AU661659B2 - Anti-thrombotic peptide and pseudopeptide derivatives - Google Patents

Description

i OPI DATE 17/11/92 AOJP DATE 2I/11/92 APPLN. ID 80896/91 I 1111 1111 IIii PCT NUMBER PCT/US91/02471 I I1lIl11 in.llrl1lllli II AU9180896 54e 3 (51) International Patent Classification 5 (11) International Publication Number: WO 92/18117 A61K 31/195, 31/215, 37/02 C07C 279/00, C07K 5/06 (43) International Publication Date: 29 October 1992 (29.10.92) C07K 5/08 (21) International Application Number: PCT/US91/02471 (74) Agents: BALOGH, Imre, (Jim); Rhone-Poulenc Rorer Inc., 500 Arcola Road, P.O. Box i200, Collegeville, PA (22) International Filing Date: 11 April 1991 (11.04.91) 19426-0107 (US) et al.

(71) Applicant (for all designated States except US): RHONE- (81) Designated States: AT (European patent), AU, BE (Euro- POULENC RORER INTERNATIONAL (HOLD- pean patent), CA, CH (European patent), DE (Euro- INGS), INC.[US/US]; 40 H p Di, E- pean patent), DK (European patent), ES (European pa- -49958(US). /tre corpo-Je Cen,-er- tent), FR (European patent), GB (European patent), GR /,yAer- A/a,-rk- So,/e 7//e (European patent), IT (European patent), JP, LU (Euro- (72) Inventors; and 3 /,o3 S"/a/ea o/ pean patent), NL (European patent), SE (European pa- Inventors/Applicants (for US only) KLEIN, Scott, I. [US/ tent), US.

US]; 436 Mill Grove Drive, Audubon, PA 19403 (US).

MOLINO, Brucc, F. [US/US]; 2828 North Ford Drive, Hatfield, PA 19440 DENER, Jeffrey, M. [US/US]; Published 251 West DeKalb Pike, Apt C-1010, King of Prussia, PA With international search report.

19406 CZEKAJ, Mark [US/US]; 206 Crest Drive, Sellersville, PA 18960 BECKER, Michael, R. [US/ US]; 62 Church Road, Norristown, PA 19401 (US).

(54)Title: ANTI-THROMBOTIC PEPTIDE AND PSEUDOPEPTIDE DERIVATIVES (57) Abstract Disclosed are novel peptide and pseudopeptide derivatives that inhibit platelet aggregation and thrombos formation in mammalian blood thereby being useful in the prevention and treatment of thrombosis associated with certain disease states, such as, Myocardial infraction, stroke, peripheral arterial disease and disseminated intravascular coagulation.

7j~ FOR THiE 1I1IRPOSES OF INFORMATION ONLY Codes used to identify States party to the PCI on [he front pages of pamphlets publishing international ap~plicatio~ns under the 110'.

AT Aut(ri AU Ausualia~ Bit Bartadou BE HeLgiuin OF BurL ma iVh.

BC Btmmtgaria Bi Benurin BR HrjA1i CA ('ammad,, CF (ci ural African Repohtint CG Congo CH swilirloomd CI C 6 1c &ttoiir CM Cameurooni CS ('tct,.,..lwia DE (ierm~my DiC tjLiurt Es spin Ft tinlandl FRi irammru GA Gaburm Cit Uniid K.iigdom CN (jolca CR Greece Hut 11migary Ir Italy ii' Japan 1Kp D)mnwralic People's Repnublic of rea KR Repuhtic of Koirea 1.i i lefihictiln LK Sri Lw.a LLI I ocwuntmurg N1(; Monacuo MC Madagascar Mt. Mal1i MN Monugolia MR Mauritania MW Malawi NL Nethuerlands~ NO Norway PL Pouland RO Romania RU Ru-sian Federation SE) Stodan SE Sweden SN Senegalt SO Soviet Onion TD (Chad TC Togo us United States of Amnerica

F-

i eYII~ ''f WO 92/18117 PCt/US91/02471 ANTI-THROMBOTIC PEPTIDE AND PSEUDOPEPTIDE DERIVATIVES Background of the Invention 7 T his t -Gafien 13l t a-ol-at-a Of a-1S- LIW. H- 1.4s 7 -E 1. Field of the Invention This invention relates to novel compounds having anti-thrombotic activity. More particularly, the invention relates to novel peptide and pseudopeptide derivatives 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.

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, fibrinogen, fibronectin, and von 35 Willebrand factor. Fibrinogen is a co-factor for platelet aggregation, fibronectin supports platelet attachments and spreading reactions, and von Willebrand Sfactor is important in platelet attachment to and spreading on subendothelial i .d j 1 i WO 92/18117 PCT/US91/02471 2 matrices. The binding sites for fibrinogen, fibronectin and von Willebrand factor have been located on the platelet membrane glycoprotein complex Ilb/Illa.

Adhesive glycoproteins, 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/lla 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.

1 0 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.

2. Reported Developments 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 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 dodecapeptide, His-His-Leu-Gly-Gly-Ala-Lys-GIn-Ala-Gly-Asp- Val, sequence. Synthetic small peptides containing the RGD or dodecapeptide units show activity: they bind to the platelet receptor and competitively inhibit binding of fibrinogen, fibronection and von Willebrand factor as well as inhibiting 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 peptide and pseudopeptide derivatives which inhibit platelet aggregation and subsequent thrombus formation.

i i LI

J.

WO 92/18117 PCJ7/US91/02471 $jmary of the Invention The present invention comprises novel peptide and pseudopeptide derivatives of the general formula:

YC-N-CH-C-Z

I I

(CH

2 )m-CO 2

H

V

wherein: V is R2,-NH NH-R 2 N j

NH-R

2 -NHkNH or -C=N; W is -(CH 2 -CH=CH-(CH2)n-, -(CH 2 )n-CH=CH-, or Y is -CH=CH-CH 2

-CH

2 CH=CH-, -CH=CH-, 0

II

N-N

-(CH

2

(CH

2 )n -CH: 0

II

2-Xl- or -Xi-CH2- wherein X 1 is 0, NH or S; WO092/18117 PCT/US9I/02471 4 -N

-N

Zs-OR1 1

R

2 R7 a D- or L-amino acid or its corresponding carboximide, a synthetic amnino acid of the formula

-NR,-C-R

4 a dipeptide or a dipeptide isostere of the formula

NH-CH-V

1

-CH-R

3

M

5

R

4 m is1, 2 or3; n isOto 6; R1 and R 2 are independently H, alkyl, aryl, aralkyl or ally[; -CON -CON R3 isH, -CO 2 H, -C0 2

R

1

-CONH

2 R2orR7

R

4 and R 5 are independently H, alkyl, cycloalky!, cycloalkylmethyl, j(C 2 )p-CH

(CH

2 )P0 X2 X2 X2/X 2 NP -(CH 2 )p-j p- WO 92/18117 PCr/US91/02471 X 2 _(CH2)p E Q\;

N

I

MI

I

-N

-(CH2 X 2 K41N2

X

2

-(CH

2 )p'ORI, -(CH 2 )pSRI, -(CH 2 )p-NRiR 2

NH

-(CH 2 )p-NH-C

N

NH

2 0 11

*(CH

2 )-C-NRlR 2

NH

2

-(CH

2 )p-CO 2 Ri or p is 0 to 8;

R

6 and R 7 form a ring with the nitrogen to which they are attached and 1 0 are -(OH 2 4

(OH

2 5

-(OH

2 6

-CIH

2

CH

2 00H 2

CH

2

-CH

2

CH

2

NR

1

OH

2 or R2,-NH-C-R 1 S1 X 2 is H, CI, Br, F, -NO 2

NH

-C

NH

2

NH

-NH-C

NH

2 I C R_ alkyl, phenyl, -00 2

R

1

-CF

3 or -NHSO 2

R,;

0 11

-C-N-

V1 is Vi i 0 H2)n -CH=CH- -CH 2 -NH- -CH 2 -0p- 1 6 0

II

-CH

2 or and pharmaceutically acceptable salts thereof.

Detailed Description of the Invention In accordance with the present invention, novel compounds are provided which inhibit platelet aggregation by inhibiting fibrinogen binding and other adhesive glycoproteins involved in platelet aggregation and blood clotting to activated platelets. Compounds of the present invention, as tested by methods predictive of anti-thrombotic activity, are believed to be useful in 1 0 the prevention and treatment of thrombosis associated with certain disease 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 1 5 cancerous diseases since they may interfere with adhesive interactions between cancer cells and the extracellular matrix (Joum. of Biol. Chem., Vol.

262, No. 36 1987, pp. 17703-17711; Science, Vol. 233, 1986, pp. 467-470; and Vol. 57, 59-69, Apr. 1989).

As used above and throughout the description of this invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings: "Alkyl" means a saturated aliphatic hydrocarbon group which may be straight or branched and having about 1 to about 20 carbon atoms in the chain.

Branched means that a lower alkyl group such as methyl, ethyl or propyl. is attached to a linear alkyl chain. Preferred alkyl groups are the "lower alkyl" groups which are those alkyl groups having from 1 to about 6 carbons. Alkyl may be substituted by other moieties such as halogen or alkoxy.

"Halogen" means Cl, Br, I or F.

"Alkcxy" means an alkyl-O- group. Lower alkoxy groups are preferred.

Exemplary groups include methoxy, ethoxy, n-propoxy, i-propoxy and n-butoxy.

WO 92/13117 PCT/US91/02471 7 "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 preferrably substituted or unsubstituted phenyl or naphthyl.

Aryl substituents include hydrogen, alkyl, alkoxy, amino, halo, aryl, aryloxy, carboalkoxy, nitro, dialkylamino, trifluoromethyl, thioalkyl and carbamoyl.

"Aralkyl" means an alkyl group substituted by an aryl radical, wherein 1 0 "aryl" means a phenyl or phenyl substituted with one or more substituents which may be alkyl, alkoxy, amino, nitro, carboxy, carboalkoxy, cyano, alkylamino, halo, hydroxy, hydroxyalkyl, mercapto, alkylthio, acyl or carbamoyl.

Exemplary groups include benzyl and phenethyl.

O

II

1 5 "Carboalkoxy" means an alkyl-O-C group. Preferred carboalkoxy groups are those in which the alkyl group is lower alkyl.

"Alkylamino" means an alkyl-NH- group. Preferred groups are lower alkylamino groups.

"Alkylthio" means an alkyl-S- group. Preferred groups are lower alkylthio.

0

I"I

"Acyl" means an alkyl-C- group. Preferred acyl groups are those in which the alkyl group is lower alkyl.

D- and L-amino acids include: Asp, Arg, Ala, Asn, Cys, Gly, Glu, Gin, His, lie, Leu, Lys, Met, Orn, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

3 0 Stereoisomers and diastereomers of the compounds covered by the general formula also constitute a part of the present invention and intended to be covered by the appended claims.

i 1 WO 92" J WO 92, /18117 PC/US9/02471 The invention also comprises pharmaceutical cormpositions useful for the prevention and treatment of thrombosis comprising an aforesaid compound in a pharmaceutically acceptable carrier.

Another aspect of this invention comprises a method for the prevention and treatment of thrombosis associated with the aforesaid diseases.

The compounds of the present invention may be readily prepared by standard solid phase or solution phase peptide synthesis techniques using 1 0 starting materials and/or intermediates available from chemical supply companies such as Aldrich and Sigma or may be synthesized by standard organic chemical techinques. Paulsen, G. Merz, V. Weichart, "Solid-Phase Synthesis of 0-Glycopeptide Sequences", Angew. Chem. Int. Ed. Engl. 27 (1988); H. Mergler, R. Tanner, J. Gosteli, and P. Grogg, "Peptide Synthesis by a 1 5 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, "Solid Phase Synthesis after 25 years: The Design and Synthesis of Antagonists of Glucagon", Makromol. Chem. Macromol. Symp. 19, 31(1988)).

The solid phase method is represented schematically as follows: 0

II

Solid support -o-C-Al-NH-P

II

Solid support -o-c-A-NH 2

RI

Deprotect 1) Coupling

P-NH-A

2

-COOH

2) Deprotect 2) Deprotect I1 p- WO 92/18117 PCT/US91/02471 0 0 II II 1) Coupling Solid support--O-C-A-NH-C-A 2

-NH

2 I IR S-y-COOH w

V-P

2) Deprotect and Cleave Final Product wherein: the solid support may be, but is not limited to, p-alkoxybenzyl alcohol resin; 0

I

P-NH-A-C-

Ri is a protected amino acid derivative;

I

V-P is an N- protected derivative of an amino, amidino or guanidino acid.

In the process of making the desired compound, the amino acid 1 5 derivatives are added one at a time to the insoluble resin to give the desired dipeptide resin derivative, then the amino or guanidino acid derivative is coupled to the N-terminal of the chain. Any reactive functional groups of these derivatives are blocked by protecting groups to prevent cross reactions during the coupling procedures. These protecting groups include, but are not limited to, tertiary butoxycarbonyl (BOC), carbobenzoxy (CBZ), benzyl, t-butyl, WO 92/18117 PCT/US91/02471 9-fluorenylmethoxycarbonyi (FMOC) and methoxy-2,3,6-trimethylbenzenesulfonyl (MTR).

Upon completion of each coupling reaction, the N-terminal amino protecting group is removed by standard procedures and the deprotected amino group coupled to a derivative having a free carboxylic acid function.

The procedure is repeated until the desired product derivative is formed. The final product is obtained by deprotection and cleavage of the product from the resin by standard techniques.

Alternatively, the compounds of the present invention may be prepared in solution, without using a solid support. In a manner that is similar to the solid phase synthesis, starting with a protected amino acid derivative with a free N-terminal amino group, the protected derivatives are coupled, then 1 5 deprotected using standard procedures.

The invention will now be explained further by the following illustrative examples: EXAMPLE 1 N-[3-(2-auanidinoethyl)benzovll-L-aspartvl-L-valine A. A solution of 2.0 g of 3-trifluoromethylphenylacetonitrile in 5 ml of ether was added dropwise to a solution of 0.50 g of lithium aluminum hydride in ml of ether, while cooling at 0 0 C. The mixture was then stirred at room temperature for four hours and then quenched by sequential addition of 0.5 ml of water, 0.5 ml of 15% sodium hydroxide solution and 1.5 ml of water. The mixture was filtered and the filtrate dried over magnesium sulfate. The filtered solution was acidified with a 1N hydrogen chloride solution in ether and the solid which precipitated was collected to give 2-(3-trifluoromethylphenyl)ethyl amine hydrochloride.

B. 1.38 g of 2-(3-trifluoromethylphenyl)ethyl amine hydrochloride was heated at 100C in 3.5g concentrated sulfuric acid for three hours according to the method of Nikolaus, or disclosed in U.S. Patent 3,792,048, which is incorporated herein by reference. The cooled solution was diluted with 100 ml

L

F- WO 92/18117 PCT/US91/02471 11 of ether and the resulting precipitate collected to give 3-(2-aminoethyl)benzoic acid as the sulfate salt.

C. 1.38 g of the amine salt product from Example 1B was dissolved in 10 ml water and 1N sodium hydroxide solution was added to bring the pH up to 7.

The guanidine was then prepared essentially by the method of Miller, et. al., Synthesis. 777(1986), which is incorporated herein by reference. To the amine solution was added 0.849g of potassium carbonate, then 0.76g aminoiminomethanesulfonic acid was added, portionwise, over 10 minutes. The mixture 1 0 was stirred at room temperature for four hours. Upon reduction of the volume by half, in vacuo. a precipitate formed which was collected and recrystallized from water. The solid was suspended in 20% aqueous tetrahydrofuran. 1 N hydrogen chloride in ether was added to give a homogeneous solution which was evaporated in vacuo. The residue was crystallized from methanol/ether to 1 5 give 3-(2-guanidinoethyl)henzoic acid hydrochloride.

D. 0.89 g N-(9-fluorenylmethoxycarbonyl)-L-valine-p-alkoxybenzyl alcohol resin ester (containing approximately 0.5 mmol of amino acid) was deprotected by shaking with 10 ml of a solution of 20% piperidine in dimethylformamide for one hour. The mixture was filtered and the resin derivative washed with methylene chloride to give L-valine-p-alkoxy benzyl alcohol resin ester.

E. The product from Example 1D was shaken with 0.822g N-FMOC-Laspartic acid-P-t-butyl ester, 0.38 g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 0.270g 1-hydroxybenzotriazole (HOBT) and 0.28 ml of triethylamine in 10 ml of dimethylformamide for two hours. The mixture was filtered and the resin derivative washed with methylene chloride.

The resin derivative was then deprotected as in Example 1D to give L-aspartyl- P-t-butyl ester-L-valine-p-alkoxybenzyl alcohol resin ester.

F. 0.25g 3-(2-guanidinoethyl)benzoic acid hydrochloride was shaken with the product from Example 1 E, 0.191g EDC, 0.135g HOBT and 0.14 ml triethylamine in 10 ml of dimethylformamide for two hours. The mixture was filtered and washed with methylene chloride. The p-t-butyl ester blocking group was removed, and the product cleaved from the resin, by treating with trifluoroacetic acid (10 ml) for two hours. The resin was removed by filtration and the filtrate diluted with water, washed with ethyl acetate and p.- WO 92/18117 PC/US91/02471 12 lyophilized to give N-3-[2-(guanidinoethyl)benzoyl]-L-aspartyl-L-valine as the trifluoroacetate salt.

EXAMPLE2 N-r4-(2-guanidinoethvlbenzovll-L-aspartvl-L-valine A. When 4-trifluoromethylphenylacetonitrile was treated in a manner similar to that in Example 1A, 2-(4-trifluoromethylphenyl)ethyl amine hydrochloride was obtained.

B. When the amine from Example 2A was treated in a manner similar to that in Example 1B, 4-(2-aminoethyl)benzoic acid sulfate was obtained.

1 5 C. When the benzoic acid derivative from Example 2B was treated in a manner similar to that in Example 2C, 4-(2-guanidinoethyl)benzoic acid hydrochloride was obtained.

D. 0.272g 4-(2-guanidinoethyl)benzoic acid hydrochloride and L-aspartylp-t-butyl ester-L-valine-p-alkoxybenzyl alcohol resin ester (prepared from N-FMOC-L-valine-p-alkoxybenzyl resin ester as in Example 1D,E) were reacted together in the presence of 0.214g EDC, 0.151g HOBT, and 0.16 ml triethylamine in 10 ml of dimethylformamide in a manner similar to that of Example 1F. The product was deprotected and cleaved from the resin as in Example 1F to give N-[4-(2-guanidinoethyl)benzoyl]-L-aspartyl-L-valine trifluoroacetate.

EXAMPLE 3 N-(3-auanidinobjnzoyl)-L-aspartyl-L-valine A. 3-guanidinobenzoic acid was prepared from 3-aminobenzoic acid by the method of Miller, et. al., cited in Example 1C. The guanidine was treated with ethereal hydrogen chloride to give 3-guanidinobenzoic acid hydrochloride.

r? WO 92/18117 13PCT/US91/02471 B. 3-guanidinobenzoic acid hydrochloride was treated in a manner similar to that in Examples 1 F and 2D to give N-(3-guanidinobenzoyl)-L-aspartyl-Lvaline.

EXAMPLE 4 N-(4-guanidinomethylbenzoyl)-L-aspartyl-L-vali ne A. 4-guanidinomethylbenzoic acid hydrochloride was prepared from 1 0 4-aminomethylbenzoic acid in a manner similar to that of Example 3A.

I-B. 4-guanidinomethylbenzoic acid hydrochloride was treated in a manner similar to that of Examples 1 F and 2D to give N-(4-guanidinomethylbenzoyl)-Laspartyl-L-valine trifluoroacetate.

EXAMPLE N-(3-auanidinomethylbenzgyfl-L-asoartl-L-valin-e A. 3-guanidinomethylbenzoic acid hydrochloride was prepared from 3-aminomethylbenzoic acid in a manner similar to that of Example 3A.

B. 3-guanidinomethylbenzoic acid hydrochloride was treated in a manner similar to that of Examples 1 F and 2D to give N-(3-guanidinomethylbenzoyl)-Laspartyl-L-valine as the trifluoroacetate salt.

EXAMPLE 6 N-(4-guanidinobenzoyl)-L-asoartvl-L-valine A. 4-guanidinobenzoic acid hydrochloride was prepared from LI 4-aminobenzoic acid in a manner similar to that of Example 3A.

B. 4-guanidinobenzoic acid hydrochloride was treated in a manner similar to that of Examples 1 F and 2D to give N-(4-guanidinobenzoyl)-L-aspartyl-Lvaline as the trifluoroacetate salt.

WO 92/18117 PCT/US91/02471 14 EXAMPLE 7 N-r3-(2-aminoethyl)benzovll-L-asDartvl-L-valine A. One equivalent of 3-(2-aminoethyl)benzoic acid (prepared as in Example 1 B) is stirred with one equivalent of di-t-butyl-dicarbonate in the presence of two equivalents of sodium carbonate in tetrahydrofuran/water The reaction mixture Is evaporated to remove the tetrahydrofuran and the aqueous is acidified with dilute hydrochloric acid. The product is extracted 1 0 into ethyl acetate and the solution is dried, then evaporated to give N-tertbutoxycarbonyl-3-(2-aminoethyl)benzoic acid.

B. If N-BOC-3-(2-aminoethyl)benzoic acid is substituted for 3-(2-guanidinoethyl)benzoic acid hydrochloride in Example 1F, and treated similarly, then 1 5 N-[3-(2-aminoethyl)benzoyl]-L-aspartyl-L-valine is obtained as the trifluoroacetate salt.

EXAMPLE 8 N-(4-uanidinohenylacetoyl)-L-aspartyl-L-valine A. If 4-aminophenylacetic acid is substituted for 3-(2-aminoethyl)benzoic acid in Example 1C, and treated similarly, then 4-guanidinophenylacetic acid hydrochloride is obtained.

B. If 4-guanidinophenylacetic acid hydrochloride is substituted for the benzoic acid in Example 1 F and treated similarly, then N-(4-guanidinophenylacetoyl)-L-aspartyl-L-valine is obtained as the trifluoroacetate salt.

EXAMPLE 9 N-(4-aminophenylacetoyl)-L-aspartyl-L-valine A. If 4-aminophenylacetic acid is substituted for the benzoic acid in Example 7A, and treated similarly, then N-tert-butoxycarbonyl-4-aminophenylacetic acid is obtained.

1 f 1 WO 92/18117 PCT/US91/02471 B. If N-BOC-4-aminophenylacetic acid is substituted for 3-(2-guanidinoethyl)benzoic acid hydrochloride in Example 1F, and treated similarly, then N-(4-aminophenylacetoyl)-L-aspartyl-L-valine is obtained as the trifluoroacetate salt.

EXAMPLE 4-Guanidinocinnamovy-L-aspartvl-L-valine trifluoroacetate 1 0 A. To a solution of 5g (25 mmol) of 4-aminocinnamic acid hydrochloride in ml of water containing 6.92g (50 mmol) of potassium carbonate was added 3.11g (25 mmol) of aminoiminomethanesulfonic acid (AIMSA) portionwise at room temperature. The reaction mixture was stirred 24 hours at room temperature and the product was filtered off and washed with cold water. The 1 5 white solid was dissolved in 1N aqueous hydrochloric acid and concentrated in vacgo to give 4-guanidinocinnamic acid hydrochloride as a white solid.

B. A solution of 0.27g (1.12 mmol) of 4-guanidinocinnamic acid hydrochloride, 0.21g (1.12 mmol) of EDC, 0.15g (1.12 mmol) of HOBT and 0.16 ml of triethylamine in 10 ml of DMF was added to the L-aspartyl-p-t-butyl ester- L-valine-p-alkoxybenzyl alcohol resin (prepared as described in Example 1E) and shaking was continued for 1 hour. The solution was removed and the resin was washed with DMF (2 x 20 ml) and methylene chloride (3 x 10 ml).

A solution containing 9.5 ml of trifluoroacetic acid, 0.5 ml of water and several drops of 1,2-ethanedithiol was added to the resin and shaking was continued for 2 hours. The resin was filtered off and washed with trifluoroacetic acid. The filtrate was concentrated in vacuo. The residue was taken up in S^ acetic acid and washed with ethyl acetate (3 x 50 ml). The aqueous layer was lyophilized to a white powder which was purified by reverse phase (RP) HPLC using a C-18 column and a methanol/water gradient to give 4guanidinocinnamoyl-L-aspartyl-L-valine trifluoroacetate.

EXAMPLE 11 4-Guanidinobenzovl-N-ethylalycvl-L-asDartyl-L-valine trifluoroacetate WO 92/18117 PCT/US91/02471 A. To 14.8 g of a 50% aqueous solution of glyoxylic acid was added 50 ml of water. The resulting solution was cooled to 0°C and treated with 10 ml of a solution of ethylamine in water added by dropwise addition over minutes. The reaction mixture was transferred to a Parr bottle, then palladium on carbon was added and the reaction vessel was shaken under hydrogen at 44 psi for 24 hours. The reaction mixture was filtered through a celite pad and the filtrate was concentrated in vacuo to give a tan oil. The oil was treated with 1N aqueous HCI and concentrated in vacuo to give a solid which was recrystalized from acetic acid.

3.65 g of N-ethyl glycine hydrochloride was stirred in 35 ml of water.

This was treated with 8.31 g of sodium carbonate and cooled to 0°C, followed by the dropwise addition of 6.77 g of 9-fluorenylmethyl chloroformate in 15 ml of tetrahydrofuran (THF). The reaction mixture was allowed to slowly warm to 1 5 room temperature and stirred for 24 hours. The THF was removed in vacuo and the residue was diluted with water and extracted with ether. The aqueous fraction was acidified to pH 2 with 1N aqueous HCI and extracted with ethyl acetate. The organic extracts (ethylacetate) were dried, filtered and concentrated to give NaFMOC-N -ethyl glycine as a white solid. All FMOC protected substituted glycines were made by this procedure simply by substituting another amine for ethyl amine in this procedure.

I

B. L-aspartyl-p-t-butyl ester-L-valine-p-alkoxybenzyl alcohol resin ester was prepared as described in Example 1E and treated with 0.33g of FMOC-Nethyl glycine, from the preceding procedure, 0.191g EDC, 0.135g HOBT and 0.14 ml of triethylamine in 10 ml of DMF for two hours. The mixture was iltered and washed with methylene chloride. The FMOC protecting group was removed by the procedure described in Example 1D to give N-ethyl glycyl-Laspartic acid-P-t-butylester-L-valine-p-alkoxybenzyl alcohol resin ester.

C. 0.204g of 4-guanidinobenzoic acid hydrochloride prepared as described in Example 6A was dissolved in 5 ml of DMF and treated with 0.10g of triethylamine. The solution was cooled to 0°C and 0.25g of bis(2-oxo-3oxazolidinyl)phosphinic chloride (BOP-CI) was added. The reaction mixture was stirred at 0°C for 5 minutes than the peptide resin from Example 11B was added. Shaking was continued for 2 hours at room temperature. Applying the procedure for removal of the peptide from the resin described in Example 1 F, iA WO 92/18117 PCT/US91/02471 17 resulted in 4-guanidinobenzoyl-N-ethyl glycyl-L-aspartyl-L-valine as the trifluoroacetate salt.

EXAMPLE 12 4-Guanidinocinnamovl-N-ethvl alvcyl-L-asoartvl-L-valine This compound was prepared in a similar manner to the compound prepared in Example 11, by replacing 4-guanidinobenzoic acid hydrochloride in Example 11C with 4-guanidinocinnamic acid hydrochloride (prepared as described in Example EXAMPLE 13 4-Guanidinohomocinnamovl-L-asoartvl-L-valine A. 4-Guanidinophenethyl alcohol was prepared from 4-aminophenethyl alcohol using the procedure described in Example 1C.

B. To a solution of 1.28g (7.15 mmol) of 4-guanidinophenethyl alcohol in 7.15 ml of a 4N aqueous sodium hydroxide and 35.75 ml of acetone was added 1.8g (7.15 mmol) of 4-methoxy-2,3,5-trimethylbenzenesulfonylchloride (Mtr protecting group) in 7.15 ml of acetone, dropwise at 0°C. After 4 hours acetone was removed in vacuo and the residue was diluted with water and brought to pH<5 with 1N aqueous hydrochloric acid. The mixture was extracted with ethyl acetate and the organic extracts were dried, filtered and concentrated to give 4-(N9-Mtr-guanidino)-phenethyl alcohol.

.,il 1.75g (4.47 mmol) of the 4-(Ng-Mtr-guanidino)-phenethyi alcohol was added in a single portion to 1.44g (6.7 mmol) of pyridinium chlorochromate in ml of methylene chloride. The resulting mixture was allowed to stir for 2 hours at room temperature and was then filtered through silica gel using ethyl acetate in hexanes as eluent. The recovered aldehyde (1.20g 3.1 mmol) was dissolved in 40 ml of chloroform and 1.03g (3.1 mmol) of methyl triphenylphosphoranylidine acetate was added in a single portion. The resulting solution was heated at reflux for 24 hours. Chloroform was removed in vacuo and the residue was taken up in ether and filtered. The filtrate was i WO 92/18117 PCT/US91/02471 18 concentrated in vacuo and subjected to flash chromatography to provide 4-(N9- Mtr-guanidino)-homocinnamic acid methyl ester.

The methyl ester (1.07g, 2.4 mmol) was dissolved in 16 ml of methanol and 4 ml of 1N aqueous sodium hydroxide. The solution was allowed to stir 3 hours at 60 0 C. Methanol was removed in vacuo and the residue was diluted with water, brought to pH<2 with 1 N aqueous hydrochloric acid and extracted with ethyl acetate. The organic extracts were dried, filtered and concentrated to provide 4-(Ng-Mtr-guanidino)-homocinnamic acid.

C. 4-(Ng-Mtr-guanidino)-homocinnamic acid, when substituted for 4guanidinocinnamic acid in Example 10B, was coupled to L-aspartyl-P-t-butyl ester-L-valine-p-alkoxybenzyl alcohol resin (which was prepared as described in Example 1E). Tne peptide was cleaved from the resin and deprotected 1 5 when the 95% trifluoroacetic acid/5% water/5% ethanediol method of Example was employed and the reaction time was extended to 24 hours. The isolation and purification of the peptide proceeded as described in Example to give 4-guanidinohomocinnamoyl-L-aspartyl-L-valine trifluoroacetate was a white powder.

EXAMPLE 14 4-(lmidazol-1-vl)-cinnamovl-L-asoartyl-L-valine trifluoroacetate A. 4-(lmidazol-1-yl)-cinnamic acid hydrochloride salt was prepared according to the procedure of Lizuka, et al. described in U.S. Patent No.

4,226,878.

B. The 4-(imidazol-1-yl)-cinnamic acid hydrochloride salt when substituted for 4-guanidinocinnamic acid hydrochloride in Example 10B gave 4-(imidazol- 1-yl)-cinnamoyl-L-aspartyl-L-valine trifluoroacetate.

EXAMPLE 4-Guanidinocinnamovl-L-aspartvl-L-leucine amide trifluoroacetate I WO 92/18117 PCT/US91/02471 19 A. 0.50g of N-t-butoxycarbonyl(BOC)-L-leucine-p-methyl benzhydrylamine (MBHA) resin (containing 0.71 mmol of amino acid per gram of resin) was shaken with 50% trifluoroacetic acid in methylene chloride for 1 hour to remove the BOC group. The mixture was filtered and the resin washed successively with methylene chloride (CH 2

CI

2 50% CH 2

CI

2 in dimethylformamide (DMF), triethylamine in CHgCI 2 50% CH 2 C1 2 in DMF and finally CH 2

CI

2 to give L-leucine MBHA resin.

B. The resin from Example 14A was shaken with 0.45g of N-BOC-P- 1 0 cyclohexyl ester-.-aspartic acid, 0.19g of HOBT, 0.27g of EDC and 198 P1 of triethylamine in 10 ml of DMF for 2 hours. The mixture was filtered and the resin was washed with DMF (4 x 10 ml) followed by treatment with 10 ml of trifluoroacetic acid in CH 2 C12 for 1 hour. The resin was filtered and then washed with the same sequence of solvents listed in Example 14A to give L- 1 5 aspartic acid-P-cyclohexyl ester-L-leucine-MBHA resin.

C. The resin from Example 14B was shaken with 0.34g of 4-guanidinocinnamic acid, HCI, 0.19g of HOBT, 0.27g of EDC and 198 p.I of triethylamine in ml of DMF overnight. The resin was filtered and then washed with DMF and

CH

2

CI

2 The peptide was cleaved from the resin and deprotected at the same time by treatment with hydrofluoric acid and lyophilized to give 200 mg of crude product. This was taken up into 150 ml of water, filtered and the filtrate was washed with ethyl acetate. The aqueous portion was then frozen and lyophilized to give a white powder which was purified by reverse phase HPLC using a C-18 reverse phase column and a methanol/water gradient. The purified fractions were lyophilized to give 4-guanidinocinnamoyl-L-aspartyl-Lleucine amide trifluoroacetate as a white powder.

Utilizing analogous procedures described in Examples 1-15 the following compounds are made: 4-Guanidinocinnamoyl-L-aspartyl-L-norvaline trifluoroacetate.

4-Guanidinocinnamoyl-N-ethyl-glycyl-L-aspartyl-L-valine ditrifluoroacetate.

4-Guanidinocinnamoyl-L-aspartyl-L-norleucine trifluoroacetate.

WO 92/18117 20PCr/US91/02471 4-uaiincnnmylsrcsl--sprylLlecnetifu20ctae 4-Guanidinocinnamoyl-sacoyl-L-aspartyl-L-Ieuine trfluoroacetate.

N 4-GuanidinocinnamoyI-L-sarcosy-L-aspartyI-L-isoleucine trifluoroacetate.

4-Guanidinocin namoyl-sarcosyl-L-aspartyl-L-arginine ditrifluoroacetate.

4-Guanidinohomocinnamoyl-aspartyI valine trifluoroacetate.

4-(Imidazol-1 -yI)-cinnamoyl-L-aspartyl-L-valine trifluoroacetate.

i1 5 4-Guanidi nocinn olaprylNmmty-DLtytpa trifluoroacetate.

4-Guanidinoci nnamoyl-L-aspartyi-LhI'istidi ne ditrifluoroacetate.

4-Guanidinoci nnamoyl-L-aspartyl-D-valine trifluoroacetate.

I 4-Guanidinoci nnamoyI-L-aspartyI-L-fP-(2-naphthyI)alanine trifluoroacetate.

125 4-Guanidir.oci nnamoyl-sarcosyl-L-aspartyl-L-valine trifluoroacetate.

A-Guanidi nobenzoyl-N-ethylg Iycyl-L-aspartyl-L-vali ne trifluoroacetate.

4-Amidi nobenzoyI-sarcoy-L-asparty-L-vei ne trifluoroacetate.

4-Guanidi nobenzoyl-N-propylglycyl-L-aspartyl-L-vali ne trifluoroacetate.

4-(4-Guanidinophenyl) butyryl-L-aspartyl-L-vali ne irifluoroacetate.

4-Guanidinobenzoyl-sarcosyl-L-asparty-L-valine trifluoroacetate.

3-Guanidinobenzoylglycyl-L-aspartyl-L-valine trifluoroacetate.

WO 92/18117 PCT/US9I/02471 21 4-Guanidinobenzoylglycyl-L-aspartyl-L-valine trifluoroacetate.

a-Cyano-para-toluyl-L-aspartyl-L-valine.

4-Guanidinocinnamoyl-L-aspartic acid-a-isobutyi amide trifluoroacetate.

4-Guanidinocinnamoyl-L-aspartyl-L-tryptophan amide hydrogen fluoride.

4-Guanidinocin namoyl-L-aspartyl-L-Iysine amide dihydrogen fluoride.

4-Guanidi nomethyl cinnamoyl-L-aspartyl-L-valine trifl uo roacetate.

4-Guanidinocin namoyi-L-aspartyl-L-leucine-amide hydrogen fluoride.

L 4-Guanidnocin namoyl-L-aspartyl-L-valine amide trifluoroacetate.

3-Guanidi foci nnamoyl-L-aspartyl-L-vali ne trifluo roacetate.

4-Guanidi nod nnamoyl-L-aspartyl-L-proline trifluoroacetate.

4-Guanidinocin namoyl-L-aspartyl- L-argi nine ditrifluoroacetate.

4-Amidinoci nnamoyl-L-aspartyl-L-vali ne trifluoroacetate.

4-Guanidi nocinnamoyl-L-aspartyl-L-asparagine trifluoroacetate.

4-Guanidinoci nnamoyl-D-aspartyl-L-valine trifluoroacetate.

4-Guanidi nocinnamoyl-L-aspartyl-L-aspartic acid trifluoroacetate.

4-Guanidi foci nnamoyl-glycyl-L-aspartyl-L-vali ne trifluoroacetate.

4-Guanidi noci nnamoyl-L-aspartyl-L-lysine ditrifluoroacetate.

4- (4-Guanidinophenyl)butyry-L-aspartyl-L-vali ne trifluoroacetate.

-7- WO 92/181 17 PCT/US91/02471 22 4-Dimethylaminocinnamoyl-L-aspartyl-L-valine.

4-Aminoci nnamoyl-L-aspartyl-L-vali ne trifluoroacetate.

4-Guanidinocinnamoyl-L-aspartyl-L-threonine trifluoroacetate.

4-Guanidinocinnamoy-L-aspartyl-L-trypbophan trifluoroacetate.

S 1 0 4-Guanidinophenylthioacetoy!-L-aspartyl-L-valine trifluoroacetate.

4-Guanidinocinnamoyl-L-aspartyl-L-seri ne trifluoroacetate.

4-Guanidinoci nnamoyl-L-aspartyl-L-tyrosine trifluoroacetate.

4-Guanidinophenoxyacetoyl-L-aspartyl-L-vai ne trifluo roacetate.

4'-Guanidinooxaniloyl-L-aspartyl-L-valine trifluoroacetate.

4-Guanidinoci nnamoyl-L-aspartyl-L-Ieuci ne trifluoroacetaie.

4 -Guanidinocinnamoyl-L-aspartyl-L-isoleucyl-L-argi nine ditrifluoroacetate.

4-Guanidinocin namoyl-L-aspartyl-L-argi nyl-L-isoleucine.

4 -GuanidinocinnamoyI-N-(ethyI)-glycyl-LaspartylLisoleucine trifluoroacetate.

4 -GuanidinocinnamoyI-N-(ethyl)-glycyl-L-aspartyl..Leucine trifluoroacetate.

nopentyl)benzoyll-L-aspartyl-L-vali ne.

[2-(6'-Guanidinohexyl)benzoyl]-L-aspartyl-'L-valine.

nopent-1 '-enyl)benzoyl]-L-aspartyl-L-valine.

LI

WO 92/18117 PCT/US91/02471 23 j--[2-(6'-guanidinohex-l1-enyl)benzoyl]-L-aspartyl-L-valine.

[2-(5'.GC-uanidinopent-1 '-ynyl)benzoyl]-L-aspartyl-L-valine.

(6'-Guanidi no hex-1 '-ynyl) be nzoyl]-L-aspartyl-L-vali ne.

[2(4Gaiioetlpey~ctlLapry--aie 1 0[2-('-guanidinout-eyl)phenyl]acetyl-L-aspartyl-L-vaine.

ii uanidi nopet- 1 nyl)phe nyl]acetyl-L-aspartyl- L-vali n e.

15-2('gaiiout1'ey~hnlaeylLapry--aie [2-(5'-Guanidinopent-1 -ynyl)phenyl]acetyl-L-aspartyl-L-vali ne.

[2-(4'-Guanidinobut-1 '-ynyI)phenyI]acetyI-L-C-spnaryI-L-valine.

4-Guanidinocinnamoyl-L-aspartyl-D-a-benzyl-arginine-(D-ornithine).

4 -Guanidinocinnamoyl-L-aspartyl-L-a-benzyl-arginine-(L-ornithine).

Compounds of the present invention were tested for inhibition of platelet aggregation using the following procedures: I. Inhibition 2f Radioaed iL2fil Fibrinogen Binding Asay 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 thcz albumin densitygradient technique. In each experimental mixture platelets in modified Tyrode's buffer are stimulated with human a-thrombin at 22-25 0 C for minutes (3.125 x 1011 platelets per liter and thrombin at 0.1 NIH units/mI).

Hirudin is then added at a 25-fold excess for 5 minutes before addition of theI radiolabeled ligand and any competing ligand. After these additions, the final platelet count in the mixture is 1 x 101 M/iter. After incubation for an additional WO 92/18117 PCT/US91/02471 24 minutes at 22-25 0 C, bound and free ligand are separated by centrifuging p. of the mixture through 300 gI of 20% sucrosa at 12,OOOxg 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 (IC 50 each compound is 1 0 tested at 0.176pgmol/1iter (60pg/ml). The IC 50 is derived by plotting residual fibrinogen binding against the logarithm of the sample compound's concentration.

II. Inhibition of Fibrinogen Mediated Platelet AQgreaation, which is 1 5 essentially based on the method described in Blood, Vol. 66, No. 4, Oct. 1985, pp. 846-952, and is as follows.

Human Platelets were isolated from freshly drawn whole blood and were suspended in 0.14 mol/L NaCI, 2.7 mmol/L K11, 12 mmol/L NaHC0 3 0.42 mmol/L Na 2 HP0 4 0.55 mmol/L glucose, and 5 mmolL Hepes, pH 7.35 at 2 x 108 platelets/ml. The suspension was incubated at 37 0 C. An aliquot of 0.4 ml of platelet suspension was activated by human thrombin at a final concentration of 2pg/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 of fibrinogen. Platelet aggregation was then recorded by an aggregometer. Rate of aggregation was used to calculate ICso.

Representative results of platelet aggregation inhibition are shown in Table I.

TABLE I 3 -(2-Guanidinoethyl)t L-valine 4 -(2-Guanidinoethyl)t L-valine 1 5 4-Guanidinocinnamo' 4-Guanidinomethyiph L-valine 3-(4-Guanidinopheny L-vali ne 4-(3-Guanidinopropyl L-valine uniioheya 4-Guanidinopenzylc *no inhibition of 1251-.

)enzoyl-L-aspartyl- )enzoyI-L-aspartylyl-L-aspartyl-L-vali ne e nyl acetoyl- L-as partykl)propanoyl-L-aspartyl- )benzoyl-L-aspartyletoyl-L-aspartyl-L-valine aspartyl-L-valine Inhibition of M-fI-Fibrinogen Binding LQsLuM 115.0 10.9 2.0 4.5 44.0 20.0 Inhibition of Fibrinogen Mediated Platelet Aggregation 33.1 7.7 2.8 14.0 7.6 inhibition at 25 uM 37.0 85.0 42.0 65.0 83.3 43.0 33.0 librinogen binding observed at concentrations of 50 jiM or lower.

TABLE I (continued) Inhibition of .l 2 51-Fibrinogen Binding 3-Guanidinomethylbenzoyl-L-aspartyl-L-valine 4-Guanidinomethylbenzoyl-L-aspartyl-L-valine 3-Guanidinobenzoyl-L-aspartyl-L-valine 1 5 3-(Guanidinomethyl)phenylacetoyl-L-aspartyl- L-valine 3-(3-Guanidinopropyl)benzoyl-L-aspartyl- L-valine 4-Guanidinocinnamoyl-D-aspartyl-L-vali ne 4-Guanidinocinnamoyl-L-aspartyl-D-valine 3-Guanidinocinnamoyl-L-aspartyl-L-valine 4-Guanidinocinnamoyl-L-glutamyl-L-valine Inhibition of Fibrinogen Mediated Platelet Aggregation 30.2 inhibition a 53.0 16.0 6.3 15.2 22.0 52.0 38.0 30.0 >200.0 >200.0 -50.0 25.0

ON

(I)

no inhibition of 25 1-Fibrinogen binding observed at concentrations of 50 jiM or lower.

I

4-Amidinocinnamoyl-L-aspartyl-L-valine 4-(lmidazol-I -yl)-cinnamoyl-L-aspartyl- L-valin e 4-Guanidinocin namoyl-glycyl-L-aspartyl- 1 5 L-valine 4-Guanidinocinnamoyl-sarcosyl-L-aspartyl- L-valine 4-Guanidinohomocinnamoyl-L-aspartyl- L-valine 4-Guanidinocinnamoyl-L-aspartyl-L-leucine 4-Guanidinocinnamoyl-L-aspartyl-L-teucine amide 4-Guanidinocinnamoyl-L-aspartyl-L-arginine 4-Guanidinocinnamoyl-L-aspartyl-L-tryptophan TABLE I (continued) Inhibition of ?-al-Fibrinogen Binding i1LIM1 50.0 >200.0 5.0 0.10 Inhibition of Fibrinogen Mediated Platelet Aggregation 6.6 2.1 %inhibition atj 36 98 54.0 4.8 30.0 0.34 0.38 0.66 0.80 *no inhibition of 125 1-Fibrinogen binding observed at concentrations of 50 jiM or lower.

TABLE I (continued) Inhibition of 1 2 51-Fibrinogen BNr-jing 4-Guanidinocinnamoyl-L-aspartyl-L-tryptophan 8.2 1 0 amide 4-Guanidinocinnamoyl-L-aspartyl-L-phenylalanine 1 .5 4-G uan idi noci n namoyl- L-aspartyl- L-aspa rag ine 34.0 4-Guanidir,-cinnamoyl-L-aspartyI-L-serine 5.6 4-Guanidinocinnamoyl-L-aspartyl-L-tyrosine 2.2 4-Guanidinocinnamoyl-L-aspartyl-L-alanine 10.0 4-G uanidinocinnamoyl-L-aspartyl-L-aspartic acid 47.0 4-Guanidinocinnamoyl-L-aspartyl-L-Iysine 4.0 4-Guanidinocinnamoyl-L-aspartyl-L-lysine amide 10.5 4-Guanidinocinnamoyl-L-aspartyl-L-histidine (W.

Inhibition of Fibrinogen Mediated Platelet Agcjregation 4.2 0.85 25.0 28.0 1.3 5.9 >75.0 1.6 11.4 4.0 inhibition at uM 93 97 92 22 87 *no inhibition of 125 1-Fibrinogen binding observed at concentrations of 50 4tM or lower.

.1 -~.AAd ts#~ TABLE I (continued) Inhibition of 15-Fibrinogen Binding 4-Guanidinobenzoyl-glycyl-L-aspartyl-L-valine 6.4 3-Guanidinobenzoyl-glycyl-L-aspartyl-L-valine 4-Guanidinobenzoyl-sarcosyl-L-aspartyl-L-valine 3.2 1 5 4-Amidi nobenzoyl-sarcosyl-L-aspartyl-L-valine 110.0 4-Guanidinobenzoyl-N-ethylglycyl-L-aspartyl- 0.17 L-valine 4-Guanidinobenzoy-N-propylglycyl-L-aspartyl- 0.46 L-valine 4-(4-Guanidi nophenyl)-butyryl-L-aspartyl- 5.8 L-valine Inhibition of Fibrinogen Mediated Platelet Aggregation 23.4 1.2 1.1 7.9 1.8 inhibition a 17 97 86 94 *no inhibition of 125 1-Fibrinogen binding observed at concentrations of 50 [iM or lower.

WO 92/18117 PCT/US91/02471 The compounds of the present invention may be orally or parenterally administered to mammals. The compound 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, compounds 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 as.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.

~fi~t ~;1 (i

Claims (19)

1. A compound of the formula $0 0 Y-C -N -CH -C-Z H (CH 2 )M-CO 2 H w or a pharmaceutically acceptable salt thereof wherein: RN N-R 1 R2-NH NH-R 2 R, NH-fl 2 NH NH or -C=N; W is -CH=CH-(CH 2 -(CH2)n-CH=CH-, or Y is -(CH 2 -CH=CH-CH 2 -CH 2 CH=CH-, -CH=CH-, 0 0 20 RiR N-N -(CH 2 N (CH 2 )n -CH 2 -Xl- or -Xj-CH 2 wherein X 1 is 0, NH or S; WO 92/18117 WO 9218117PCr/US91/02471 N R-, Z is -OR1, R2, R 1 aD- or L-amino acid or its corresponding carboximide, a synthetic amino acid of the formula R 3 -NR 1 -Co-R 4 I a dipeptide or a dipeptide isostere of the formula -NH-CH-V 1 -CH-R 3 R 5 R m is 1, 2 or 3; n is 0to 6; R, and R 2 are independently H, alkyl, aryl, aralkyl or allyl; R3 is H, -CO2H, -CO2131, -CONH 2 2or R-1 N R 7 R 4 and R 5 are independently H, alkyl, cycloalkyl, cycloalkylmethyl, X2 -0 (CH 2 )p F- I WO 92/181 17 PCT/US91/02471 fl- N. x 2 -(CH2)p-4 N I KI X 2 (CH 2 NH-/0 \NH 2 -(CH 2 )pORI, -(CH 2 )pSR1, -(CH 2 )p-NRi R 2 NH0 -(CH 2 1 NH 2 -(0H 2 )p-CO 2 Rl or -H 2 )p-C-NRlR 2 p is 0 to 8; R 6 and R7 form a ring with the nitrogen to which they amp attached and are independently -(OH 2 4 (OH 2 5 -(OH 2 -CH 2 CH 2 OCH 2 CH 2 CH 2 -CH 2 -CH 2 -CH 2 CH 2 NRlCH 2 or -N\ R 2 0 11 -NH-C-R 1 -SR1, X 2 is H, Cl, Br, F, -OR1 1 -NO 2 NH -C NH 2 NH -NH-C NH 2 0 11 C-N alkyl, phenyl, -00 2 R 1 -OF 3 or -NH S0 2 Rj; and 0 -N V, is ,i isH)n -CH=OH- -CH 2 -NH- -0H 2 -O- I _II WO 92/18117 PCT/US91/02471 0 II -CH 2 or -C-CH 2

2. A compound of Claim 1 wherein said D- or L-amino acid is selected from the group consisting of: Asp, Arg, Ala, Asn, Cys, Gly, Glu, Gin, His, lie, Leu, Lys, Met, Orn, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

3. A compound of claim 1 wherein: R 1 and R 2 are independently hydrogen or phenyl; R 3 is H or-CO 2 H; R 4 and R 5 are independently H, alkyl or cyc!oalkyl; mis 1; n is O; p is 1; and Re and R 7 are -(CH 2 4 forming a ring with the nitrogen to which they are attached.

4. A stereoisomeric compound of claim 1. A diastereomeric compound of claim 1.

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

7. A method of preventing or treating thrombus formation in a mammal comprising the administration of a composition of claim 6. a i g i Ij

8. A compound of claim 1 which is: i~ I- -1; WO092/181!7 35PCT/US9/02471 N,.{3-(2-guanidinoethyl)benzoyl]-L-p-sparty-L-valine; N -4 -(2-guanidi noethyl)be nzoyl]-L-aspartyl-L-valine; N-(3-guanidi nobenzoyl)-L-aspartyl-L-vali ne; N-(4-guanidinomethylbenzoyl)-L-aspartyl-L-valine; or N-(3-guanidinomethylbenzoyl)-L-aspartyl-L-valine.

9. A compound of claim 1 which is: N-(4-guanidinobenzoyl)-L-aspartyl-L-valine; N-[3-(2-ami noethyl)benzoyl]-L-asparty-L-vali ne; N-(4-guanidinophenylacetoyl)-L-aspartyl-L-valine; N-(4-aminophenylacetoyl)-L-aspartyl-L-valine; or 4-guanidi foci flnamoyl-L-aspartyl-L-valine trifluoroacetate. A compound of claim 1 which is: 4-Guanidi nobenzoyl-N-ethyl g Iycyl-L-aspartyl-L-vali ne trifluoroacetate; 4-guanidinocinnamoyi-N-ethyl glycyl-L-aspartyl-L-valine; 4-Guanidinohomocinnamoy-L-aspartyl-L-valine; 4-(imidazol-1 -yi)-cinnamoyl-L-aspartyl-L-valine trifluoroacelate; or 4-guanidi foci nnamoyl-L-aspartyl-L-leuci ne amide trifluoroacetate.

11. A compound of claim 1 which is: WO 92/18117 WO 9218117PCIZ/US91/02471 4-Guanidinocinnamoyl-L-aspartyl-L-norvaline trifluoroacetate; 4-Guanidinocin namoyl-N-(ethyl)-glycyl-L-aspartyl-L-valine ditrifluoroacetate; 4-Guanidinocinnamoyl-L-aspartyl-L-norleucine trifluoroacetate; 4-Guanidinoci nnamoyl-sarcosyl-L-aspartyl-L-leucine trifluoroacetate; or 4-Guanidi nocinnamoy-p3-alanyI-L-aspartyl-L-vali ne trifluoroacetate.

12. A compound of claim 1 which is: 4-Guanidinocinnamoyl-L-sarcosyl-L-aspartyl-L-isoleucine trifluoroacetate; 4-Guanidinocin namoyl-sarcosyl-L-aspartyl-L-argi nine ditrifluoroacetate; 4-Guanidinohomoci nnamoyl-aspartyI valine trifluoroacetate; 4-(lmidazol-1 -yl)-cin namoyl-L-aspartyl-L-valine trifluoroacetate; or 4-undncnaoiasatlNmmty-,L-tryptophan trifluoroacetate.

13. A compound of claim 1 which is: 4-Guanidi noci nnamoyl-L-aspartyl-L-histidine ditrifluoroacetate; 4-Guanidi nocinnamoyl-L-aspartyl-D-valine trifluoroacetate; 4-Guanidinocinnamoyl-L-asparty-L-P3-(2-naphthyl)alanine trifluoroacetate; 4-G uanidi nocinnamoyl-sarcosyl-L-aspartyl-L-vali ne trifluo roacetate; or 4-Guanidinobenzoy-N-ethylg lycyl-L-aspartyl-L-vali ne trifluoroacetate. 4 [1 I I WO92/18117 PCF/US91/O2471 37

14. A compound of claim 1 which is: 4-Amidinobenzoyl-sarcosyl-L-aspariyl-L-valine trifluoroacetate; ft 4-Guanidi nobenzoyl-N-propylg lycyl-L-aspartyl-L-vali ne trifluoroacetate; 4 -(4-Guanidinophenyl)butyryl-L-aspartyl-L-valine trifluoroacetate; 4-Guanidinobenzoyl-sarcosyl-L-aspartyl-L-valine trifluoroacetate; or 3-Guanidi nobenzoylglycyl-L-aspartyl-L-vali ne trifluoroacetate. A compound of claim 1 which is: 4-Guanidi nobenzoylglycyl-L-aspary-L-vali ne trifluo roacetate; a-Cyan o-para-to Iuy I-L-as partyI- L-vali1ne; 4-Guanidi nociniiamoyl-L-aspartic acid-cx-isobutyl amide trifluoroacetate; 4-Guanidi nocin,"namoyl- L-aspartyl-L-tryptophan amide hydrogen fluoride; or 4-Guanidinocinnamoy-L-aspartyl-L-lysine amide dihydrogen fluoride.

16. A compound of claim 1 which is: 4-Guanidinomethyl cm namoyl-L-aspartyl-L-valine trifluoroacetate; 4-Guanidinocinnamoyl-L-aspartyl-L-leucine-amide hydrogen fluoride; 4-G uanidnocin namoyl-L-aspartyl-L-valine amide trif luo ro acetate; 3-Guanidi foci nnamoyl-L-aspartyl-L-valine trifluo roacetate; orj 4-Guanidi foci nnamoyl-L-aspartyl-L-proline trifluo roacetate. WO 92/ 18117 PC7/US91/02471 38

17. A compound of claim 1 which is: 4-Guanidinocinnamoyl-L-aspartyl-L-arginine ditrifluoroacetate; rfuraeae 4-GAidinocinnamoyl-L-aspartyl-L-asprne trifluoroacetate; 4-G uanidi nocinnamoyl-D-aspartyl-L-asali ne trifluoroacetate; 4-undncnaoLI-satlLvln tilooct;o 4-Guanidinocinnamoyl-L-aspartyl-L-aspartic acid trifluoroacetate. 18~. A compound of claim 1 which is: 4-Guanidi nocinnamoyl-g lycyl-L-aspartyl-L-valine trifluo roacetate; 4-Guanidinoci nnamoyl-L-aspartyl-L-lysine ditrifluoroacetate; 4-(4-Guanidinophenyl)butyryl-L-aspartyl-L-vali ne trifluoroacetate; Vt 4-Di methylaminocinnamoyl-L-aspartyl-L-valine; or 4-Aminocinnamoyl-L-aspartyl-L-vali ne trifluoroacetate.

19. A compound of claim 1 which is: 4 u n d n c n a olI- s at]L t r o i n r f u r a e a e 4-Guanidinoci nnamoyl-L-aspartyl-L-treponine trifluoroacetate; 4-Guanidinophe nylthioacetoyl-L-aspartyl-L-valine trifluoroacetate; 4-G uanidi nocin namoyl-L-aspartyl-L-serine trifluoroacetate; or 4-Guanidinocinnam oyl-L-aspartyl-L-tyrosine trifluoroacetate. WO 92/18117 WO 9218117PCJ7/US91/02471 A compound of claim 1 which is: 4-Guanidi nophenoxyacetoy-L -aspartyl-L-vali ne trifluoroacetate; 4'-Guanidinooxaniloyl-L-aspartyl-L-valine trifluoroacetate; 4-Guanidinocinnamoyl-L-aspartyl-L-leucine tnifluoroacetate; 4-Guanidinocinnamoyl-L-aspartyl-L-isoleucyl-L-arginine ditrifluoroacetate; or 4-Guanidinocin namoyl-L-aspartyl-L-arginyl-L-isoleucine.

21. A compound of claim 1 which is: 4-Guanidi nocin namoyl-N-(ethyl)-g Iycyl-L-aspartyl-L-isoleucine trifluoroacetate; 4-Guanidinocinnamoyl-N-(ethyl)-glycyl-L-aspartyl-L-Ieucin~e trifluoroacetate; (2-(6'-Guanidinohexyl)benzoyl]-L-aspartyl-L-valine; or E.-[2-(5'-guanidinopent-1 '-enyl)benzoyl]-L-aspartyl-L-valine.

22. A compound of claim 1 which is: E-[2-(6'-gua -,idinohex-l1'-e nyl)benzoyl]-L-aspartyl-L-valine; uanidinopent-1 '-ynyl) benzoyl]-L-aspartyl-L-valine; uanidino hex-1 l-ynyl)benzoyl]-L-aspartyl-L-vali ne; or WO 92/18117 PCT/US9/02471 [2-(4'-Guanidinobutyl)phenyl]acetyl-L-aspartyl-L-valine.

23. A compound of claim 1 which is: E-[2-(5'-guanidinopent-l1 -enyl)phenyl]acetyl-L-aspartyl-L-valine; E-[2-(4'-guanidinobut-1 '-enyl)phenyl]acetyl-L-aspartyl-L-valine; or [2-(5'-Guanidinopent-1 '-ynyl)phenyliacetyl-L-asparty-L-vali ne. 4

24. A compound of claim 1 which is: [2-(4'-Guanidinobut-1 '-ynyl)phenyl]acetyl-L-aspartyl-L-valine; 4-Guanidinocinnamoyl-L'aspartyl-D-x-benzyl-arginine-(D-ornithine); or 4-Guanidinocinnamoyl-L-aspartyl-L-a-benzyl-arginine-(L-ornithine). __I INTERNATIONAL SEARCH REPORT international Application NoPCT/US91/02471 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, Indicate all) According to International Patent Classification (IPC) or to both National C ,sirnation and IPC A61K 31/195,31/215, 37/02; C07C .279/00 C07K 5/06, 5/08 US.Cl: 514/19, 563; 530/331; 560/34; 562/439 II. FIELDS SEARCHED Minimum Documentation Searched 4 Classification System Classification Symbols U.S. 514/18, 19, 563; 530/331; 560/34; 562/439 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched III. DOCUMENTS CONSIDERED TO BE RELEVANT 14 Category Citation of Document, if; with indication, where appropriate, of the relevant passages Relevant to Claim No. id A US, A, 4,105,789 (Ondetti et al.) 1-24 08 August 1978, see abstract. A US, A, 4,379,764 (Fujii et. al.) 1-24 12 April 1983, see abstract. A US, A, 4,634,715 (Greenlee et al.) 1-24 06 January 1987, see abstract. A US, A, 4,870,207 (Umezawa et al.) 1-24 26 September 1989, see entire document. A US, A, 4,956,504 (Takeuchi et al.) 1-24 11 September 1990, see abstract. SSpecial categories of cited documents: 15 later document published after the international filing date document defining the general state of the art which is not or priority date and not in conflict with the application but cited to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but published on or after the international document of particular relevance; the claimed invention filing date cannot be considered novel or'cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another document of particular relevance: the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 2 17 July 1991 08 OCT 1991 International Searching Authority I Signature of Authorized Officer rs ISA/US Lester L. Lee Form PCT/ISA/210 (second sheet) (May 1986) .i