polynuclear aromatic hydrocarbon radical which can be substituted or

unsubstituted in one or more positions. Examples of aryl groups include phenyl, naphthyl, anthranyl, phenanthranyl, azulyl and the like which can be substituted with one or more of the substituents. Aryl is preferably substituted or unsubstituted phenyl or naphthyl. Aryl substituents include hydrogen, alkyl, alkoxy, amino, halo, aryl, eryloxy, carboalkoxy, nitro, dialkylamino,

trifluoromethyl, thioalkyl and carbamoyl. "Aralkyl" means an alkyl group substituted by an aryl radical. The preferred aralkyl groups are lower alkyl groups substituted by phenyl or substituted phenyl. The most preferred aralkyl group is benzyl. In accordance with the present invention, novel compounds are provided which inhibit platelet aggregation by inhibiting fibrinogen binding to activated platelets and other adhesive glycoproteins involved in platelet aggregation and blood clotting. Compounds of the present invention, as tested by methods predictive of anti- thrombotic activity, are believed to be useful in the prevention and treatment of thrombosis associated with certain diseased states, such as myocardial infarction, stroke, peripheral arterial disease and disseminated intravascular coagulation.

The present compounds may also be useful for the treatment of certain diseases associated with abnormal cell growth since they may interfere with adhesive

interactions between abnormal cells and the extracellular matrix (Journ. of Biol. Chem., Vol. 262, No. 36 1987, pp. 17703-17711; Science, Vol. 233, 1986, pp. 467-470; and Cell, Vol. 57, 59-69, Apr. 1989).

The compounds of the present invention may be readily prepared by standard solid phase or solution phase peptide synthesis using starting materials and/or readily

available intermediates from chemical supply companies such as Aldrich or Sigma, (H. Paulsen, G. Merz, V.

Weichart, "Solid-Phase Synthesis of O-Glycopeptide

Sequences", Angew. Chem. Int. Ed. Engl. 27 (1988); H.

Mergler, R. Tanner, J. Gosteli, and P. Grogg, "Peptide Synthesis by a Combination of Solid-Phase and Solution Methods I: A New Very Acid-Labile Anchor Group for the Solid-Phase Synthesis of Fully Protected Fragments.

Tetrahedron letters 29 , 4005 (1988); Merrifield, R.B., "Solid Phase Peptide Synthesis after 25 Years: The Design and Synthesis of Antagonists of Glucagon", Makromol. Chem. Macromol. Symp. 19, 31 (1988)).

We prefer to use the solid phase method schematically represented as follows: solid support

solid support

solid support

wherein: the solid support may be, but is not limited to, p-alkoxy benzyl resin; and- P is an N-protected amino acid. In the synthetic process of making the desired compound the amino acid derivatives are added one at a time to the insoluble resin until the total sequence has been built up on the resin. The functional groups of the amino acid derivatives are protected by blocking groups to prevent cross reaction during the coupling procedure.

These blocking groups include α-tertiary butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ), benzyl, t-butyl, 9-fluor- enylmethyloxycarbonyl (FMOC), 2-(trimethylsilyl)ethyl, and 4-methoxy-2,3,6-trimethylbenzenesulfonyl. Upon completion of the coupling reaction a functional group is deprotected by standard methods to give an active α-amino function which, in turn, is reacted with a protected amino acid derivative having a free α-carboxyl function thereon.

This procedure is repeated until the desired peptide or pseudopeptide is formed. The compound is then deprotected and removed from the solid support by standard procedures to obtain the final product.

Alternatively, the compounds of the present invention may be prepared in solution, i.e., without using a solid support. In a manner that is similar to the solid phase synthesis the protected amino acid derivatives or analogs are coupled by using standard procedures, then deprotected to yield the desired final compound.

The invention will now be further explained by the following illustrative examples:

EXAMPLE 1

L-Arginyl-L-Aspartyl-L-Valine

1.0g of N-(9-fluorenylmethyloxycarbonyl)-L-valine p- alkoxybenzyl alcohol resin ester (containing 0.56 mmole of amino acid) is shaken with 20 ml of 20% (v/v) piperidine in methylene chloride for 1 hour to remove the FMOC group. The mixture is filtered and the resin washed with

methylene chloride. The deprotected resin is treated with 0.92g of N-FMOC-L-aspartic acid-β-t-butyl ester in 15 ml of dimethylformamide in the presence of 0.43g 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 0.31 ml triethylamine, and 0.30g 1-hydroxybenzo- triazole (HOBT), for 1 1/2 hours. This is filtered, washed with methylene chloride, and the resulting resin treated with 20% piperidine in methylene chloride as above to remove the FMOC group. The resulting resin derivative is then treated as above with 1.36g N-α-FMOC-N-ω-(4- methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine in the presence of triethyl-amine, EDC, and HOBT. The FMOC group is removed as above. The peptide is removed from the resin by treating with 20 ml of 95% trifluoroacetic acid for two hours. The arginine residue is deprotected by overnight treatment with concentrated trifluoroacetic acid. The resulting solution is diluted with 0.5% acetic acid, washed with 3 portions of ethyl acetate, then lyophilized to give L-arginyl-L-aspartyl-L-valine as the ditrifluoroacetate salt; m.p. 90-95ºC. EXAMPLE 2

L-Arginylglycyl-L-Aspartyl-α-Isobutylamide

A. 1.16g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.93 ml of triethylamine are stirred together in 50 ml of methylene chloride for 10 minutes. 2.5g N-α-(FMOC)-L-aspartic acid β-t-butyl ester, 0.60 ml isobutylamine and 0.82g hydroxybenzotriazole

(HOBT) are added and the solution stirred at room

temperature overnight. The solution is diluted with ethyl acetate, washed twice with water and dried over magnesium sulfate. The filtered solution is evaporated in vacuo to give 2.2g N-α-(FMOC)-L-aspartic acid isobutyl amide β- butyl ester. B. The amide obtained in 2A is dissolved in 20% (v/v) piperidine in methylene chloride and stirred at room temperature for 2 hours. The solution is evaporated in vacuo and the residue dissolved in ethyl acetate and this solution is washed with 10% sodium bicarbonate solution, dried over sodium sulfate, filtered and evaporated to give 1.7g L-aspartic acid-α-isobutyl amide-β-t-butyl ester.

C. 0.67g N-FMOC glycine and 0.55g of the amide obtained in 2B are treated under the conditions of 2A to give N-α-(FMOC) -glycyl-L-aspartic acid isobutyl amide-β- butyl ester.

D. The product obtained in 2C is treated as in 2B to remove the FMOC protecting group to give glycyl-L- aspartic acid isobutyl amide-β-butyl ester.

E. 0.40g of the product of 2D and 0.78g N-α-t-BOC- N-ω-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine are treated as in 2A with 0.29g EDC, 0.17g HOBT and 0.18 ml triethylamine to give N-α-BOC-N-ω-methoxy-2,3,6- trimethylbenzenesulfonyl)-L-arginylglycyl-L-aspartic acid isobutyl amide-β-butyl ester. F. 0.35g of the product obtained in 2E is treated with concentrated trifluoroacetic acid in the presence of two drops of ethanedithiol overnight. The solution is diluted with 0.5% acetic acid and washed with 4x100 ml of ethyl acetate. The aqueous solution was lyophilized to 0.19g of a white solid, L-arginylglycyl-L-aspartyl-α- isobutylamide as the ditrifluoroacetate salt; m.p. 90- 95°C.

EXAMPLE 3

L-Ornithylglycyl-L-Aspartyl-Valine

A. 1.27g L-valine t-butyl ester and 2.5g N-α-FMOC- L-aspartic acid β-t-butyl ester are treated as in 2A in the presence of 1.16g EDC, 0.93g triethylamine and 0.82g hydroxybenzotriazole. The resulting product is then deprotected as in 2B to give L-aspartyl-β-t-butyl ester-L- valine-α-t-butyl ester.

B. 1.1g of the product obtained from 3A is treated with N-α-FMOC-glycine in the presence of 0.60g EDC, and 0.43g of triethylamine in methylene chloride as in 2A and the resulting product deprotected in 20% piperidine in methylene chloride as in 2B to give 0.65g glycyl-L- aspartyl-β-t-butyl ester-L-valine-α-t-butyl ester.

C. 0.25g of the product from 3B is treated with 0.23g N-α-t-BOC-N-δ-CBZ-ornithine in 5ml of methylene chloride in the presence of 0.12g EDC, 0.80g HOBT and 0.09 ml triethylamine as in 2A to give 0.45g N-α-t-BOC-N-δ-CBZ- L-ornithγl-glycyl-L-aspartyl-β-t-butyl ester-L-valine-α-t- butyl ester. D. The benzyloxycarbonyl protecting group on the product compound of 3C is removed by dissolving 0.45g of the protected compound in 20 ml of cyclohexene and adding O.lOg 10% palladium on carbon and heating at reflux, under nitrogen, for 2 hours. The resulting solution is

filtered, evaporated, and chromatographed on silica gel in chloroform/methanol/water 90:10:3 to give 0.25g N-α-t-BOC- L-ornithyl-glycyl-L-aspartyl-β-t-butyl ester-L-valine-t- butyl ester. E. 0.23g of the product obtained in 3D is dissolved in 5 ml trifluoroacetic acid with 3 drops of ethanedithiol added. The solution is stirred for 7 hours, evaporated, and the residue partitioned between ethyl acetate and 0.5M acetic acid. The aqueous portion was separated and lyophilized and the resulting solid purified by HPLC to give L-ornithyl-glycyl-L-aspartyl-L-valine as the

ditrifluoroacetate salt; m.p. 122-25°C. EXAMPLE 4

L-Arginylsarcosyl-L-Aspartyl-L-Valine N-α-FMOC-sarcosine was substituted for N-α-FMOC- glycine and the resulting product was treated with

piperidine in methylene chloride as in Example 1 to remove the FMOC group. The corresponding product was obtained. Treating this product with the arginine derivative of Example 1, cleaving the resulting peptide from the resin and deprotecting as in Example 1 gave L-arginylsarcosyl-L- aspartyl-L-valine as the ditrifluoroacetate salt; m.p. 145°C (dec). EXAMPLE 5

L-Arginylglycyl-L-Aspartyl-L-(N-Methyl)Valine

A. 1g of p-alkoxybenzylalcohol resin (0.5-1 mmole/g of resin), 0.706g of N-FMOC-N-methyl-L-valine, 0.382g EDC, 0.270g HOBT, and 0.28 ml triethylamine are combined in 15 ml of dimethylformamide and shaken for 2 hours. The mixture is filtered and the resin washed with DMF. The resin is treated as above for a second time, then shaken with 0.28 ml glacial acetic acid, 0.955g EDC, and 0.7 ml triethylamine in DMF and deprotection effected with 20% piperidine in methylene chloride as in 1A. This gives N- Methyl-L-valine-p-alkoxybenzyl resin ester. B. L-aspartic acid, glycine and L-arginine are coupled and deprotected, sequentially, as in the previous examples and the peptide removed from the resin to give L- arginylglycyl-L-aspartyl-L-(N-methyl)valine as the

ditrifluoroacetate salt which decomposes at 153°C. EXAMPLE 6

L-Arginylglycyl-L-Aspartyl Glycine Starting with N-FMOC-glycine-p-alkoxy benzyl resin ester, sequentially coupling L-aspartic acid, glycine and arginine, deprotecting and removing the peptide as in the above examples, L-arginylglycyl-L-aspartyl glycine was obtained as the ditrifluoroacetate salt; m.p. 85-90°C.

EXAMPLE 7

N-(L-Arginyl-2-Aminoethyl)-L-Aspartyl-L-Valine A. 1.18g EDC and 0.86 ml of triethylamine are combined in 20 ml of methylene chloride and stirred for 10 minutes. 2.0g N-α-CBZ-L-aspartic acid β-t-butyl ester, 0.83g HOBT, 1.30g L-valine-t-butyl ester and 0.86 ml triethylamine were added and the solution stirred

overnight. The solution is diluted with ethyl acetate and washed with 10% citric acid solution, 10% sodium carbonate solution, water, then dried over sodium sulfate,

evaporated to give 1.9g N-α-CBZ-L-aspartyl-t-butyl ester- L-valine-t-butyl ester.

B. 2.2g of N-α-CBZ-glycine methyl ester is

dissolved in 50 ml of anhydrous toluene and cooled to - 78°C, under nitrogen. To this is added 13 ml of 1.5M diisobutyl aluminum hydride in toluene over a period of 1 hour. The solution is stirred for an additional hour at - 78°C, then quenched by addition of 50 ml 5% hydrochloric acid solution. The solution is extracted with ethyl acetate which is washed with water and dried over sodium sulfate, evaporated to give 1.55g N-α-CBZ-2- aminoacetaldehyde.

C. The product from 7A is deprotected as in 3D to give L-aspartyl-t-butyl ester-L-valine-t-butyl ester. D. 1.55g of the aldehyde from 7B, 3.4g of the product from 7C, 1.64g sodium acetate, 1.23g sodium cyanoborohydride and lg of 3 angstrom molecular sieves are stirred together in 100 ml methanol for 3 days. The solution is filtered and 5 ml of 5% hydrochloric acid is added. The solution is diluted with water and adjusted to pH 9 with 10% sodium carbonate, then extracted with water, and dried over sodium sulfate. The solution is evaporated and the residue purified by flash chromatography in ethyl acetate/hexane, 1:1, to give 1.1g N-CBZ-aminoethyl-L- aspartyl-β-t-butyl ester-L-valine-t-butyl ester.

E. The CBZ group is removed from the product of 7D as in 3D to give N-aminoethyl-L-aspartyl-t-butyl ester-L- valine-t-butyl ester.

F. The product from 7E is coupled with N-α-t-BOC-N- ω-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine as in 2D and the resulting product deprotected as in 2E to give N-(L-arginyl-2-aminoethyl)-L-aspartyl-L-valine as the tritrifluoroacetate salt; m.p. 91-5°C.

EXAMPLE 8 L-Arginylglycyl-L-Aspartic Acid α-Benzyl Ester

A. lg of N-t-BOC-L-aspartic acid α-benzyl ester is treated with 0.366g of 2-(trimethylsilyl)ethanol in the presence of 0.592g EDC, 0.419g HOBT and 0.43 ml

triethylamine in 20 ml of methylene chloride for 2 hours. The product is isolated as in 2A to give N-t-BOC-L- aspartic acid α-benzyl ester-β-2-(trimethylsilyl) ethyl ester. B. The product of 8A is deprotected by treating with 10 ml of trifluoroacetic acid in 30 ml of methylene chloride for 2 hours at room temperature. The mixture was cooled to 0°C and 20 ml of saturated sodium carbonate solution is added dropwise. The layers are separated and the organic layer dried over magnesium sulfate, filtered, evaporated to give L-aspartic acid-α-benzyl ester-β-2- (trimethylsilyl) ethyl ester. C. The product of 8B and N-t-BOC glycine are coupled in a manner similar to that described in the previous examples to give BOC-glycyl-L-aspartic acid-α- benzyl ester-β-2-(trimethylsilyl) ethyl ester. D. The BOC group is removed from the product of 8C as in 8B to give glycyl-L-aspartic acid-α-benzyl ester-β- 2-(trimethylsilyl)ethyl ester.

E. The product from 8D is coupled to N-α-BOC-N-ω- (4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine as in 2D to give N-α-BOC-N-ω-(4-methoxy-2,3,6-trimethyl- benzenesulfonyl)-L-arginyl-glycylaspartic acid α-benzyl ester-β-2-(trimethylsilyl) ethyl ester. F. 0.30g of the product obtained in 8E is stirred with 5 ml of trifluoroacetic acid at room temperature for 24 hours. The reaction mixture is then stirred with 0.5 N acetic acid and washed with ethyl acetate. The aqueous layer is lyophilized to give L-arginylglycyl-L-aspartic acid α-benzyl ester ditrifluoroacetate; m.p. 85-7°C.

EXAMPLE 9

N-(6-Aminohexanoyl)-L-Aspartyl-L-Valine

A. 1.0g of N-(9-fluorenylmethoxycarbonyl)-L-valine p-alkoxybenzyl alcohol resin ester (containing

approximately 0.56 mmol of amino acid) was deprotected by shaking with 10 ml of a solution of 20% piperdine in dimethylformamide for 1.5 hours. The mixture was filtered and the resin derivative washed with methylene chloride to give L-valine p-alkoxybenzyl resin ester. B. The product from Example 1A was shaken with 0.92g of N-FMOC-L-aspartic acid β-t-butyl ester, 0.30g of 1-hydroxybenzotriazole (HOBT), 0.43 g of 1-(3-dimethyl- aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.32 ml of triethylamine in 10 ml of dimethylformamide for 2 hours. The mixture was filtered and the resin washed with methylene chloride. The resin derivative was then deprotected as in Example 1A to give L-aspartyl-β-t-butyl ester-L-valine p-alkoxybenzyl resin ester.

C. 2.00g of 6-aminohexamoic acid and 3.23g of sodium carbonate were dissolved together in 30 ml of water. The solution was cooled in an ice bath and 3.32g of di-t-butyldicarbonate in 15 ml of tetrahydrofuran was added. The mixture was stirred at room temperature for 5 hours, then diluted with 400 ml of water and extracted with ether. The aqueous solution was acidified to pH 2 with hydrochloric acid and extracted with ethyl acetate. The ethyl acetate layer was dried over magnesium sulfate, filtered and evaporated in vacuo to give N-tert-butoxy- carbonyl-6-aminohexamoic acid.

D. The product from Example 1B was shaken with 0.52g of N-BOC-6-aminohexanoic acid, 0.30g of HOBT, 0.43g of EDC and 0.32 ml of triethylamine in 10 ml of

dimethylformamide for 17 hours. The mixture was filtered and the resin derivative washed with methylene chloride. The peptide derivative was deprotected and cleaved from the resin by treating with 10 ml of 95% trifluoroacetic acid for 2 hours. The resin was filtered off and the filtrate diluted with 50 ml of 0.5 N acetic acid. The aqueous solution was washed with 4x25 ml of ethyl acetate, filtered, then lyophilized to give N-(6-aminohexanoyl)-L- aspartyl-L-valine as the trifluoroacetate salt; m.p. 75- 85°C. EXAMPLE 10

N-(7-Aminoheptanoyl)-L-Aspartyl-L-Valine A. When 7-aminoheptanoic acid was substituted for

6-aminohexanoic acid and treated in a manner similar to that in Example 1C, N-tert-butoxycarbonyl-7-aminoheptanoic acid was obtained. B. L-aspartyl-β-t-butyl ester-L-valine p-alkoxybenzyl resin ester (prepared from l.Og of N-FMOC-valine p- alkoxybenzyl resin ester as in Examples 1A and B) was treated with 0.55g of N-BOC-7-aminoheptanoic acid, with 0.30g of HOBT, 0.43g of EDC and 0.32 ml of triethylamine in 10 ml of dimethylformamide in a manner similar to that in Example 1D to give N-(7-aminoheptanoyl)-L-aspartyl-L- valine as the trifluoroacetate salt.

EXAMPLE 11

N-(7-Guanidinoheptanoyl)-L-Aspartyl-L-Valine

A. 7-Guanidinoheptanoic acid was prepared

essentially by the method of Miller, et al, Synthesis. 777 (1986), which is incorporated herein by reference. 0.50g of 7-aminoheptanoic acid was dissolved in a solution of 0.475g of potassium carbonate in 3.5 ml of water. 0.427g of aminoiminomethanesulfonic acid was added portionwise over 10 minutes and the mixture stirred at room

temperature for 24 hours. The resulting solid was

collected by filtration. The guanidine was dissolved in diluted hydrochloric acid and the solution evaporated in vacuo. Two portions of 2-propanol were evaporated from the residue to give 7-guanidinoheptanoic acid

hydrochloride.

B. L-aspartyl-β-t-butyl ester-L-valine p-alkoxy- benzylalcoholester (prepared from 1.0g of N-FMOC-L-valine p-alkoxybenzylalcohol ester resin as in Examples 1A and B) was treated with 0.50g of 7-guanidinoheptanoic acid hydrochloride, 0.30g of HOBT, 0.43g of EDC and 0.32 ml of triethylamine in 10 ml of dimethylformamide in a manner similar to that in Example ID to give N-(7-guanidino- heptanoyl)-L-aspartyl-L-valine as the trifluoroacetate salt; m.p. 75-80ºC.

EXAMPLE 12 N-(8-Guanidinooctanoyl)-L-Aspartyl-L-Valine

A. 8-guanidinooctanoic acid hydrochloride was prepared from 8-aminooctanoic acid in a manner similar to the process used in Example 3A.

B. 0.40g of 8-guanidinooctanoic acid hydrochloride, L-aspartyl-β-t-butyl ester-L-valine p-alkoxybenzyl resin ester (prepared in the same manner as in Examples 1A and B), 0.22g of HOBT, 0.32g of EDC and 0.24 ml of

triethylamine were shaken in 10ml of dimethylformamide and treated as in Example ID to give N-(8-guanidinooctanoyl)- L-aspartyl-L-valine as the trifluoroacetate salt.

EXAMPLE 13

If 6-guanidinohexanoic acid hydrochloride is

substituted for 7-guanidinohexanoic acid hydrochloride in Example 3B, N-(6-guanidinohexanoyl)-L-aspartyl-L-valine can be prepared.

EXAMPLE 14

A. If 8-aminooctanoic acid is substituted for 6- aminohexanoic acid in Example IC, N-tert-butoxycarbonyl-8- aminooctanoic acid can be prepared.

B. If N-BOC-8-aminooctanoic acid is substituted for N-BOC-6-aminohexanoic acid in Example 1D, N-(8-amino- octanoyl)-L-aspartyl-L-valine can be prepared as the trifluoroacetate salt.

EXAMPLE 15

8-Guanidinooct-2-Enoyl-L-Aspartyl-L-Valine

A. 4g of 6-amino-1-hexanol is dissolved in 50 ml of 10% aqueous tetrahydrofuran and the solution cooled to 0°C. 7.46g of di-tert-butyldicarbonate in 25 ml of

tetrahydrofuran is added dropwise and the resulting mixture stirred for 3 days at room temperature. The solvent is evaporated in vacuo and the residue dissolved in ethyl acetate. The ethyl acetate solution is washed with water, dried over magnesium sulfate and evaporated in vacuo to give 7.4g of N-tert-butoxycarbonyl-6-amino-1- hexanol.

B. To a solution of 8.8g of pyridinium

chlorochromate in 250 ml of methylene chloride is added 8.8g of 3 Angstrom molecular sieves. A solution of 7.4g of N-tert-butoxycarbonyl-6-amino-1-hexanol in 50 ml of methylene chloride is added dropwise and the mixture stirred at room temperature for 2 hours. The reaction mixture is filtered through silica gel, washing with 40% ethylacetate in hexane, and the filtrate evaporated in vacuo to give 6-N-tert-butoxycarbonylaminohexanol.

C. 1g of 6-N-tert-butoxycarbonylaminohexanol and 1.54g of methyl (triphenylphosphoranylidene) acetate are combined in 25 ml of chloroform and the solution refluxed for 2 hours. The solvent is then removed in vacuo and the residue taken up in ether and allowed to stand in the freezer overnight. The resulting suspension is filtered, the filtrate evaporated and the residue flash

chromatographed in 20% ethyl acetate in hexane to give methyl-8-N-tert-butoxycarbonylamino-2-octenoate. D. A solution of 3.2g of methyl 8-N-tert- butoxycarbonylamino-2-octenoate in 25 ml of methanol and 25 ml of 1 Normal aqueous sodium hydroxide is heated at reflux for 2 hours. The methanol is removed in vacuo and the aqueous solution acidified with IN hydrochloric acid. The resulting mixture is extracted with ethyl acetate. The organic solution is dried over magnesium sulfate and evaporated to give 8-N-tert-butoxycarbonylamino-2-octenoic acid.

E. 3g of 8-N-tert-butoxycarbonyl-amino-2-octenoic acid is dissolved in 30 ml of trifluoroacetic acid and the solution stirred at room temperature for 1 hour, then evaporated in vacuo to give 8-amino-2-octenoic acid as the trifluoroacetate salt.

F. 3.1g of 8-amino-2-octenoic acid trifluoroacetate is added to 30 ml of water and the pH adjusted to 7 with IN sodium hydroxide solution. 1.9g of potassium carbonate is added, then 1.75g of aminoiminomethane- sulfonic acid is added, portionwise, over 10 minutes. The mixture was stirred for 5 hours at room temperature and the resulting solid collected by filtration. The solid is dissolved in diluted hydrochloric acid and the solution evaporated and two portions of 2-propanol evaporated from the residue to give 8-guanidino-2-octenoic acid

hydrochloride.

G. L-aspartyl-β-t-butylester-L-valine p- alkoxybenzyl resin ester (prepared from 0.6g of N-FMOC- valine p-alkoxybenzyl resin ester as in Examples 1A and B) is treated with 0.33g of 8-guanidino-2-octenoic acid hydrochloride in the presence of 0.184g of HOBT, 0.26g of EDC and 0.19 ml of triethylamine in 10 ml of

dimethylformamide in a manner similar to that in Example ID to give 8-guanidinooct-2-enoyl-L-aspartyl-L-valine as the trifluoroacetate salt. By using methods analogous to that used in Examples 1 through 15, the following compounds were made:

N-(5-guanidino-2-aminopentyl)glycyl-L-aspartyl-L- valine tritrifluoroacetate; m.p. 90-95°C:

2-[N-(5-guanidinopentanoyl)glycyl-L-aspartyl]-

1,2,3,4-tetrahydroisoquinoline:

L-arginyl-glycyl-L-N-methylaspartyl-L-valine

ditrifluoroacetate:

N-(5-guanidinopentanoyl)glycyl-L-aspartylphenethyl- amide; m.p. 90-100°C:

N-(5-aminopentanoyl)glycyl-L-aspartyl-L-valine trifluoroacetate; m.p. 95-99°C:

N- (5-guanidinopentanoyl) glycyl-L-aspartyl-L-valine dihydrochloride; m.p. 60-70 ° C:

8-Guanidino-2,2-dimethyloctanoyl-L-aspartyl-L-valine trifluoroacetate:

9-Guanidino-non-2-(E)-enoyl-L-aspartyl-L-valine trifluoroacetate:

8-Guanidino-oct-3-(E)-enoyl-L-aspartyl-L-valine trifluoroacetate:

5-Guanidinovaleroyl-L-aspartyl-L-valine

trifluoroacetate:

9-Aminononanoyl-L-aspartyl-L-valine trifluoroacetate :

9-Guanidinononanoyl-L-aspartyl-L-valine

trifluoroacetate:

11-Guanidinoundecanoyl-L-aspartyl-L-valine

trifluoroacetate:

10-Guanidinodecanoyl-L-aspartyl-L-valine

trifluoroacetate:

8-Guanidino-2 (R, S) -ethyloctanoyl-L-aspartyl-L-valine trifluoroacetate:

Compounds of the present invention were tested for inhibition of platelet aggregation using the following procedures:

I. Inhibition of Radiolabeled (¹²⁵I) Fibrinogen Binding Assay, which is essentially based on the method described in Proc. Natl. Acad. Sci. USA Vol. 83, pp. 5708- 5712, Aug. 1986, and is as follows.

Platelets are washed free of plasma constituents by the albumin density-gradient technique. In each

experimental mixture platelets in modified Tyrode's buffer are stimulated with human α-thrombin at 22-25°C for 10 minutes (3.125 x 10" platelets per liter and thrombin at 01 N1H units/ml). Hirudin is then added at a 25-fold excess for 5 minutes before addition of the radiolabeled ligand and any competing ligand. After these additions, the final platelet count in the mixture is 1 x 10"/liter. After incubation for an additional 30 minutes at 22-25°C, bound and free ligand are separated by centrifuging 50μl of the mixture through 300μl of 20% sucrose at 12,000xg for 4 minutes. The platelet pellet is then separated from the rest of the mixture to determine platelet-bound radioactivity. Nonspecific binding is measured in

mixtures containing an excess of unlabeled ligand. When binding curves are analyzed by Scatchard analysis, nonspecific binding is derived as a fitted parameter from the binding isotherm by means of a computerized program. To determine the concentration of each inhibitory compound necessary to inhibit 50% of fibrinogen binding to

thrombin-stimulated platelets (IC₅₀), each compound is tested at 6 or more concentrations with ¹²⁵I-labeled fibrinogen held at 0.176μmol/liter (60μg/ml). The IC₅₀ is derived by plotting residual fibrinogen binding against the logarithm of the sample compound's concentration.

II. Inhibition of Fibrinogen - Mediated Platelet Aggregation, which is essentially based on the method described in Blood, Vol. 66, No. 4, Oct. 1985, pp. 946- 952, and is as follows.

Human Platelets were isolated from freshly drawn whole blood and were suspended in 0.14 mol/L NaCl, 2.7 mmol/L K11, 12 mmol/L NaHCO₃, 0.42 mmol/L Na₂HPO₄, 0.55 mmol/L glucose, and 5 mmol/L Hepes, pH 7.35 at 2 x 10⁸ platelets/ml. The suspension was incubated at 37°C. An aliquot of 0.4 ml of platelet suspension was activated by human thrombin at a final concentration of 2μg/ml of thrombin for one minute. After one minute the reaction was stopped by a thrombin inhibitor. Serial dilution of the compound being tested was then added to the activated platelet, the reaction was allowed to proceed for one minute, followed by the addition of human fibrinogen at a final concentration of 60μg/ml of fibrinogen. Platelet aggregation was then recorded by an aggregometer. Rate of aggregation was used to calculate IC₅₀. Representative results of platelet aggregation inhibition are shown in Table I.

The compounds of the present invention may be orally or parenterally administered to mammals. The compounds may be incorporated into pharmaceutical formulations having excipients suitable for these administrations and which do not adversely react with the compounds, for example, water, vegetable oils, certain alcohols and carbohydrates, gelatin and magnesium stearate. The pharmaceutical formulations containing an active compound of the present invention may be made into: tablets, capsules, elixirs, drops or suppositories for enteral administration; and solutions, suspensions or emulsions for parenteral administration.

In general, a compound of this invention is

administered in dosages of approximately 1 to 200 mg per dosage unit or higher. The daily dosage is approximately 0.02-5 mg/kg of body weight. It is to be understood, however, that the particular dose for each patient usually depends on very diverse factors, such as the age, body weight, general condition of health, sex, diet and the like of the patient, on the time and route of

administration, on the rate of excretion, on the

combination of medicaments and on the severity of the disease.

Having described the invention, it will be apparent to one of ordinary skill in the art that changes and modifications can be made thereto without departing from the spirit and scope of the invention as set forth herein.

Claims (36)

WHAT IS CLAIMED IS:

1. A compound of the formula

or a pharmaceutically acceptable salt thereof wherein: X is H,

Y is H,

alkyl,

cycloalkyl,

aralkyl,

-R¹

-NR¹R² or R²;

A, B and D are independently: -NR²,

CH₂O or

CH₂- NR² ;

K is H,

CH₃, C ;

₃

CH₂- OH,

CH₃CH - OH,

R¹ and R² are independently:

H,

alkyl ,

cycloalkyl,

aryl, or

aralkyl;

Z is OR¹, NH₂ or NR¹R²; and m is 0-9;

2. A pharmaceutical composition for the prophylaxis or treatment of abnormal thrombus formation in a mammal comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of claim 1.

3. A method of preventing or treating thrombus formation in a mammal comprising the administration of the composition of claim 2.

4. A compound of the formula

or a pharmaceutically acceptable salt thereof wherein:

Y is H,

alkyl,

cycloalkyl,

aralkyl,

- R¹

- NR¹R² or - R²;

A and D are independently: - NR²,

CH₂O or

CH₂-NR²;

K is H,

CH₃,

CH₂ -OH,

CH₃CH-OH,

R¹ and R² are independently

H,

alkyl ,

cycloalkyl ,

aryl , or

aralkyl ;

Z is OR¹, NH₂ or NR¹R²; and m is 0-9.

5. A pharmaceutical composition for the prophylaxis or treatment of abnormal thrombus formation in a mammal comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of claim 4.

6. A method of preventing or treating thrombus formation in a mammal comprising the administration of the composition of claim 5.

7. L-arginyl-L-aspartyl-L-valine.

8. N-(L-arginyl-2-aminoethyl)-L-aspartyl-L-valine.

9. N-(5-guanidino-2-aminopentyl)glycyl-L-aspartyl-L- valine.

10. N-(6-aminohexanoyl)-L-aspartyl-L-valine.

11. N-(7-aminoheptanoyl)-L-aspartyl-L-valine.

12. N-(7-guanidinoheptanoyl)-L-aspartyl-L-valine .

13. N-(8-guanidinooctanoyl)-L-aspartyl-L-valine.

14. N-(6-guanidinohexanoyl)-L-aspartyl-L-valine.

15. 8-Guanidinooct-2-enoyl-L-aspartyl-L-valine.

16. 8-Guanidino-2,2'-dimethyloctanoyl-L-aspartyl-L-valine trifluoroacetate.

17. 9-Guanidino-non-2-(E)-enoyl-L-aspartyl-L-valine

trifluoroacetate.

18. 8-Guanidino-oct-3-(E)-enoyl-L-aspartyl-L-valine

trifluoroacetate.

19. 5-Guanidinovaleroyl-L-aspartyl-L-valine

trifluoroacetate.

20. 9-Aminononanoyl-L-aspartyl-L-valine trifluoroacetate.

21. 9-Guanidinononanoyl-L-aspartyl-L-valine

trifluoroacetate.

22. 11-Guanidinoundecanoyl-L-aspartyl-L-valine

trifluoroacetate.

23. 10-Guanidinodecanoyl-L-aspartyl-L-valine

trifluoroacetate.

24. 8-Guanidino-2(R,S)-ethyloctanoyl-L-aspartyl-L-valine trifluoroacetate.

25. A compound of the formula

and pharmaceutically acceptable salts thereof wherein:

X is H or

Y is H,

alkyl,

cycloalkyl,

aralkyl, or - R²;

A, B and D are independently: - NR²,

E is H,

CH₃, or

R¹ and R² are independently:

H,

alkyl; and m is 2-8; provided that when X is NH₂, then: m is 3;

E is at least one radical in A, B and D is not

26. A pharmaceutical composition for the prophylaxis or treatment of abnormal thrombus formation in a mammal comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of claim 25.

27. A method of preventing or treating thrombus formation in a mammal comprising the administration of the composition of claim 26.

28. A compound of the formula

and pharmaceutically acceptable salts thereof wherein:

X is H or Y is H,

alkyl,

cycloalkyl,

aralkyl, or

A and B are independently: F is or CH₂;

G is OR₂,

1

( -

(

R¹ and R² are independently:

H or

alkyl ; m is 2-8; and

n is 0-5;

provided that when X is NH₂, then: m is 3;

F is

G is OH; and

that one radical in A and B is not

NH.

29. A pharmaceutical composition for the prophylaxis or treatment of abnormal thrombus formation in a mammal comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of claim 28.

30. A method of preventing or treating thrombus formation in a mammal comprising the administration of the composition of claim 29.

31. L-ornithylglycyl-L-aspartyl-valine.

32. L-arginylsarcosyl-L-aspartyl-L-valine.

33. L-arginylglycyl-L-aspartylglycine.

34. L-arginylglycyl-L-aspartic acid α-benzyl ester.

35. 2-[N-(5-guanidinopentanoyl)glycyl-L-aspartyl]- 1,2,3,4-tetrahydroisoquinoline.

36. N-(5-aminopentanoyl)glycyl-L-aspartyl-L-valine.