AU673497B2 - Anti-thrombotic peptides and pseudopeptides - Google Patents

Description

OPI DATE 02/11/92 APPLN. ID 20143 92 AOJP DATE 10/12/92 PCT NUMBER PCT/IJS92/02637 yR7./Q7

TREA

INTERN/-. TY (PCT) (51) International Patent Classification 5 nte onal ublication Number: WO 92/17196 A61K 37/02, C07K 5/06, 5/08 Al C07K 5/10, 7/06 (43) International Publication Date: 15 October 1992 (15.10.92) (21) International Application Number: (22) International Filing Date: Priority data: 677,006 28 March Parent Application or Grant (63) Related by Continuation

US

Filed on PCT/US92/02637 30 March 1992 (30.03.92) S1991 (28.03.91) 677,006 (CIP) 28 March 1991 (28.03.91), (71) Applicant (for all designated States except US): RHONE- POULENC RORER INTERNATIONAL (HOLD- INGS) INC. [US/US]; 4"-bw (72) Inventors; and Inventors/Applicants (for US only) KLEIN, Scott, 1, (US/ US]; 1612 Meadowview Lane, Mont Clare, PA 19453 MOLINO, Bruce, F. [US/US]; 2825 North Ford Drive, Hatfield, PA 19440 CZEKAJ, Mark [US/ US]; 206 Crest Drive, Sellersville, PA 18960 (US).

GARDNER, Charles [US/US]; 644 South 5th Avenue, Royersford, PA 19468 DECKER, Michael, R. [US/ US]; 62 Church Road, Norristown, PA 19401 DE- NER, Jeffrey, M. [US/US]; 251 West DeKalb Pike, Apt.

C-1010, King of Prussia, PA 19406 PELLETIER, Jeffrey, C. [US/US]; 1308 Lakeview Drive, Lansdale, PA 19446 (US).

(74) Agents: DARKES, Paul, R. et al.; Rhone-Poulene Rorer Inc., 500 Arcola Road, P.O. Box 1200, Collegeville, PA 19426 (US).

(81) Designated States: AT, AT(European patent), AU, BB, BE (European patent), BF (OAPI patent), BG, BJ (OAPI patent), BR, CA, CF (OAPI patent), CG (OAPI patent), CH, CH (European patent), CI (OAPI patent), CM (OAPI patent), CS, DE, DE (European patent), DK, DK (European patent), ES, ES (European patent), FI, FR (European patent), GA (OAPI patent), GB, GB (European patent), GN (OAPI patent), GR (European patent), HU, IT(European patent), JP, KP, KR, LK, LU, LU (European patent), MC (European patent), MG, ML (OAPI patent), MN, MR (OAPI patent), MW, NL, NL (European patent), NO, PL, RO, RU, SD, SE, SE (European patent), SN (OAPI patent), TD (OAPI patent), TG (OAPI patent), US.

Published With international search report.

i I/ (54)Title: ANTi-THROMBOTIC PEPTIDES AND PSEUDOPEPTIDES k 104 nr (57) Abstract 'l 4T O^ This 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 disease states such as myocardial infarction, stroke, peripheral arterial disease and disseminated intravascular coagulation, to pharmaceutical compositions including such compounds and to their use in inhibiting thrombus formation and platelet aggregation in mammals.

311 r o0 A 'D E C.

CLenu\ ftA ~ra WO 92/17196 PCT/US92/02637 1 ANTTHROMBOTIC PEPTIDES AND PSEUDOPEPTIDES BACKGROUND OF INVENTION 1. Field of the Invention This invention relates to 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 and are useful in the prevention and treatment of thrombosis associated with 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 vhole 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 Willebrand factor. Fibrinogen is a co-factor for platelet aggregation, while 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 protein rrnplex known as glycoprotein lib/llia.

WO 92/17196 PCTfUS92/O26,1' 2 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/llla binding site accessible to fibrinogen. Compounds within the scope of the present invention block the fibrinogen receptor, thus inhibiting platelet aggregation and subsequent thrombus formation and when administered in the form of pharmaceutical compositions comprising such compounds are useful for the prevention 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 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 GPilb/llla 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. Bio. Chem. 1985, 260, 3931-36; and Gartner, et al., J. Biol. Chem. 1987, 260, 11,891-94).

Indolyl compounds containing guanidinoalkanoyl- and guandinoalkenoyl- aspartyl moieties are reported to be platelet-aggregation inhibitors by Tjoeng, et al., U.S. Patent Nos. 5,037,808 and 4,879,313.

U.S. Patent No. 4,992,463 (Tjoeng, et issued February 12, 1991, discloses generically that a series of aryl and aralkyl guanidinoalkyl peptide mimetic compounds exhibit platelet aggregation inhibiting activity and discloses specifically a series of mono- and dimethoxy phenyl peptide mimetic compounds and a biphenylalkyl peptide mimetic compound.

WO 92/17196 PCT0US92/02637 3 U.S. Patent No. 4,857,508 (Adams, et issued August 15, 1980, discloses generically that a series of guandinoalkyl peptide derivatives containing terminal aralkyl substituents exhibit platelet aggregation inhibiting activity and discloses specifically a series of O-methyl tyrosine, biphenyl, and naphthyl derivatives containing a terminal amide functionality.

Haverstick, et al., in Blood66 946-952 (1985), disclose that a number of synthetic peptides, including arg-gly-asp-ser and gly-arg-gly-aspser, are capable of inhibiting thrombin-induced platelet aggregation.

Plow, et al., in Proc. Natl. Acad. Sci. USA 79, 3711-3715 (1982), disclose that the tetrapeptide glycyl-L-prolyl-L-arginyl-L-proline inhibits fibrinogen binding to human platelets.

French Application No. 86/17507, filed December 15, 1986, discloses that tetra-, penta- and hexapeptide derivatives containing the -arg-gly-aspsequence are useful as antithrombotics.

U.S. Patent No. 4,683,291 (Zimmerman, et issued July 28, 1987, discloses that a series of peptides, comprised of from six to forty amino acids, which contain the sequence -arg-gly-asp- are platelet binding inhibitors.

European Application Publication No. 0 319 506, published June 7, 1989, discloses that a series of tetra-, penta-, and hexapeptide derivatives containing the -arg-gly-asp- sequence are platelet aggregation inhibitors.

Cyclic peptide analogues containing the moiety Gly-Asp are reported to be fibrinogen receptor antagonists in U.S. Patent No. 5,023,233.

Peptides and pseudopepiides containing amino-, guanidino-, imidizaloyl, and/or amidino- alkanoyl, and alkenoyl moieties are reported to be antithrombotic agents in pending United States applications serial nos.

07/677,006, 07/534,385, and 07/460,777 filed on March 28, 1991, June 7, 1990, and January 4, 1990, respectively, as well as in U.S. Patent No.

4,952,562, and in International Application No. PCT/US90/05448, filed September 25, 1990, all assigned to the same assignee as the present invention.

WO 92/17196 PCT/US92/02637 4 Peptides and pseudopeptides containing amino- and guanidino- alkyland alkenyl- benzoyl, phenylalkanoyl, and phenylalkenoyl moieties are reported to be antithrombotic agents in pending United States application serial no. 07/475,043, filed February 5, 1990, assigned to the same assignee as the present invention.

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

SUMMARY OF THE INVENTION Compounds of the present invention are described by Formula I 3 0 A- CH 2 C)-B -C-CH2--D-CH 'Z M l I 'k 'h 2 m2

R

2 R4 (CHn

COOH

Formula I wherein: A is cyano, -N oNRs "NH C-(NH)--R6 Rs, x x' or N40 RR I R6 B and D are independently -CH2-NH-, -CH 2

-CH

2 0 R, 0 0 11

CH-CH

2

-CH-CH

2

-O-

-C-NR, I I I S Rll R7 Y r7 or Y, (CH,)q B may also be 5-tetrazol-l-yl, -CR7=CRs-, -CC- or Z is -ORa, nitrogen-containing heterocyclyl, a D- or L-isomer of an aamino acid bonded at the a-nitrogen, a dipeptide bonded at the N-terminal a- NVO 92/17196 WO 9217196PCT/US92/02637 H-V>C-Rt amino acid, or -NRaRx, where Rx is H or where V is 0 0 11 1I C-NRg C-CH 2 -CH=CH-, -CH 2 NH-, -CH 2 or

-CH

2 Re and Rf are independently H, alkyl, cycloalkyl, cycloalkylmethyl, or -(CH2)s- Rz where Rz is nitrogen-containing heterocyclylcarbonyl, -COORn, NH NH 11 11 -ORn, SRn-, -NRnRo, -NH-C-NH 2

-C-NH

2 phenyl, substituted phenyl naphth-1-yI, naphth-2-yI, substituted napth-1-yl, substituted naphth-2-yl, 1,1diphenylmethyl, 1, 1 -di (substituted phenyl)methyl, N-Rn substituted indol-2-yl, 1 0 N-Rn substituted indol-3-yI, substituted (N-Rn substituted) indol-2-yI, substituted (N-Rn substituted) indol-3-yi, quinolin-2-yl, quinolin-3-yl, quinolin-4yl, substituted quinolin-2-yl, substituted quinoli n-3-yI, substituted quinolin-4-yl, N- Rn substituted imidazol-2-yl, N-Rn substituted imidazol-4-yI, N-Rn substituted substituted N-Rn substituted imidazol-2-yI, substituted N-Rn 1 5 substituted imidazol-4-yl, substituted N-Rn substituted imidazol-5-yl, imidizol-1 yl, or substituted imidazol-1 -yI; RI-ia, Ra, Rg, Rk, Rm-p, Rq, and Rs are independently H, alkyl, cycloalkyl, cycloalkylmethyl, aryl, substituted aryl, aralkyl or substituted aralkyl; Rt is -COOH, -COOlik, carbamoyl, N-containing heterocyclyl or 0 11 C-NRkRm; Rn is R1io or Yj; 0 11 Yj is H, amino or -NH-C-RP it being understood that Yj may be the same or different for B and D; x, x"'and are independently 0 or 1; ml and M2 are independently 0 to 9; hl, h 2 and k are independently 0 or 1; n is i to 3; q is 1 to 5; and p and s are independently 0 to 6; WO 92/17196 PCT/US92/02637 6 provided that when A is guanidino, and B and D are then Z is other than aralkylamino or substituted aralkylamino; and when A is guanidino, B and D are and Z is -NRaRx where Rx Re is Rf and Rt is -C(O)NH 2 and P hydrogen, then Rf is other than benzyl, substituted benzyl, naphthylmethyl or substituted napthylmethyl; and

O

R1 R, j A -CH 2 ,H C(B )C H2- D-CHOC mlhl k h2 m I

R

2

R

4

(H

2 )n when COOH is arginylglycyl-aspartyl, then Z is other than a naturally occurring amino acid or a dipeptide composed of two naturally occurring amino acids; and pharmaceutically acceptable salts thereof.

Additionally, this invention relates to pharmaceutical compositions comprising such compounds, and to methods of treatment of abnormal thrombus formation in mammals comprising the administration of such compounds and pharmaceutical compositions DETAILED DESCRIPTION OF THE INVENTION As used above, and throughout the description of this invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings: "Nitrogen-containing heterocyclyl" means about a 4- to about a membered nitrogen-containing monocyclic or multicyclic ring system in which one or more of the other atoms in the ring or rings may be an element other than carbon, for example nitrogen, oxygen or sulfur and further that the heterocycle is bound at the nitrogen atom. Preferred nitrogen-containing heterocyclyl groups include pyrrolidin-1-yl, piperidin-1-yl, homopiperidin-1-yl, WO 92/17196 PCT/US92/02637 7 morpholin-4-yl, 1,2,3,4-tetrahydroisoquinolin-2-yl, piperazin-1-yl. In the case of piperazin-1-yl, the nitrogen at the 4-position preferably may be substituted by alkyl, cycloalkyl, cycloalkylmethyl, aryl, substituted aryl, aralkyl or substituted arakyl.

"a-amino acid" means a synthetic or naturally occurring amino acid.

Preferred a-amino acids are the naturally occurring amino acids, i.e, glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, proline, hydroxyproline, aspartic acid, aspargine, glutamine, glutamic acid, histidine, arginine, ornithine, and lysine.

"Dipeptide" means a-aminoacyl-a-aminoacid.

"Carboxy" means a -COOH group.

0

II

"Carbamoyl" means a -C-NH 2 group.

"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 straight or branched alkyl groups are the "lower alkyl" groups which are those alkyl groups having from 1 to about 6 carbons.

"Cycloalkyl" means a saturated carbocyclic group having about 3 to about 8 carbon atoms. Preferred cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

NH

II

"Guanidino" means an -NH-

C

-NH

2 group.

"Aryl" means a phenyl or naphthyl group.

"Substituted aryl" means a phenyl or naphthyl group substituted by one or more aryl group substituents which may be the same or different, where "aryl group substituent" includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxy, alkoxy, WO 92/17196 C/S226 PCr/LIS92/02637 8 aryloxy, aralkoxy, hydroxyalkyl, acyl, formyl, carboxy, alkenoyl, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamnino, aroylamino, carbamoyf, alkylcarbamoyl, dialkylcarbamoyl, arylcarbamoyl, aralkylcarbamoyl, alkylsulfonyl, alkylsulfinyl, arylsu Ifonyl, arylsulfinyl, aralkylsulfonyl, aralkylsulfinyl, or -NRR' where R and R' are independently hydrogen, alkyl, aryl, or aralkyl.

"Substituted"- phenyl naphth-1 -yl, naphth-2-yl 1,1 -diphenylmethyl, 1 ,1 di (substituted phenyl)methyl, indol-2-yi, indol-3-yl, quinolin-2-yi, qui nolin-3-yl, quinolin-4-yl, imidazol-2-yI, imidazol-4-yl, imidazol-5-yl, and imidazol-1-yl means that these groups are substituted by an aryl group substituent.

Preferred aryl group substitutents for these groups are hydrogen, halo, nitro, trihalomethyl, phenyl, alkyl, nitrogen-containing heterocyclyl carbonyl, nitrogen-containing heterocyclyl carbonylalkyl, amidino, guanidino, -N~qRs, 0 11 0 -CN-Rq 11 1 -SRq, -COORq, NHSO2Rq, -NHCRq, or Rs where Rq and Rs are independently H, alkyl, cycloalkyl, cycloalkylmethyl, aryl, substituted aryl, aralkyl or substituted aralkyl.

"Aralkyl" means an alkyl group substituted by an aryl radical. Preferred aralkyl groups include benzyl and phenethyl.

"Substituted aralkyl" means an aralkyl group substituted on the aryl portion by one or more aryl group substituents.

A preferred class of compounds of the present invention is described by Formula I wherein: B and D are independently -CH 2

-CH

2

-CH

2 0 R9 0

-CH-CH

2 -N -HC 2 -0- Rf I I I I I

XC.NR

9 hH) H 7 YJ R 7 or Y

-CH-CH-

B may also be .,CR 7

CR

8 -CC- or 0 H2)q WO 92/17196 WO 9217196PCT/!US92/02637 and Rz is nitrogen-containing heterocyclylcarbonyl, -COORn, -ORn, -SRn-, NH NH 11 11 -NRnRo, -NH-C-NH 2 or 0C-NH 2 Another preferred class of compounds of the present invention is described by Formula I wherein: B and D are independently -CH 2

-CH

2 -0H 2 0 ~R 9 0

CNR

9 4 H\ jj- -CH-CH 2 -CH-0H 2

-O-

C gf I II I I

R

11 h7, R 7 or Y1

-OH-OH-

B may also be -CR 7

=CR

8 -CC- or 0

H

2 )q and Rz is nitrogen-containing heterocyclylcarbonyl, -COORn, -ORn, NH NH I1 II SRn-, -NRnRO, -NH-C-NH 2 or -C-NH 2 and Z is -ORa, pyrrolidin-1 -yl, piperidin-1 -yl, homopiperidin-1 -yl, morpholin-4yi, piperazin-1-yl, a D- or L-isomer of an a-amino acid bonded at the axnitrogen, a dipeptide bond~ed at the N-terminal a-amino acid, or -NRaRx where

R

Ris H or Another preferred class of compounds of the present inveintion is described by Formula 11 NH 0 0 0 NF1 2 -C-NH-+oH 2 2

-O-NH-CH-C-Z

COGH

Formula 11 WO 92/17196 927196PUS92/02637 wherein: ml is 2 to 9; and Z is phenethylamino or 1 ,2,3,4-tetrahydroisoquinolin-2-yl.

Still another preferred class of compounds of the present invention is describedJ by Formula I wherein: B is 5-tetrazofyl-1 -yl; and Rz is -CH 0 W2 W ,7w N

NJ

Hn HII or W wherein: W1 and W 2 are independ~ently hydrogen, halo, nitro, trihalornethyl, phenyl, alkyl, nitrogen-con'taining heterocyclyl carbonyl, nitrogen-containing heterocyclyl carbonylalkyl, amidino, guanidino, -NRqRs, 0 11 0 -C-NRq 1I 1 -SRq. -COORq, -NHS02iq, -NHCRq, or ;i and Rn, Rq and Rs are independently H, aIyl, cycloalkyl, cycloalkylmethyl, aryl, substituted aryl, aralky'. or substituted aralkyl.

WO 92/171966 PCT/US92/0263- 11 A more preferred class of compounds of the present invention is described by Formula III 0 0

II

I

CCH

COOH

Formula III wherein: -N N

R,

A is guanidino or ml is 1 to 9; m2 is 0 or 1; and o C-NR9- CH B is -CH=CH- or A still mc t preferred class of compounds of the present invention is described by the more preferred class of compounds wherein A is guanldino.

A most preferred class of compounds of the present invention is described by the still more preferred class of compounds wherein Z is a D- or L-isomer of an a-amino acid bonded at the a-nitrogen, or Z is a dipeptide bonded at the N-terminal a-amino acid.

Another most preferred class of compounds of the present invention is described by the still more preferred class of compounds wherein Z is a D- or L-isomer of an a-amino acid bonded at the a-nitrogen.

WO 92/17196 WO 9217196PCT/US92/02637 12 A particularly preferred special embodiment of the present invention is described by the still more preferred class of compounds wherein Z is a D- or L-isorner of an ct-amino acid bonded at the ax-nitrogen, said a-amino acid being selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, serine, thweonine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, proline, hydroxyproline, aspartic acid, aspargine, glutamine, glutamic acid, histidine, arginine, ornithine, and lysine.

.A most preferred special embodiment of the present invention is described by the still more preferred class of compounds wherein Z is a D- or L-isomer of an a-amino acid bonded at the a-nitrogen, said a-amino acid being selected from the gro,,Ip consisting of valine, leucine, isoleucine, and arginine.

1 5 Representative compounds of the present invention include: ntanoyl-N-(ethyl)-g lycyi-L-aspartyl- L-leucine; 6-guanidinohexanoyl-N,'ethyl)-glycyl-L-aspartyl-L-leucine; 6-guanidinohexanoyl-N-(ethyl)-giycyl-L-aspartyl-L-isoleucine; 6-guanidi nohexanoyl-sarcosyl-L-aspartyl-L-leucine; 6-guanidinohexanoyl,-N-(ethyl)-glycyl-L-aspartyl-L-valine; 6-guanidi nohexanioyl-sarcosyl-L-aspartyl-L-vali ne; nine; 8-guanidinooct-2-enoyl-L-aspartyl-L-valine; 9-guanidi non onanoyi-L-aspartyl-L-iso leucine-4-guanidin obutyl amide; 9-guanidinononanoyl-L-aspartyl-L-leucine; WO 92/17196 PCT/US92/02637 13 9-guanidinononanoyl-L-aspartyl-L-arginine; 9-guanidinononanoyl-L-aspartyl-L-arginine-isobutyl ester; 9-guanidinononanoyl-L-aspartyl-L-leucyl-arginine; 9-guanidinononanoyl-L-aspartyl-Lvalyl-arginine; N-[N-(9-guanidinononanoyl-L-aspartyl)-2-aminobutanoy]-L-arginine; 9-guanidinononanoyl-L-aspartyl-L-alanyl-arginine; 9-guanidinononanoyl-L-aspartyl-L-norleucyl-arginine; 9-guanidinononanoyl-L-aspartyl-D-homoisoleucyl-L-arginine; 9-guanidinononanoyl-L-aspartyl-L-phenylalanyl-L-arginine; or N-(9-guanidinonnanoyl-L-aspartyl)-3-amino-2-sec-butylpropionyl-Larginine of the ditrifluoroacetate salt thereof; or pharmaceutically acceptable salts thereof.

Compounds of the present invention contain asymmetric centers. These asymmetric centers may independently be in either the R or S configuration.

The present invention comprises the individual stereoisomers and mixtures thereof.

The compounds of the present invention may be useful in the form of the free base or acid or in the form of a pharmaceutically acceptable salt thereof.

All forms are within the scope of the invention.

Where the compound of the present invention is substituted with a basic moiety, acid addition salts may be formed and are simply a more convenient form for use; and in practice, use of the salt form inherently amounts to use of the free base form. The acicd which can be used to prepare the acid addition WO 9)2/17196( PCT/US92/02637 14 salts include preferably those which produce, when combined with the free base, pharmaceutically acceptable salts, that is, salts whose anions are nontoxic to the animal organism in pharmaceutical doses of the salts, so that the beneficial antithrombotic properties inherent in the free base are not vitiated by side effects ascribable to the anions. Although pharmaceutically acceptable salts of said basic compounds are preferred, all acid addition salts are useful as sources of the free base form even if the particular salt, per se, is desired only as an intermediate product as, for example, when the salt is formed only for purposes of purification, and identification, or when it is used as intermediate in preparing a pharmaceutically acceptable salt by ion exchange procedures. Pharmaceutically acceptable salts within the scope of the invention are those derived from the following acids: mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid; and organic acids such as acetic acid, citric acid, lactic acid, tartaric acid. malonic acid, methanesufonic acid, ethanesulfonic acid, benzenesulfonic acid, ptoluenesulfonic acid, cyclohexylsulfamic acid, quinic acid, and the like. The corresponding acid addition salts comprise the following: hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartarate, malonate, methanesulfonate, ethanesulfoncte, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate, respectively.

The acid addition salts of the compounds of this invention are prepared either by dissolving the free base in aqueous or aqueous-alcohol solution or other suitable solvents containing the appropriate acid and isolating the salt by evaporating the solution, or by reacting the free base and acid in an organic solvent, in which case the salt separates directly or can be obtained by concentration of the solution.

Where the compound of the invention is substituted with an acidic moiety, base addition salts may be formed and are simply a more convenient form for use; and in practice, use of the salt form inherently amounts to use of the free acid form. The bases which can be used to prepare the base addition salts include preferably those which produce, when combined with the free acid, pharmaceutically acceptable salts, that is, salts whose cations are nontoxic to the animal organism in pharmaceutical doses of the salts, so that the beneficial antithrombotic properties inherent in the free acid are not vitiated by side effects ascribable to the cations. Pharmaceutically acceptable salts within WO 92/17196 W 279CKT/S92/02637 the scope of the invention are those derived from the following bases: sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, Nbenzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, tetramethylammonium hydroxide, and the like.

Metal salts of compounds of the present invention may be obtained by contacting a hydroxide, carbonate or similar reactive compound of the chosen metal in an aqueous solvent with the free acid form of the compound. The aqueous solvent employed may be water or it may be a mixture of water with an organic solvent, preferably an alcohol such as methanol or ethanol, a keone such as acetone, an aliphatic ether such as tetrahydrofuran, or an ester such as ethyl cetate. Such reactions are normally conducted at ambient temperature but they may, if desi'ed, be conducted with heating.

Amine salts of compounds of the present invention may be obtained by contacting an amine in an aqueous solvent with the free acid form of the compound. Suitable aqueous solvents include water and mixtures of water with alcohols such as methanol or ethanol, ethers such as tetrahydrofuran, nitriles such as acetonitrile, or ketones such as acetone. Amino acid salts may be similarly prepared.

Compounds of this invention may be prepared in accordance with the reaction sequences described below, or can be prepared by methods known in the art. The starting materials used in the preparation of compounds of this invention are known or are commercially available, or can be prepared by known methods or by specific reaction schemes described herein.

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, Paulsen, G. Merz, V. Weichart, "Solid- Phase Synthesis of O-Glycopeptide Sequences", Angew. Chem. Int. Ed. Engl.

27 (1988); H. Morgler, R. Tanner, J. Gosteli, and P. Grogg, "Peptide Synthesis by a Combination of Solid-Phase and Solution Methods I: A New Very Acid- WO) 92/17196 PCrI US92/0263- 16 Labile Anchor Group for the Solid-Phase Synthesis of Fully Protected Fragments. Tetrahedron letters 4005 (1988); Merrifield, "Solid Phase Peptide Synthesis after 25 Years: The Design and Synthesis of Antagonists of Glucagon", Makromol. Chem. Macromol. Symp. 12, 31 (1988)).

A preferred method of preparing compounds of the present invention is by the solid phase method schematically represented as follows: solid support -X 1 -N -P Deprotect I P R solid support -X 1 NH Coupling II P R 0

II

solid support -X 1 -NH -C -X 2 -N -P I I I I P R P R wherein: the solid support may be, but is not limited to, p-alkoxy benzyl resin, -Xn-N-P I I and P R is a 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 N-a-tertiary butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ), benzyl, t-butyl, 9-fluorenylmethyloxycarbonyl (FMOC), 2-(trimethylsilyl)ethyl, and 4-methoxy-2,3,6trimethylbenzenesulfonyl. Upon completion of the coupling reaction a functional group is deprotected by standard methods to give an active a-amino function which, in turn, is reacted with a protected amino acid derivative having a free carboxyl function thereon. This procedure is repeated until the desired peptide or pseudopeptide is formed. The compound is then deprotected and WO 92/17196 PCT/US92/02637 17 removed from the solid support by standard procedures to obtain the final product.

In another preferred method, 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 the protected amino acid derivatives oi analogs are coupled by using standard procedures, then deprotected to yield the desired final compound.

It may also be desirable or necessary to prevent cross-reaction between other chemically active substituents on reactants. The substituents may be protected by standard blocking groups which may subsequently be removed or retained, as required, by known methods to afford the desired products or intermediates (see, for example, Green, "Protective Groups in Organic Synthesis", Wiley, New York, 1981). Selective protection or deprotection may also be necessary or desirable to allow conversion or removal of existing substituents, or to allow subsequent reaction to afford the final desired product.

The invention is further explained by the following illustrative examples.

In the examples, when the carboxyl terminus of a compound ends in an amino acid other than valine, the synthetic process starts with the use of an appropriate commercially available N-a-FMOC-amino acid p-alkoxybenzyl alcohol resin ester. When such is not available, the appropriate N-a-FMOC protected amino acid p-alkoxybenzyl alcohol resin ester is prepared by the procedure of E. Givalt, et a. (Int. J. Peptide Protein Res. 1989, 33, 368).

Subsequent treatment of the starting materials is described in Example 1.

EXAMPLE 1 L-Arginyl-L-Aspartyl-L-Valine 1 3 of N-(9-fluorenylmethyloxycarbonyl)-L-valine p-alkoxybenzyl alcohol resin ester (containing 0.56 mmole of amino acid) is shaken with 20 ml of 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-B-t-butyl ester in 15 ml of dimethylformamide in the presence of 0.43g 1-(3- WO 92/171966 PCT/US92/02637 18 dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 0.31 ml triethylamine, and 0.30g 1-hydroxybenzotriazole (HOBT), for 1 1/2 hours. This is filtered, washed with methylene chloride, and the resulting resin treated with piperidine in methylene chloride as above to remove the FMOC group.

The resulting resin derivative is then treated as above with 1.36g N-a-FMOC- N-co-(4-methoxy-2,3,6-trimethylbenzenesuifonyi)-L-arginine in the presence of triethylamine, EDC, and HOBT. The FMOC group is removed as above. The peptide is removed from the resin by treating with 20 mi 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 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. 90-950C.

EXAMPLE 2 L-Arginvlglvcyl-L-Aspartvl-a-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-a-(FMOC)-L-aspartic acid B-tbutyl 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 .ac.u to give 2.2g N-a-(FMOC)- L-aspartic acid isobutyl amide 8-butyl ester.

B. The amide obtained in Example 2A is dissolved in 20% (v/v) piperidine in methylene chloride and stirred at room temperature for 2 hours.

The solution is evaporated in yuo 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-a-isobutyl amide-1-t-butyl ester.

C. 0.67g N-a-FMOC glycine and 0.55g of the amide obtained in 2B are treated under the conditions of Example 2A to give N-a-(FMOC)-glycyl-Laspartic acid isobutyi amide-B-butyl ester.

WO 92/17196 WO 92/7 96 CT/ US92/02637 19 D. The product obtained in Exampie 20 is treated as ini Example 2B to remove the FMVOC protecting group to give glycyl-L-aspartic acid isobutyl amide-13-butyl ester.

E. 0.40g of the product of Example 2D and 0.78g N-xc-t-BOO-N-CO(4methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine are treated as in Example 2A with 0.29g EDC, 0.17g HOBT and 0.18 ml triethylamine to give N-a-BOO..N- (4-co-methoxy-2 ,3,6-trimethylbenzenesulfonyl)-L-argi nylglycyl-L-aspartic acid isobutyl amide-13-butyl ester.

F. 0.35g of the product obtained in Example 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 4x1l00 ml of ethyl acetate. The aqueous solution was lyophilized to 0.1 9g of a white 1 5 so, L-arg inylg lycyl-L-aspartyl-a-isobutylamide as the ditrifluoroacetate salt; m.p. 90..951C.

EXAMPLE 3 L-OrnItbylglv spartyl-Valine A. 1 .27g L-valine t-butyl ester and 2.5g N-ca-FMOC-L-aspartic acid 13t-butyl ester are treated as in Example 2A in the presence of 1. 1 6g EDO, 0.93g triethylamine and 0.82g hydroxybenzotriazole. The resulting product is then deprotected as in Example 2B to give L-aspartyl-13-t-butyl ester-L-valine-xc-tbutyl ester.

B. 1 .1g of the product obtained from Example 3A is treated with N-oa- FMOO-glycine in the presence of 0.60g EDO, and 0.43_q of triethylamine in methylene chloride as in Example 2A rind the resulting product deprotected in piperidine in methylene chloride as in Example 26 to give 0.65g glycyl-Laspartyl-13-t-butyl ester-L-valine-ca-t-butyl ester.

C. 0.25g of the product from Example 3B is treated with 0.23g N-ca-t- BOC-N-B-CBZ-ornithine in 5mI of rrethylene chloride in the presence of 0.1 2g EDO, 0.8g HOBT and 0.09 ml triethylamine as in 2A to give 0.45g N-a-t-BOC- -OBZ-L-ornithyl-g lycyl-L-aspartyl-B1-t-butyI ester-L-valine-cx-t-butyl ester.

WO 92/17196 7PC/ LIS92/O263-7 D. The benzyloxycarbonyl protecting group on the product compound of Example 3C is removed by dissolving 0.45g of the protected compound in ml of cyclohexene and adding 0.10g 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-a-t-BOC-L-ornithyl-glycyl-L-aspartyl- -t-butyl ester-Lvaline-t-butyl ester.

E. 0.23g of the product obtained in Example 3D is disiolved 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 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 0

C.

EXAMPLE 4 L-Arginvlsarcosyl-L-Asartv-L-Valine N-a-FMOC-sarcosine is substituted for N-a-FMOC-glycine and the resulting product is treated with piperidine in methylene chloride as in Example 1 to remove the FMOC group. The corresponding product is 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 Larginylsarcosyl-L-aspartyl-L-valine as the ditrifluoroacetate salt; m.p. 1450C (dec.).

EXAMPLE L-Arginvlglycyc-L-Aspartyl-L-(N-Methyl)Valine A. ig of p-alkoxybenzylalcohol resin (0.5-1 mmole/g of resin), 0.706g of N-FMOC-N-methyl-L-valine, 0.382g -0C, 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 WO 92/17196 PCT/IS92/02637 21 acid, 0.955g EDC, and 0.7 ml triethylamine in DMF and deprotection effected with 20% piperidine in methylene chloride as in Example 1. 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 ditrifluoroactate salt which decomposes at 153°C.

EXAMPLE 6 L-Arainvlalvyvl-L-AsDartvl Glycine Starting with N-a-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 is obtained as the ditrifluoroacetate salt; m.p. 85-90oC.

EXAMPLE 7 N-(L-Arainvl-2-Aminoethyl)-L-Asoartl-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 g N-a-CBZ-L-aspartic acid 1-tbutyl ester, 0.83g HOBT, 1.3 g 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-ca- CBZ-L-aspartyl-t-butyl ester-L-valine-t-butyl ester.

B. 2.2g of N-a-CBZ-glycine methyl ester is dissolved in 50 ml of anhydrous toluene and cooled to -780C, 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 -780C, 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-a-CBZ-2-arninoacetaldehyde.

VWO 92/17196 PCU/ tS92/02637 22 C. The product from Example 7A is deprotected as in Example 3D to give L-aspartyl-t-butyl ester-L-valine-t-butyl ester.

D. 1.55g of the aldehyde from Example 7B, 3.4g of the product from Example 7C, 1.64g sodium acetate, 1.23g sodium cyanoborohydride and 1g 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 die, ted 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-B-t-butyl ester- L-valine-t-butyl ester.

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

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

EXAMPLE 8 L-Arginylglvyvl-L-Aspartic Acid a-Benzyl Ester A. 1g of N-t-BOC-L-aspartic acid c-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 Example 2A to give N-t-BOC-L-aspartic acid a-benzyl ester-13-2- (trimethylsilyl)ethyl ester.

B. The product of Example 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 is cooled to 0°C and 20 ml of saturated sodium carbonate solution is added dropwise. The layers are separated and the WO 92/17196 I"CT/ US92/02637 23 organic layer dried over magnesium sulfate, filtered, evaporated to giive L- ,,qrartic acid-a-benzyl ester- 1-2-(trimethylsilyl)ethyl ester.

C. The product of Example 8B and N-t-BOC glyfdne are cu.Ipled in a manner similar to that described in the previous examples to give BOC-gI ,Icyl- L-aspartic acid-cz-benzyl ester- r-2-(trimethylsi lyl)ethyl ester.

D. The BOO group is removed from the product of Example 80 as in Example 8B to give glycyl-L-aspartic acid-a-benzyl ester-3-2- 1 0 (trimethylsilyl)ethyl ester.

E. The product from Example 8D is coupled to N-a-BC-N-co-(4methoxy-2,3,6-trimethylbenzenesulfonyl)-L-argi nine as in 2D to give N-c-BOO- N-co-(4-methoxy-2,3,6-tri methyl-benzenesulfony)-L-argi nyl-glycylaspartic acid 1 5 cx-benzyl ester- 1-2-(trimethylsilyl)ethyl ester.

F. 0 30g of the product obtained in Examples 8E is stirred with 5 ml of trifluoroacetic acid at room temperature for 24 hours. The reaction mixture is then stirred witt 0.5 N acetic acid and washed with ethyl acetate. The aqueous layer is lyophilized to give L-arginylglycyl-L-aspartic acid a-benzyl ester ditrifluoroacetate; m.p. 85-71C.

EXAMPLE 9 N-(6-Aminohexanoyl)L-ASpartvl-L-Vallne A. 1 g of N-(9-fluo-renylmethoxycarbonyl)-L-vali ne p-alkoxybenzyl alcohol resin ester (containing approximately 0.56 mmol of amino acid) is deprotected by shaking with 10 ml of a solution of 20% piperdine in dimethylformamide for 1.5 hours. The mixture Is filtered and the resin derivative washed with methylene chloride to give L-valine p-alkoxybenzyl resin ester.

B. The product from Example 9A is shaken with 0.92g of N-a-FMOO- L-aspartic acid B-t-butyl ester, 0.3 g of 1 -hydroxybenzotriazole (HOBT), 0.43 g of 1 -(3-di methyl-aminopropyl) .3*ethylcarboclii mide hydrochloride (EDO) and :2 ml of triethylamine in 10 ml of dimethys"(ormamide for 2 1hours. The mixture M'O 92/17196 9217196PC'f/ US92/02637 24 is filtered and the resin washed with methylene chloride. The resin derivative is than deprotected as in Example 1 to give L-aspartyl-13-t-butyl ester-L-valine p-alkoxybenzyl resin ester.

0. 2 g of 6-aminoherzanoic acid and 3.23g of sodium carbonate are disso!ved together in 30 ml of water. The solution is cooled in an ice bath and 3.32g of di-t-butylIdicarbo nate in 15 ml of tetrahydrofuran is added. The mixture is stirred at room temperature for 5 hours, then diluted with 400 ml of water ind extracted with ether. The aqueous solution is acidified to pH 2 with 1 0 hyr. -,chloric acid and extracted with ethyl acetate, The ethyl acetate layer is d,,isod over magnesium sulfate, filtered and evaporated in yg. to give N-ate ri-butoxy-carbonyl-6-aminohexanoic acid.

D. The product from Example 9B is shaken with 0.52g of N-o)-BOC-6aminohexanoic acid, 0.3 g of HOBT, 0,43g of EDO and 0.32 ml of riethylamine 10 ml of dimethylformamide for 17 hours. The mixture is filtered and the resin derivative washed with methylene chloride. The peptide derivative is dceprotected and cleaved from 1, resin by treating with 10 ml of 95%/1 t.-ifluoroacetic acid for 2 hours. The resin is filtered off and the filtrate diluted with 50 ml of 0.5 N acetic acid. The aqueous so,)tion 1, wash~'d with 425 ml of ethyl acetate, filtered, then lyophilized to give N-(6-aminoh ,,anoyl)-Laspartyl-L-valine as the trifluoroacetate saht; m.p. 75-850C.

EXAMPLE N-(7-Aminoheptanov-.L-Aspaivl-L-Valine A. When 7-aminoheptanolo acid is sobstituted for 6-aminohexanoic acid and treated in a manner sirniiiar to that in Example 90, N-co-tertbutoxycarbonyl-7-aminohoptanoic acid is obtained.

B. L-aspartyl-B-t-butyl ester-L-valine p-alkoxy-benzyl resin ester (prepared from 1 g of N-FMOC-vr,,ine p-alk~oxybenzyl resin ester as in Examples 1lA and B) is treated with 0.55g of N-BOC-7-aminoheptanoic acid, with 0.3 g of HOBT, 0.43g of EDO and 0.32 ml of triethylamine in 10 ml of cl~imethylformamide in a manner similar to that in Example 9D to give N-(7am.,inoheptanoyl)-L-aspartyl-L-valine as the trifluoroacetate salt, WO 92/17196 WO 9217196PCT/ US92/02637 EXAMPLE 11 N-(7-G.uanidi noheptanoyl-L-Aspagrtyl-L-Vali ne A. 7-Guanidinoheptanoic acid is prepared essentially by the method of Miller, et al, Sytegs 777 (1986), which is incorporated herein by reference. 0.50g of 7-aminoheptanoic acid is dissolved in a solution of 0.475g of potassium tcarbonate in 3.5 ml of water. 0.427g of amincilminome'thanesulfonic acid is added portionwise over 10 minutes and the mixture stirred at room temperature for 24 hours. The resulting solid is collected by filtration. 'The guanidine is dissoived in diluted hydrochloric acid and the solution evaporated LQyco Two portions of 2-propanol are evaporated from the residue to give 7-guanidinoheptanoic acid hydrochloride.

B. L-aspartyl-B-t-butyl ester-L-valine p-alkoxy-benzylalcoho lester (prepared from 1 g of NWFMOC-L-valine p-alkoxybenzylah.ohal ester resin as ir Examples 9A and B) is treated with 0.50 of 7-guanidinoheptanoic acid hydrochioride, 0.3 g of HOBT, 0.43g of EDC and 0.32 ml of triethylamine in ml of dimethylformamide in a manner similar to that in Example 1 D to give N- (7-guanidino-heptanoyl)-L-aspartyl-L-valine as the trifluoroacetate salt; m.p.

75-80 0

C.

EXAMPLE 12 N-(8-Guanidlnooctanyl -L-.AsoartvI-L-ValIng A. 8-guanidiriooctanolc acid hydrochloride is prepared from 8aminooctanolo, acid in a mar'ner similar to the process used in Example 11 A.

B. 0.4 g of 8-guanidinooctanoic acid hydrochloride, L-aspartyl-B-tbutyl ester-L-valine p-alkoxybenzyl resin ester (prepared in the same manner as in Example 0.22g of HOBT, 0.32g of EDO and 0.24 ml of triethylamine are shaken In 1lOmi of dimethylformamide and treated as in Example 9D to give N-(-guanidinooctanoyl)-L-aspartyl-L-valine as the trifluoroacetate salt.

WO 92/17196 PCr/UrS92/263'7 26 EXAMPLE 13 If 6-guanidinohexanoic acid hydrochloride is substituted for 7-guanidinoheptanoic acid hydrochloride in Example 11B, N-(6-guanidinohexanoyl)-Laspartyl-L-valine is prepared.

EXAMPLE 14 A. If 8-aminooctanoic acid is substituted for 6-aminohexanoic acid in Example 9C, N-tert-butoxycarbonyl-8-aminooctanoic acid is prepared.

B. If N-c-BOC-8-aminooctanoic acid is substituted for N-co-BOC-6aminohexanoic acid in Example 9D, N-(8-amino-octanoyl)-L-aspartyl-L-valine is prepared as the trifluoroacetate salt.

EXAMPLE 8-Guanidinooct-2-Enoyl-L-A rtyl-L-Valine A. 4g of 6-amino-1-hexanol is dissolved in 50 ml of 10% aqueous tetrahydrofuran and the solution cooled to OC. 7.46g of di-tertbutyldicarbonate in 25 ml of tetrahydrofuran is added dropwise and the resulting mixture stirred for 3 days at room temperature. The solvent is evaporated in vac and the residue dissolved in ethyl acetate. The ethyl acetate solution is washed with water, dried over magnesium sulfate and evaporated in acu to give 7.4g of N-tert-butoxycarbonyl--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 ethylacetate in hexane, and the filtrate evaporated in yvQg to give 6-N-tertbutoxycarbonylaminohexanal.

C. 1g of 6-N-tert-butoxycarbonylamninohexanal and 1.54g of methyl(triphenylphosphorany!idene) acetate are combined in 25 ml of WO' 92/171966 PCT/US92/02637 27 chloroform and the solution refluxed for 2 hours. The solvent is then removed in vac 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-2octenoate in 25 ml of methano! and 25 ml of 1 Normal aqueous sodium hydroxide is heated at reflux for 2 hours. The methanol is removed in vcuo and the aqueous solution acidified with 1N hydrochloric acid. The resulting mixture is extracted with ethyl acetate. The organic solution is dried ovor magnesium sulfate and evaporated to give 8-N-tert-butoxycarbonylamino-2octenoic 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 auo to give 8-amino-2-octenoic acid as the trifluoroacetate salt.

F. 3.1g of 8-amino-2-ctenoic acid trifluoro-acetate is added to 30 ml of water and the pH adjusted to 7 with 1N sodium hydroxide solution. 1.9g of potassium carbonate is added, then 1.75g of aminoiminomethane-sulfonic acid is added, portionwise, over 10 minutes. Th: mixture is 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-2octenoic acid hydrochloride.

G. L-aspartyl-B-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 1D to give 8-guanidinooct-2-enoyl-L-aspartyl-L-valine as the trifluoroacetate salt.

WO 92/17196 PCT/US92/0263 28 EXAMPLE 16 6-Guanidinohexanovl-N-Ethylglycvl-L-Aspartvl-L-Valine A. If 6-aminohexanoic acid is substituted for 7-aminoheptanoic acid in Example 11A, 6-guanidinohexanoic acid is prepared.

B. To 14.8 g of a 50% aqueous solution of glyoxylic acid is added ml of water. The resulting solution is cooled to 0 C and treated with 10 ml of a 70% solution of ethylamine in water added by dropwise addition over minutes. The reaction mixture is transferred to a Parr bottle, then palladium on carbon is added and the reaction vessel is shaken under hydrogen at 44 psi for 24 hours. The reaction mixture is filtered through a celite pad and the filtrate is concentrated in vacuo to give a tan oil. The oil is treated with 1N aqueous HCI and concentrated in vacuo tc give a solid which is recrystalized (rom acetic acid.

3.65 g of N-ethyl glycine hydrochloride is stirred in 35 ml of water. This is treated with 8.31 g of sodium carbonate and cooled to 0°C, followed by the dropwise addition of 6.77 g of 9-fluorenylmothyl chloroformate in 15 ml of tetrahydrofuran (THF). The reaction mixture is allowed to slowly warm to room temperature and stirred for 24 hours. The THF is removed in vacuo and the residue is diluted with water and extracted with ether. The aqueous fraction is acidified to pH 2 with 1N aqueous HCI and extracted with ethyl acetate. The organic extracts (ethylacetate) are dried, filtered and concentrated to give N-a- FMOC-N-a-ethyl glycine as a white solid. All FMOC protected substituted glycines are made by this procedure simply by substituting the appropriate amine for ethyl amine in this procedure.

C. N-a-FMOC-N-a-ethyl glycine is substituted for N-a-FMOC glycine and the resulting product is treated with piperidine in methylene chloride as in Example 1 to remove the FMOC groups. N-a-Ethyl glycyl-L-aspartyl-B-t-butyl ester-L-valine-p-alkoxybenzyl alcohol resin ester is obtained.

D. A solution of 0.44 g of 6-guanidinohexanoic acid hydrochloride in ml of DMF is treated with 0.23 g of triethylamine. The solution is cooled to 0°C and 0.57 g of N,N-bis[2-oxo-3-oxzolinyl]phosphorodiamidic chloride (BOP- WO 92/17196 PC3 JS92/0263" 29 Cl) is added in a single portion. The reaction mixture is stirred at 00C for minutes and then 1 g of N-a-Ethyl glycyl-L-aspartyl-B-t-butyl ester-L-valine-palkoxybenzyl alcohol resin ester is added. The reaction mixture is shaken for 2 hours al room temperature. The procedure for removal of the peptide from the resin is the same as that described in Example 1. The trifluoroacetate acid solution is diluted with 0.5% acetic acid, washed with 3 portions of ethyl acetate, then lyophilized to give 6-guanidinohexanoyl-N-ethyl glycyl-L aspartyl-L-valine as a white powder.

EXAMPLE 17 6-(Imidazol-1 -vl-Hefxanoy-N-Ethvl Gvcyl-L-Aspartvl-L-Valine A. A solution of 10 g (51 mmol) of 6-bromohexanoic acid in 100 ml of methanol is treated with anhydrous HCI gas for 5 minutes at .,om temperature.

Concentiation in va.Cu gives the methyl ester.

A 50 mL round bottom flask is charged with 8 g (38.29 mmol) of 6bromohexanoic acid methyl ester, 5.7 g (84.24 mmol) of imidazole and 20 mL of THF. The resulting mixture is heated at reflux for 24 hours. Solvent is removed in vacuo and the residue is purified by flash chromatography using methanol/ethyl acetate.

6-(imidazol-1-yl)-hexanoic acid methyl ester is treated with 1N aqueous HCI for 24 hours at reflux to provide, after concentration in vacu. 6-(imidazol- 1-yl)-hexanoic acid hydrochloride.

All corresponding compounds are prepared in a similar fashion starting from the appropriate c-bromohexanoic acid.

B. When 6-(imidazol-1-yl)-hexanoic acid hydrochloride is substituted for 6-guanidinohexanoic acid hydrochloride and treated in a manner similar to that in Example 16D, 6-(imidazol-1-yl)-hexanoyl-N-ethyl glycyl-L-aspartyl-Lvaline is obtained.

WO 92/17196 PCT/US92/02637 EXAMPLE 18 [4-(4'-Guanidinobutvyl etrazol-1 -yl-Acetvl-L-Aspartvl-L-Valine A. N-Phthalyl-5-Aminopentanoyl-Glycine, Methyl Ester To a stirred suspension of 5.02 g (20.3 mmol) of aminopentanoic acid and 3.04 g (24.2 mmol) of glycine methyl ester hydrochlcride in 100 ml of dry tetrahydrofuran is added 3.27 g (24.3 mmol) of HOBT, 4.65 g (24.3 mmol) of EDC, and 4.94 g (48.8 mmol; 6.80 ml) of triethyl amine in that order. The resulting suspension is stirred for 18 hours, then partitioned between 250 ml of ethyl acetate and 100 ml of water. The layers are separated and the aqueous phase is extracted with 250 ml of ethyl acetate The combined organic layers are washed with 150 ml of 10% aqueous sodium carbonate, then with two 125 ml portions of saturated aqueous sodium chloride. The organic phase is dried (MgSO 4 and concentrated invacuo to give 7.39 g of a white solid. Recrystallization of the product from dichloromethane-hexane gives 6.22 g of the title compound as a fluffy solid.

B. [4-(N-Phthalyl-4'-Aminobutyl)Tetrazol-1-yl]-Acetic Acid, Methyl Ester.

To a stirred suspension of 3.7 g (11.6 mmol) of the protected dipeptide in 65 ml of benzene is added 3.5 g (16.8 mmol) of phosphorus pentachloride in one portion. The suspension is stirred for 2 hours during which time the solids had dissolved. The reaction mixture is concentrated in.yvacu to give a light brown oil which is dissolved in 60 ml of a hydrazoic acid solution in benzene.

This solution is stirred for 24 hours, concentrated in vacuo and the residue is purified by extraction and chromatography as described before to give 2.89 g of the desired tetrazole ester as an oil which crystallizes on standing.

C. [4-(N-Phthalyl-4'-Aminobutyl)Tetrazol-1-yl]-Acetic Acid.

To a stirred suspension of 2.5 g (7.29 mmol) of the above ester in 99 ml of methanol, cooled in an icewater bath, is added 1.4 g (33.7 mmol) of lithium hydroxide monohydrate in 33 ml of water. The suspension is warmed to 300C 'WO 9)2/17196 PCT/US92/02637 31 to dissolve the solids and the solution is stirred at room temperature for hours. Acidification of the solution with 12 ml of 6M aqueous hydrochloric acid, followed by work-up as described previously affords 2.39 g (100%) of the tetrazole acid as an unstable white foam.

D. [4-(4'-Aminobutyl)Tetrazol-1-yl]-Acetic Acid, Hydrochloride.

To a solution of 2.35 g (7.14 mmol) of the tetrazole acid in 29 ml of ethanol is added 463 mg (450 ml; 7.94 mmol) of 55% aqueous hydrazine. The suspension is heated under reflux for 2.5 hours, cooled to room temperature, and diluted with 30 ml of water. The solution is acidified with 1.36 g (22.7 mmol) of acetic acid, stirred for 2 hours, then boiled for 30 minutes. After cooling the solution in a refrigerator overnight, the precipitate is filtered and washed with 20 ml of cold water. The combined filtrate and washings are concentrated to dryness and the residue (1.98 g) is dissolved in 20 ml of aqueous ethanol and 515 mg (16.1 mmol; 5000 ml) of 55% aqueous hydrazine is added. This solution is heated for 2.5 hours, cooled to room temperature and concentrated to dryness. The residue is stirred with 15 ml of 2_ aqueous hydrochloric acid for 13 hours, then the suspension is heated under reflux for 30 minutes. The cooled suspension is filtered and the precipitate is washed with 15 ml of water. The filtrate and washings are concentrated in vacuo to give 3.05 g of the crude amine hydrochloride as a colorless semisolid. Further solid impurities are removed by filtration of the ethanol-soluble material; this gives 1.93 g of a pale yellow oil.

E. [4-(4'-Guanidinobutyl)Tetrazol-1-yl]-Acetic Acid, Hydrochloride.

To a stirred solution of 1.85 g (7.92 mmol) of the tetrazole amino acid hydrochloride in 20 ml of water is added 2.3 g (16.6 mmol) of potassium carbonate. To this solution is added 1.18 g (9.52 mmol) of aminoiminomethanesulfonic acid in small portions over a 10 minute period. This solution is stirred for 35 hours and worked up as before to give 1.4 g of the crude guanidino tetrazole acid as an off-white solid. The solid is converted to the hydrochloride by evaPoration of a dioxane-aqueous hydrochloric solution of this material to dryness.

WO 92/17196 PCr/US92/0263- 32 F. The peptide coupling of [4-(4'-guanidinobutyi)tetrazol-1-yl]-acetic acid hydrochloride to L-aspartyl-8-t-butyl ester-L-valine p-alkoxybenzyl alcohol resin ester, the cleavage of the resulting peptide from the resin, and the subsequent isolation steps is performed as described in Example 1. The guanidinobutyl)tetrazol-1-yl]-acetyl-L-aspartyl-L-valine is obtained as the trifluoroacetate salt.

EXAMPLE 19 f5-5'-GuanidinoDentvl)Tetrazol- 1-vl-Acetvl-L-Aspartvl-L-Valine The desired product is prepared by the procedure described for the preparation of [4-(4'-guanidinobutyl)tetrazol-1-yl]-acetyl-L-aspartyl-L-valine, by substituting N-phthalyl-6-aminohexanoic acid for N-phthalyl-6-aminopentanoic acid in Example 18A.

EXAMPLE 9-GuanidinononanoQy-L-aspartyl-a-benzylphenylalanine A. BOC-L-phenylalanine (4.74g, 17.9 mmol), paraformaldehyde (1.65g, 54.9 mmol), and p-toluenesulfonic acid (0.38g, 2 mmol) are dissolved in toluene (100 ml) and heated at reflux for two hours while removing water with a Dean-Stark trap. The mixture is allowed to cool, diluted with ether and the, organic phase washed with saturated sodium bicarbonate solution, brine, dried over magnesium sulfate, then concentrated inL.Y.gu to give the crude oxazolidinone. The oxazolidinone (4.38g, 15.8 mmol) is dissolved in tetrahydofuran (THF) (40 ml) and the solution cooled to -78°C under a nitrogen atmosphere. A solution of 1 M sodium bis(trimethylsilyl)amide (23 ml) in THF is added and the mixture stirred at -78 C for 30 minutes. Benzyl bromide (2.82g, 23.7 mmol) is added and stirring continued for 1.5 hours at -78°C. The mixture is quenched with ammonium chloride solution and diluted with ether.

The ether solution was washed with saturated sodium bicarbonate solution, brine, dried, and concentrated injvyu to give the crude dibenzyl oxazolidinone. The dibenzyl oxazolidinone (6.34g) is dissolved in ethanol/water (100 ml) and sodium hydroxide (1.35g) is added. The mixture is haeated at reflux for 1 hour, cooled, concentrated in vacgu and the residue WO 92/17196 PCT'/US92/02637 33 diluted with water and extracted with ethyl acetate. The aqueous layer is acidified with 3N HCI and extracted with ether/ethyl acetate The organic solution is washed with brine, dried over magnesium sulfate, filtered, and concentrated in vacuo to give BOC-2,2-dibenzyl glycine (3.29g).

B. BOC-2,2-dibenzyl glycine (3.29g, 9.25 mmol) is dissolved in a solution of methanol (18 ml) and water (2 ml) and the pH adjusted to 8 with cesium carbonate solution (11 ml). The solution is concentrated in vacuo to dryness and the residue dissolved in dimethylformamide (DMF) (25 mi) and re-concentrated twice and dried under high vacuum. The cesium salt is taken up into DMF (25 ml), benzyl bromide (1.74g, 10.2 mmol) is added and the mixture stirred at room temperature for 16 hours. The mixture is concentrated jn ,acuo and the residue diluted with ether. The organic phase is washed witn water, brine, dried over magnesium sulfate, and concentrated i.nva=yo. The crude product is purified by flash chromatography, eluting with 10% ethyl acetate in hexane to give BOC-2,2-dibenzyl glycine benzyl ester.

C. BOC-2,2-dibenzyl glycine benzyl ester is deprotected (trifluoroacetic acid) as in Example 8 and coupled (BOP-CI) to N-BOC-Laspartic acid-p-benzyl ester essentially in the manner of Example 16. The resulting dipeptide is, in turn, deprotected (TFA) and coupled (EDC) essentially in the manner of Example 2 to 9-nitroguanidinononanoic acid. Subsequent hydrogenation (H 2 Pd/C) gives the desired product, which is isolated as the trifluoroacetate salt, 9-guanidinononanoyl-L-aspartyl-a-benzylphenylalanine, Cal'd: 568, Found: 568.

EXAMPLE 21 9-Guanidinononanovl-L-aspartyl-(R.S-oa-isobuty.ornithine A. BOC-L-leucine is converted to racemic BOC-(2-isobutyl)-allyl glycine benzyl ester using essentially the procedures of Example 20, Steps A and B, substituting allyl bromide in the alkylation step. The benzyl ester (2.07g, 5.73 mmol) is dissolved in THF (40 ml), under nitrogen, 0.5 M 9borabicyclo[3.3.1]-nonane (9-BBN) in hexanes (46 ml, 23 mmol) is added and mixture stirred at room temperature for 16 hours. The reaction mixture is quenched with water (1 ml) and a mixture of 1N aqueous sodium hydroxide WO 92/17196 PCIYUS92/02637 34 solution (51 ml) and 30% hydrogen peroxide solution (18 ml) is added dropwise. The mixture is stirred for 1 hour at room temperature, saturated with solid sodium chloride, then extracted with ether. The organic layer is washed with saturated sodium bicarbonate solution, brine, dried over magnesium sulfate, filtered, and concentrated iny vac The crude product is purified by flash chromatography, eluting with 20% ethyl acetate/hexanes to give BOC-(2isobutyl)-3-hydroxypropyl glycine benzyl ester.

B. BOC-(2-isobutyl)-3-hydroxypropyl glycine benzyl ester (0.17g, 0.45 mmol) is dissolved in pyridine (2 ml), cooled to 0°C, and p-toluenesulfonyl chloride (0.25g, 1.31 mmol) is added. The mixture is than stirred at room temperature for 16 hours, diluted with ether, and the organic layer washed with 1N HCI, 10% copper sulfate solution, brine. The organic layer is dried over magnesium sulfate, filtered, concentrated in vacuo to give the tosylated product. The primary tosylate (0.23g, 0.43 mmol) is dissolved in DMF/water (10:1) (2 ml) and sodium azide (0.29g, 4.46 mmol) is added. The mixture is heated at 90 0 C for four hours, cooled, diluted with ether and poured into brine.

The organic layer is washed with water, brine, dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude product is purified by flash chromatography, eluting with 10% ethyl aceate in hexanes to give BOC-(2isobutyl)-3-azidopropyl glycine benzyl ester.

C. Using essentially the procedure of Example 20C, 9guanidinononanoyl-L-aspartyl-(R,S)-a-isobutylornithine is prepared from BOC- (2-isobutyl)-3-azidopropyl glycine benzyl ester, and isolated as the ditrifluoroacetate salt, Cai'd: 501, Found: 501.

EXAMPLE 22 6-Guanidinohexanovl-N-ethvl-glvcv-L-aspartyl-2.2-diethylglvcine A. Using essentially the procedure of Example 20B, BOC-2,2-diethyl glycine benzyl ester is prepared from BOC-2,2-diethylglycine.

B. Using essentially the procedures of Examples 2, 8, 16, and BOC-2,2-diethyl glycine benzyl ester is coupled (EDC) to N-BOC-L-aspartic acid-3-benzyl ester, to N-BOC-N-ethyl glycine (BOP-CI), and finally to 6-nitro WO 92/1719(' WO 2/J19;PC1'/US92/0263'.

guanidinohexanoic acid to give, after hydrogenation and deprotection as described abv, 6-guanidinohexanoyl-N-ethyl-glycyl-L-aspartyl-2 ,2diethyiglycine, which is isolated as the acetate salt.

EXAMPLE 23 9-Guanidinononanoyl-L-aspartl-(S-a-b-agjjjiem hy ete A. (S)-(2-benzyl)-allylglycine (prepared according to the method of Zydowski, at al., J. Org, Chem. 1990, 55, 5437) is protected (di-tbutyldicarbonate, sodium carbonate, THF/water, 7 days) and esterified (methyl iodide, DMF, sodium carbonate, 2 days) to give (S)-BOC-(2-benzyl)allyl glycine methyl ester.

B. Using essentially the procedure of Example 21 A, (S)-BOC-(2benzyl)allyl glycine methyl ester is converted to (S)-BOC-(2-benzyl)-3azidopropyl glycine methyl ester.

C. (S)-BOC-(2-benzyl)-3-azidopropyl glycine methyl ester (0.1 9g, 0.52 mmol) is dissolved in methanol/chloroform (15:1) (8 ml) Snd hydrogenated at atmospheric pressure over 10% palladium on carbon for 5 hours. The mixture is filtered, concentrated jaya and the residue triturated with ether/benzene and reconcentrated to give (S)",BOC-(2-benzyl)-ornithine methyl ester hydrochloride.

D. (S)-BOC-(2-benzyl)-ornithine methyl ester hydrochloride (0.2 g, 0.52 mmol) is dissolved in ethanol (10 ml) and triethylamine (0.2 ml, 1.43 mmol) and S-methylisothiourea (0.1 g, 0.74 mmol) are added and the mixture heated at ref lux for 16 hours. The solvent is removed in vacuo and the crude product purified by flash chromatography (50% ethyl acetate in hexane) to give (S)-BOC-(2-benzyl)-nitroargi nine methyl ester.

E. Using procedures analagous to those described hereinabove, BOC-(2-benzyl)-nit'-oarginine methyl ester, is coupled to N-BOC-L-aspartic acid P-benzyl ester and the resulting dipeptide coupled to 9nitroguanidinononanoic acid. The resulting product is hydrogenated and deprotected to give 9-guanidino nonanoyl-L-aspartyl-(S)-a-be nzyI arginine WO 92/17196 PCT/ US92/02637 36 methyl ester, which is isolated as the ditrifluoroacetate salt, Cal'd: 591, Found: 591.

EXAMPLE 24 9-Guanidino-L-spartyl-(S)--isobutylarginine methyl ester A. Using essentially the procedure of Example 23, Step D, (S)-BOC- (2-isobutyl)-nitroarginine methyl ester is prepared from L-'eucine.

B. Using essentially the procedures of Example 23, Step E, 9guanidino-L-aspartyl-(S)-a-lsobutylarginine methyl ester is prepared from BOC-(2-isobutyl)-nitroarginine methyl ester, and isolated as the ditrifluoroacetate salt, Cal'd: 557, Found: 557.

EXAMPLE N-(9-guanidinononanovI-L-aspartvyl-(R)-4-alinoo-4-ibutvlbutyric acid A. Sodium hydride (2.52g 60% suspension in mineral oil, washed with hexane, 63.1 mmol) is suspended in THF (200 ml) and cooled to 0 C under nitrogen. Triethylphosphonoacetate (12.5 ml, 63.1 mmol) is added over minutes and the mixture cooled to -780C, and BOC-L-leucinal in THF ml) is added over 30 minutes. After stirring at room temperature for 1 hour the excess NaH is quenched by addition of saturated ammonium chloride solution, The mixture is extracted with ethyl acetate and the organic layer washed with satruated sodium bicarbonate solution, brine, then dried over magnesium sulfate, filtered, and concentrated in vacuo The crude product was purifed by flash chromatography, eluting with 10% ethyl acetate in hexanes to give the corresponding BOC-L-leucine-za,3-unsaturated ethyl ester. The ester (1.5 g, 5.26 mmol) is dissolved in ethanol (20 ml) and hydrogenated at atmospheric pressure over 10% palladium on carbon (0.16g) for 24 hours. The mixture is concentrated i ~vacuo diluted with ether, filtered, and the filtrate dried over magnesium sulfate, filtered, and concentrated in vacuo to give (R)-BOC-4amino-4-isobutylbutyric acid ethyl ester.

NVO 92/17196 9217196PCT/ US92/02637 37 B. (R)-BOCA4-amino-4-isobutylbutyric acid ethyl coupled sequentially to N-BOC-L-aspartic acid- i-benzyll ester, and tc, guanidinononanoyl as using procsedAures desotibed hereinabove. The resulting pseudotripeptide ethyl ester (0.16g, 0.25 rnmol) is dissolved in methanol/water (6 ml), cesium carbonate (0.33g, 1.01 mmol) is added, and the mixture stirred at room temperature for 18 hours. The solvent is removed Lnyg= and the residue dissolved in ethyl acetate. The ethyl acetate is stirred with 1 N HCI, then the organic layer is washed with brine, dried over magnesium sulfate, filtered, and concentrated in-gu This oroduct was deprotected under hydrogenation conditions as described hereinabove to give N-(9-guanidinononLnoyl-L-aspartyl)-(R)-4-amino-4- .,sobutylbutyric acid, as aft trifluroacetate salt, Ca 472, Found: 472.

EXAMPLE 26 N4Nanidinonodnnovj..L aspartfl)-(R-4-min-4-iob tvbLtYrylb-Lxrlinilm (R)-BOG-4-amino-4-Isobutylbutyric acid ethyl ester is treated with sodium hydrox~de In ethanol, followed by acidification to prepare (R)-BOC-4amlno-4-isobutylbutyric acid which is coupled, using assentially the procedure of Example 2, to NGntoI-riiebny ester p-tosylate, to give the correzyonding dipeptide which is, in turn, sequertally coupled to N-BOC-Laspartic acld-p-benzyi' ester and 9-guanidinononanoic acid, then deprotected as described hereinabove to give N-[N-(9.Guanidinononanoyl-L.-asparty)-(R)- 4-amino-4-isobutylbutyryl]-L-ar- inine, as the ditrifluoroacetate salt, M. Cal'd: 628, Found: 628.

EXAMPLE 27 N-[N-(9-Guanidinononanoyl-L-gsoatW3D-(R)-sec-butyl-a-a! nobuty.L1' arginine Using essentially the procedures of Examples 25 and 26, the desired product Is prepared from BOC-D-1.oleucinal, and isolated as the ditrifluoroacetatG salt, Cal'd*: 628, Found: 628.

92/ 17 196 PCIfU S 9 2/012637 EXAMPLE 28 fR)-tLNI,-nidinnnanl-L-iDartfl-ami no]-4-isobutvlbuityla nidi ne A. (R)-BOC-4-amino-4-isobutylbutyric acid ethyl ester (0.84g, 2,92 mmol.) is dillusved in THF (5 ml) and lithium chloride (0,27g, 6.3 mmol) and sodium borohydride (0.23g, 6.3 mmol) and ethanol (9 ml) are 8added. Tha mnixture Is stirred at room temperature for 16 hours, the mixture Is cooled to 000, and 10%/ aqueous citric acid solution Is added. The mixture is concentrated in vg~ and the residue partitioned between ethyl acetate and water. Thrc organic solution is wpshed with saturated sodium bicarbonate solution, br'ine, dried over magnesium sulfate, filtered, and concentrated inyva=uo The crude product is purified by flash chromatography, eluting with 20% ethyl acetate/hexane, to give iOC-4-arnino-4-isobutylbutanol.

8. Using essentially the procedures of Egriples 21 arid 23 (mesylation, azide displacemeant, reduclton, and nitroguanylation), (R)-BOC-4,amino-4.Thsobutylbutanol, is converted to (i3)-BOC-'N-(4-amIno-4isobu~tylbutyl)nitroguariidirg., C. Subsequent coupling and deprotection as described nereinabovk? converts (Fi)-BOC-N-(4-amino-4-isobljutyibutyl)n itroguanidi ne to (R)-4-[N-(9-guanidrioonanoyl-L-aspartyl)-amno]-4-lsobutylbuto,-uaidl.~,e, which ls solated as the ditrifluroacetate salt, Cal'd: 479, Found: 479.

EXAMPLE 29 6-Guanidinohexangyl-N-etbvLQiIYcI-L-asp2aryl-(( -amino-4isobutylbutyl)augnldi ne Using procedures analagou-s to those described hereinabove, the deisired product is prepared from (R)-BOC-N-(4-amino-4isobutylbutyl)nitroguanidine, ancJ Isolated~ as the acetate salt, Cal'd: 542, Found: 542.

WO 92/17196 WO 9/1716 Ci/ 1JS92/0)2637- 39 EXAMPLE 9-QGwan idi no non an oyI-L-aspartyl-((S)-4-amI no- -ec-bwtylbutvflagUanidineQ A. Using essentially the procedures of Example 2e8, amino-4-sec-butylbutyl)nitroguanidine, Is prepared from (S)-BOC-4-ami no-4sec-butylbutyric acid ethyl ester.

B The desired product is prepared from (S)-BOC-N-(4-amino-4-secbutylbutyl)nitroguanidine, using sequential coupling and deproatectlon procedures as described hereinabove, and isolated as the ditrifluoro acetate salt, Cal'd; 499, Found: 499.

EXAMPLE 31 Vguanidinonponanol-L-aapartl-(B)-6-amino-6-sec-butylh*,,Qan2vfrj,-arainine A. Using essentially the procedure of Example 25, Step A, (R)-BOC- 6-wn',ino-6-sec-butylhexanolc acid ethyl ester Is prepared from BOC-Lisoleucinal and triethyl-4-phosphonocrotonate.

B. Using essentially the procedures of Example 26, the desired product is prepared am (R)-BOC-6-amino-6-sec-butylhexanoic acid ethyl ester, and isolated a~.s the ditrifluoroacetate salt, Gal'd: 650, Found: 656.

EXAMPLE 32 9- -gii-ononanol-! -aspatl-((R)-Q-amnino-Q-sec-butylhexl~gUanidine .00A. Using essentially the proceduires of Example 28, (R)-BOC-(6amino-6-sec-bplylhsxyl)nitroarginlnie is prepared from (R)-BO';-6-amh W, 1~hexanoic acid ethyl ester.

B. Using essentially the procedures of Example 28, the desired product is prepared from (R)-BOC- (6-ami no-6-sec-butylhexyl) nit roargi nln e, and isolated as the ditrifluoroacetate salt, Cal'd: 527, Found: 527.

WO 92/17196 PCT/US92/02637 Compounds within the scope of the present invention inhibit platelet aggregation by inhibiting fibrinogen binding to activated platelets and other adhesive glycoproteins involved in platelet aggregation and blood clotting and are 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 in humans and other mammals.

Compounds within the scope of the present inventin exhibit activities which interfere with adhesive interactions between abnormal cells and the extracellular matrix and, therefore, are believed to be useful in the treatment of disease conditions, in humans and other animals, characterized by abnormal cell proliferation which have been shown to be dependent on such adhesive interactions (see, for example, Journ. of Biol. Chem. 262 17703-17711 (1987); Science 233, 467-470 (1986); and Cell 57, 59-69 (1989)).

The compounds of this invention can normally be administered orally or parenterally, in the treatment or prevention of thrombosis associated disease states.

The compounds of this invention may be *-ulated for administration in any convenient way, and the invention includes v scope pharmaceutical compositions containing at leasit. ompound according to the invention adapted for use in human or vete'aan, medicine. Such compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers or excipients. Suitable carriers include diluents or fillers, sterile aqueous media and various non-toxic organic solvents. The compositions may be formulated in the form of tablets, capsules, lozenges, troches, hard candies, powders, aqueous suspensions, or solutions, injectable solutions, elixirs, syrups and the like and may contain one or more agents selected from the group including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a pharmace rtically accept' -le preparation.

The particular carrier and the ratio of platelet aggregation and thrombus inhibiting compound to carrier are determined by the solubility and chemical properties of the compounds, the particular mode of administration and WO 92/17196 /Cf/US92/02637 41 standard pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate and dicalcium phosphate and various disintegrants such as starch, alginic acid and certain complex silicates, together with lubricating agents such as magnesium stearate, sodium lauryl sulphate and talc, can be used in producing tablets. For a capsule form, lactose and high molecular weight polyethylene glycols are among the preferred pharmaceutically acceptable carriers. Where aqueous suspensions for oral use are formulated, the carrier can be emulsifying or suspending agents. Diluents such as ethanol, propylene glycol, glycerin and chloroform and their combinations can be employed as well as other materials.

For parenteral administration, solutions or suspensions of these compounds in sesame or peanut oil or aqueous propylene glycol solutions, as well as sterile aqueous solutions of the soluble pharmaceutically acceptable salts described herein can be employed. Solutions of the salts of these compounds are especially suited for intramuscular and subcutanleous injection purposes. The aqueous solutions, including those of the salts dissolved in pure distilled water, are also useful for intravenous injection purposes, provided that their pH is properly adjusted, they are suitably buffered, they are made isotc iic with sufficient saline or glucose and sterilized by heating or microfiltration.

The dosage regimen in carrying out the method of this invention is that which insures maximum therapeutic response until improvement is obtained and thereafter the minimum effective level which gives relief. In general, the oral dose may be between about 1 mg/kg and about 200 mg/kg, preferably between about 2 mg/kg to 100 mg/kg, and most preferably between about mg/kg and 100 mg/kg, and the i.v. dose about 0.1 mg/kg to about 20 mg/kg, preferably between about 0.5 mg/kg to 10 mg/kg, bearing in mind, of course, that in selecting the appropriate dosage in any specific case, consideration must be given to the patient's weight, general health, age, and other factors which may influence response to the drug. The drug may be administered orally 1 to 4 times per day, preferably twice daily.

The following pharmacologic tests evaluate the inhibitory activity of compounds of the present invention on fibrinogen-mediated platelet aggregation and fibrinogen binding to thrombin-stimulated platelets, and WO 9'2/17196, W0 ICr/uS9/02637 42 results of these tests correlate to the in-vivo inhibitory properties of compounds of the present invention.

The Platelet Aggregation Assay is based on that described in Blood 66 946-952 (1985). The Fibrinogen-Binding Assay is essentially that of Ruggeri, et al., Proc. Natl. Acad. Sci. USA 83, 5708-5712 (1986) and Plow, et al., Proc. Natl. Acad. Sci., USA 82, 8057-8061 (1985).

Platelet Aaaregation Assay Preparation of Fixed-Activated Platelets Platelets are isolated from human platelet concentrates using the gelfiltration technique as described by Marguerie, et al., J. Biol. Chem. 254, 5357-5363 (1979) and Ruggeri, et al., J. Clin. Invest. 72,1-12 (1983).

The platelets are suspended at a concentration of 2 x 108 cells/ml in a modified calcium-free Tyrode's buffer containing 127 mM sodium chloride, 2 mM magnesium chloride, 0.42 mM Na 2

HPO

4 11.9 mM NaHCO 3 2.9 mM KCI, mM glucose, 10 mM HEPES, at a pH of 7.35 and 0.35% human serum albumin (HSA). These washed platelets are activated by addition of human a-thrombin at a final concentration of 2 units/ml, followed by thrombin inhibitor 1-2581 at a final concentration of 40pM. To the activated platelets is added paraformaldehyde to a final concentration of 0.50% and this incubated at room temperature for 30 minutes. The fixed activated platelets are then collected by centrifugation at 650 x g for 15 minutes. The platelet pellets are washed four times with the above Tyrode's-6.35% HSA buffer and resuspended to 2 x 108 cells/ml in the same buffer.

Platelet Aggregation Assay The fixed activated platelets are incubated with a selected dose of the compound to be tested for platelet aggregation inhibition for one minute and aggregation initiated by addition of human fibrinogen to a final concentration of 250 gg/ml. A platelet aggregation profiler Model PAP-4 is used to record the platelet aggregation. The extent of inhibition of aggregation is expressed as the percentage of the rate of aggregation observed in the absence of inhibitor.

ICso, the amount of inhibitor required to reduce the aggregation rate by WO 92/17196 PC'/US92/02637 43 is then calculated for each compound (see, for example, Plow, et al., Proc. Natl. Acad. Sci., USA 82, 8057-8061 (1985)).

Fibrinooen-Biding Assay Platelets are washed free of plasma constituents by the albumin densitygradient technique of Walsh, et al., Br. J. Haematol. 281-296 (1977), as modified by T(apani-Lombardo, et al., J. Clin Invest. 76, 1950-1958 (1985).

In each experimental mixture platelets in modified Tyrode's buffer (Ruggeri, et al., J. Clin. Invest. 72,1-12 (1983)) are stimulated with human athrombin at 22-25 0 C for 10 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 (unit/unit) for 5 minutes before addition of the 12 5 1-labeled fibrinogen and the compound to be tested. After these additions, the final platelet count in the mixture is 1 x 1011/liter. After incubation for an additional 30 minutes at 22- 0 C, bound and free ligand are separated by centrifuging 50pl of the mixture through 300pl 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 (Munson, Methods Enzymc'. 92, 542-576 (1983)). To determine the concentration of each inhibitory compound necessary to 'nhibit 50% of fibrinogen binding to thrombin-stimulated platelets (IC50), 'cch compound is tested at 6 or more concentrations with 1 2 5 1-labeled fibrinogen held at 0.176p.mol/liter The IC50 is derived by plotting residual fibrinogen binding against the logarithm of the sample compound's concentration.

Compounds of the present invention exhibit marked activity in the foregoing tests and are considered useful in the prevention and treatment of thrombosis associated with certain disease states. Results of testing of compounds of the present invention by the above methods are presented in the Table I below.

The compounds listed in Table I are prepared by the methods described herein, by analogous methods, or by methods known in the art. Mass spectral WO 92/17190 PCTriUS92/0263-1 44 analysis, where .provided, is by Low Resolution Fast Atom Bombardment with the "calculated" values being (M+1 TABLE I Inhibition of 125 1-Fibrinogen Binding to Platelets 1Q~aQ~ inhibition of Fibrinogen Mediated Platelet Aggregation 1QlUbM 49 L-argffnyl-L-aspartyl-L-valine, Cal'd: *389, Found: 389 L-arginylglycyl-L-aspartyl-a-isobutyiamide ditriftuoroacetate, m.p. 90-95 0

C.

L-ornithylglycyl-L-aspartyl-L-valine ditriffiuoroacetate, m.p. 122-1 25 0

C,

Cal'd: 632, Found: 632 L-arginyIg lycyl-L-asparfic acid a-benzyl ester ditrifluoroacetate, rn.p. 85-87 0

C

L-arginyisarcosyl-L-aspartyl-L-vali ne ditrilluoroacetate, m.p. 1 45 0 C (dec), C-aId: 460, Found: 460 L-argh'4l glycyl-L-aspartyl-L-(N-methyl)valine ditrifluoroacetate, m.p. 1 53 0 C (dec) L-arginylglycyl-L-aspartyl glycine ditrifluoroacetate, m.p. 85-90 0

C,

Cal'd: 404, Found: 404 >200 100 26.5 Inhibited at S0[igfml 25.0 14 Inhibited 50% at 160 14.3 TABLE I (cont'd) Inhibition of Fibrinogen Inhibition of 125 1-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at 1C 04 100LM aQ~m N-(L-arginyl-2-aminoethyl)-L-aspartyl-L-vali ne >200 tritrifluoroacetate, m.p. 91-95 0

C,

Cal'd: 432, Found: 432 L-arginyl-g lycyl-L-N-methylaspartyl-L-valine >64ditrifluoroacetate, Cal'd: 460, Found: 460 glycyl-L-aspartyl- 68.5 >64 67 L-valine trifluoroacetate, nip. 95-99 0

C

N-58-(L-arginyl)-L-ornithyl-L-valine >1 00 tritrifluoroacetate, Cal'd: 373, Found: 373 L-arginyl-L-aspartyl-L-':aline ditrifluoroacetate, >200 0049 m.p. 90-95 0 C, Cal'd: 389, Found: 389 L-arginyl glycyl glycyl-L-valine ditrifluoroacetate, >200 >100 Cal'd: 388, Found: 388 L-arginyl glycyl-L-aspartic acid ax-benzyl 25.0 14 96 ester ditrifluoroacetate, Cal'd: 437, Found: 437 TABLE I (cont'd) Inhibition of 125 I-Fibrinogen Binding to Platelets IQauww Inhibition of Fibrinogen Mediated Platelet Aggregation 1MQULM inhibition at 100 m L-ornithyl-glycyl-L-aspartyl-L-valine ditrifluoroacetate, m.p. 122-1 25 0

C

L-valine dihydrochioride, m.p. 60-700C 5 L-arginyl-sarcosyl-L-aspartyl-L-valine ditifluoroacetate, m.p. 1 45 0 C(dec) L-arginylglycyl-L-alanyl-L-vali ne ditrifluoroacetate, Cal'd: 402, Found: 402 L-argi nylglycyl-L-aspartyl-L- (N-m ethyl)valine ditrifluoroacetate, Cal'd: 460, Found: 460 lycyl-Laspartylj-1 ,2,3,4-tetrahydroisoquinoin,, Cal'd: 447, Found: 447 N-(5-guanidinopentanoy)glycyl-Laspartylphenethylamide acetate, m.p. 90-1 0000 Inhibited 50% at 0.7 14 Inhibited 50% at Inhibited 50% at 5O jg/mi 130 92 92 17.5 >1 00 160 20.4 TABLE I (contk( Jr trifluoroacetate, Cal'd: 374, Found: 374 9-aminononanoy-L-aspartyl-L-valine triffuoroacetate, Cal'd: 388, Found: 388 9-guanidinononanoyi-L-aspartyl-L-valine trifluoroacetate, Cal'd: 430, Found: 430 1 1-guanidinoundecanoyl-L-aspartyl-L-valine trifluoroacetate, Calrd: 458, Found: 458 9* yuanidinononanoyl-L-aspartyl4.-leucine, CaI'd: 444, Found: 444 arginine ditrifluoroacetate, CaI'd: 516, Found: 516 L-Ieucine trifluoroacetate, CaI'd: 473, Found: 473 ihibition of 1 25 1-Fibrinogen Binding to Platelets Inhibition of Fibrinogen Mediated Platelet Aggregation inhibitlo:: at IQUIU 25 IM 28 19.1 10.1 96 0.59 0.23 0.39 0.54 TABLE I (cont'd) Inhibition of Fibrinogen Inhibition of 125 l-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at 1CQu{LLM.afmi 6-gua:nidinohexanoy-N-ethy-gycy-L-aspartyl- 5.4 .0.56 95.5 L-leucine trifluoroacetlate, Cal'd: 487, Found: 487 5-(lmidazol-1 -yl)-valeroylglycyl- L-aspartyl- 1.25 20.2 58 L-valine, CaI'd: 440, Found: 440 k 5-(Imidazol-1 -yl)-pentanoyl-.,arcosyl-L- 0.15 1.5 aspartyl-L-valine trifluoroacetate, Cal'd: 454, Found: 454 6-(Imkdazo-1 -yi)-hexanoy-glycyI-L-aspartyl 2.8 17.0 67 L-valine trifluoroacetate, Cal'd: 454, Found: 454 6-(Imidazol-1 -yl)-hexanoytsarcosyl-L-aspartyl, 1.25 2.8 94 Cal'd: 468, Found: 468 6-(lmidazol-1 -yl)-hexanoyl-N-ethylglycyl-L- 0.90 1.0 aspartyl-L-valine trifluoroacetate, 1 Cal'd: 482, Found: 482 8-(lmidazol-1 -yI)-octanoyl-L-aspartyl-L-valirne 0.86 11.6 78 trifiuoroacetate, Cal'd: 425, Found: 425 TABLE I (cont'd) midazol-1 -yI)-nonanoyl-L-aspartyl-L-valine, Cal'd: 439, Found: 439 9-guanidinononanoyl-L-aspartyl-L-arginine isobutyl ester, Card: 543,. Found: 543 8-Guanidinooctanoyl-L-aspartic acid-aisobutylamide trifluoroacetate, CaI'd: 372, Found: 372 1 2-Guanidinododecanoyl-L-aspartyl-L-valine triffuoroacetate, CaI'd: 472, Found: 472 9-Guanidinononanoyl-L-asf- rtyl-L-isoleucinetrifluoroacetate, Cal'd: 444, Found: 444 9-Guanidino-,ionanoyl-L-aspartLyl-L-arginineditifluoroacetate, CaI'd: 487, Found: 487 8-Guanidinooct-2-enoyl.L-aspartyl-L-arginin e ditritluoroacetate, CaI'd: 471, Found: 471 8-Guanidinooctanoyl-L-aspartyl-argini ne ditrifluoroacetate, CaI'd: 473, Found: 473 Inhibition of 125 l-Fibrinogen Binding to Platelets 50 0-625 Inhibition of Fibrinogen Mediated Platelet Aggregation inhibition at 1czMfl 2540 3.4 96 0.54 97.0 14.5 0.58 0.13 0.58 0.33 0.26 0.63 TABLE I (cont'd) Inhibition of Fibrinogen Inhibition of 1 25 l-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at Q5-Qfw25 LM 1 0-Guanidinodec-2-enoyl-L-aspartyl-L-valine 12.0 8.8 77 trifluoroacetate, Cal'd: 442, Found: 442 9-Guanidinononanoyl-L-aspartyl-arginyl-isoleucine 1.2 2.4 93 ditrifluoroacetate, Cal'd: 600, Found: 600 9-Guanidinononanoyl-L-aspartyl-isoleucyl-arginine 0.37 0.67 94 ditrifluoroacetate, CaI'd: 600, Found: 600 8-G uanidinooctanoyl-L-aspartyl-L-valin e 2.8 2.7 89 triffujoroacetate, CaI'd: 415, Found: 415 8-Guanidinooct-2-enoyl-L-aspartyl-L-valinie 0.84 0.29 96 trifluoroacetate, Cal'd: 414, Found: 414 8-Guanidino-2-(R,S)-ethyl-octanoyl-L-aspartyl- >200 L-valinu- trifluoroacetate, Cal'd: 444, Found: 444 1 0-Guanidinodecanovl-L-aspartyl-L-valine 2.5 10.3 77 trifluoroacetate, Cal'd: 444, Found: 444 9-G uanidino non-2- (E)-en oyI- L-aspartyl- L-va line 0.43 24 triflUoroacetate, Cald: 428, Found: 428 TABLE I (cont'd) Inhibition of Fibrinogen frniibition of 125 1-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at Pu 15Q1UW25 LM I1-Guanidino-undecanoyl-L-asparty,-L--valine 17.0 36 trifluoroacetate, Card: 458, Found: 458 4-Guanidinobutanoyl-glycyl-L-aspartyl-L- 10.0 21.9 43 valine trifluoroacetate, m.p. 50-52 0 C. L Cal'd: 417, Found: 417 1.0 0.52 0 trifluoroacetate, Cal'd: 445, Found: 445 6-Guanidinohexanoyl-glycyl-L-aspartyl-' -valine .46.5 trifluoroacetate, -CaI'd: 445, Found: 445 4-Guanidinobutanoyl-L-sarcosyl-L-aspartyl-L- 3.3 10.8 84 valine trifluoroacetate, m.p. 62-64 0

C,

Cal'd: 431, Found: 431 6-Guanidinohexanoyl-L-sarcosyl-L-aspartyl-L- 0.32 0.63 97 valine trifluoroacetate, m.p. 65-70 0

C,

Cal'd& 459, Found: 459 4-Guanidinobutanoy-L-f3-alanyl-L-aspartyl-L- 10.0 10.6 78 b valine trifluo ro acetate, Cal'd: 431, Found: 431 TABLE I (cont'd) In 7-Guanidinoheptanoyl-glycyl- L-aspartyl-L-valine trifluoroacetate, Cal'd: 459, Found: 459 7-Guanidinoheptanoyl-L-sarcosyl- L-aspartyl-Lvalin-r, frifluoroacetate, 473, Found: 473 6-Guanidinohexanoyl-L.-sarcosyl-L-aspartyl-Lleucine trifluoroacetate, Cal'd: 473, Found: 473 6-Guanidinohexanoyl-N-benzylglycyl-L-aspartyl- L-valine trifluoroacetate, Cal'd: 535, Found: 535 6-Guanidinohexanoyl-N-ethylglycine-L-aspartyl- L-valine trifluoroacetate, Cal'd: 473, Found: 473 6-Guanidinohexanoy-N-isobutylglycyl-L-aspartyi- L-valine trifluoroacetate, 3(Q Cal'd: 5-01, Found: 501 Binding to Platelets 6.4 Inhibition of Fibrinogen Mediated Platelet Aggregation* inhibition at 1Q LM 2-w 21.0 56 0.09 0.84 0.12 0.38 0.10 TABLE I (cont'd) Inhibition of Fibrinogen Inhibition of 125 1-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at 6-G uanidinohexanoyi-N-(2-methylpentl)glycine- >200 -10.5 86 L-aspartic acid-L-valine trifluoroacetate, Ca'.d: 529, Found: 529 6-Guanidinohexanoy-N-ethylglycyl ,-L-aspartyl-L- 0.20 1.3 97 phenylalanine trifluoroacetate,

L

Cal'd: 521, Found: 521 6-Guanidinohexanoyl-N-ethylg lycyl-L-aspartyf-L- 0.05 0.56 96 isofeucine trifluoroacetate, Cal'd: Found: 487 6-Guanidinohexanoy-N-(2-propyl)glycyl-L-aspartyl- 1.1 8.6 83 L-valine trifluoroacetate, Car'd: 487, Found: 487 6-Guanidinohexanoy-N-ethylglycyl-L-aspartyl-L- 0.24 1.4 97 arginine dlitrifluoroacetate, Cal'd: 530, Found: 530 U0 0.04 0.63 91 valine trifluoroacetate, 1 Cal'd: 447, Found: 447 TABLE I (coni'd) Inhibition of Fibrinogen Inhibition of 12 6'l-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at 6-Guanidinohexanoyl-sarcosyl-L-aspartyl-L- 0.52 2.1 97 arginine ditrilluoroacetate, Cal'd: 516, Found: 516 6-Aminohexanoy-N-athylglycyi-L-aspartyl-L- 4.0 4.1 92 V valine dlitrifluoroacetate, L NILS., Cal'd: 431, Found: 431 6-Guanidinohexanoyl-N-isopropylglycyl-L-aspartyl- 1.1 8.6 83 L-valine, Cal'd: 487, Found: 487 9-Guanidinononanoyl-L-aspartyl-L-arginine isobutyl 3.0 2.20 89 amide dlitrifluoroacetate, CaI'd: 542, Found* 542 8-Guanidinooctanoyl-L-aspartyl- L-lysine 0.78 1.6 93 bis(trifluoroacetate) salt, Cal'd: 445, Found: 445 9-(Imidazol-2-methyl-1 -yl)nonanoyl-L-aspartyl- 80 3.0 74 I L-aspartyl-L-valine trifluoroacetate, CaJ'd: 453, Found: 453 TABLE I (co nt'd) Inhibition of Fibrinogen Inhibition of 125 1-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at MUL" I51JMI 25 IM 9-Guanidinononanoyl-L-aspartyl-L-arginine 3.0 2.20 89 isobutyl amide ditrifluoroacetate, Cal'd: 542, Found: 542 8-Guanidinooctanoyl-L-asparty-L-lysine 0.78 1.6 93 bis(trifluoroacetatelsait, Cal'd: 445, Found: 445 >25 32 tetrazole, CaI~d: 440, Found: 440 1 0-(Imidazol-1 -yI)decanoyl-L-aspartyl-L-valine 20 >25 45.0 trifluoroacetate, CaI'd: 453, Found: 453 9-Guanidinononanoy-L-aspartyl-3-alanyl-L- 0.64 1.5 93.0 arginine ditrifluoroacetate, CaI'd: 558, Found: 558 .Z'G uanidi no non an oyl- L-asparty -g lycyI- L- 7.0 2.2 85.0 arginine ditrifluoroacetate, (n% Cal'd: 544, Found: 544 8-Guanidinooctanoyl-glycyl-glycine 12 trifluoroacetate, Cal'd: 316, Found: 316 TAB LE I (co ntd) Inhibition of Fibrinogen Inhibition of 125 1-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at Mufuw Q5Q13L 25 LIM 7-(lmidazol-1 -yl)heptanoyl-L-aspartyl-L- 2.0 >25 valine trifluoroacetate, CaI'd: 411, Found: 411 6-(2-Methylimidazol-1 -yl) hexanoyl-N- (ethyl)- 0.35 1.9 93.0 !clycyl-L-aspartyl-L-valine trifluoroacetate, 1lb CaI'd: 496, Found: 496 4-Guanidinobutanoy-N-(ethyl)glycyl-L- 0.45 2.7 94 aspartyl-L-vali ne trifluoroacetate, CaI'd: 445, Found: 445 7-(Irnidazol-1 -yl)heptanoy-N-(ethyl)glycyl-L- 1.3 10.8 82 aspartyl-L-valine trifluoroacetate, Cal'd: 496, Found: 496 6-Guanidinohexanoyl-sarcosyk-L-3ispartic acid- f-methylester-L-valine methylester trifluoroacetate, Cal'd: 487, Found: 487 9-Guanidinononanoyl-L-aspartyl-isoleucine-4- 0.24 5.7 95.0 guanidinobutyl amide dlitrifluoroacetate, CaI'd: 556, Found: 556 TABLE I (cont'd) Inhibition of Fibrinogen Inhibition of 125 l-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at 1~~ojuM~2 UQ~ 2M 6-G uanidinohexanoyl-sarcosyl-L-aspartic acid--- 1-methylester-L-valine trifluoroacetate, Cal'd: 587, Found: 587 6-Guanidinohexanoy-N-(ethyl)gycyl-L- 0.18 1.25 95 L aspartyl-L-tyrosine trifluoroacette, co Cal'd: 537, Found: 537 6-Guanidinohexanoyl-N-cyclopentylg lycine- 0.88 5.7 76.0 L-as party l-L-valine trifluoroacetate, CaI'd: 513, Found: 513 9-Guanidinononanoyl-L-aspartyl-(des-carboxy)- 0.52 4.4 84 L-arginine dlitrifluoroacetate, Cal'd: 443, Found: 443 8-Guanidinooctanoyl-L-aspartyl-(des-carboxy)- 0.99 1.9 91 L-arginine dlitrifluoroacetate, CaI'd: 429, Found: 429 6- Guan Win o hexa noyl-sarcosyl- L-aspartyl- L- 0.34 4.1 86.0 ly-sine dlitrifluoroacetate, Cal'd: 488, Found: 488 TABLE I (cont'd) Inhibition of Fibrinogen Inhibition of 125 1-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at l %Uw2a~M 6-Guanidinohexanoyl-sarcosyl-L-aspartyl-L- 3.6 7.3 94.0 tyrosine trifluoroacetate, Cal'd: 523, Found: 523 6-Guanidinohexanoyl-sarcosyl-L-asat-L 0.19 6.6 89.0 tryptophan ditrif luo ro acetate, sprnlL CaI'd: 546, Found: 546 6-(2-Phenylimidazol-1 -yl~hexanoyl-sarcosy-L- 0.38 5.6 84.0 aspartyl-L-valine trifluoroacetate, CaI'd: 544, Found: 544 6-(2-Ethylimidazol-1 -yl)hexanoyl-sarcosyl-L- 0.313 4.9 89.0 aspartyl-L-valine trifluoroacetate, Cal'd: 496, Found: 496 6-Guanidinohexanoy-N-(ethyl)glycyl-L-aspartyl- 0.13 1.3 91.0 L-tryptophan ditrifluoroacetate, Cal'd: 560, Found: 560 1.6 24.0 56.0 L-arginine ditrifluoroacetate, Cal'd: 572, Found: 572 TABLE I (co nt'd) Inhibition of Fibrinogen Inhibition of 125 1-Fibrinogen Mediated Platelet Aggregation* Binding to Platelets inhibition at Mad" IZ~faw25 M 9-Guanidinononanoy-L-aspartyl-f3-alanyl-L- 54 999 isoleucine trifluoroacetate, Cat'd: 515, Found: 515 9-Guanidinononanoyl-L-aspartyl-glycyl-L- 40 25.0 54.0 isoleucine trifluoroacetate, Cal'd: 501, Found: 501

C

9-Guanidinononanoyl-L-aspartyl-L-norfeucyl- 0.21 0.3 95.0 L-arginine ditrifluoroacetate, Cal'd: 600, Found: 600 9-Guanidinononanoyi-L.-aspartyl-cc-benzylphenyl- >200 >25 10.0 alanine trifluoroacetic acid salt, CaI'd: 568, Found: 568 9-Guanidinononanoyl-L-aspartyl-homo-L- 1.25 1.9 94.0 isoleucyf-L-arginine ditrifluoroacetate, CaI'd: 614, Found: 614 9-Guanidinononanoyl-L-aspartyl-D-isoleuc,1- 10.2 13.1 75.0 L-arginine ditrifluoroacetate, Cal'd: 600, Found: 600 TABLE I (cont'd) Inhibition of Fibrinogen Inhibition of 125 1Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at lc~uiuw M5ULw~ 25 M 9-Guanidinononanoyl-L-aspartyl-(R, >25 isobutyl ornithine ditrifluoroacetate, Cal'd: 501, Found: 501 L-Lysylg lycyl-L-aspartyl-L-valine dlitrifluoroacetate, Cal'd: 418, Found: 418 60 >25 9-Guanidinononanoyl-L-aspartyl-(R)-4-amino- 4-isobutylbutyryl-L-arginine dlitriffuoroacetate, Cal'd: 628, Found: 628 9-Guanidinononanoy-L-aspartyl-L-arginyl-L- 15.0 15.0 68 arginine tritrifluoroacetate, Cal'd: 643, Found: 643 9-Guanidinononanoy-L-aspartyl-acsx-diethyl- 40 >25 20.0 glycyl-L-arginine ditrifluoroacetate, Cal'd: 600, Found: 600 9-Guanidinononanoyl-L-aspartyl-L-arginine-3- 0.15 1.7 97 methylbutyl ester ditrifluoroacetate, Cal'd: 557, Found: 557 TABLESI (cont'd) Inhibition of Fibrinogen Inhibition of 25 I-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at 1~QQEM 14M 25 LM 8-Guanidinooctanoyl-L-aspartyl-L-phenyl- 0.2 0.69 96 alanine trifluoroacetate, Oal'd: 464, Found: 464 9-Guanidinononanoyl-L-aspartyl-L-arginine-2- 0.12 0.73 97 methyl-butylester ditrifluoroacetate, Cal'd: 557, Found: 557 9-Guanidinononanoyl-L-aspartyl-L-arginine- 0.26 0.82 91.0 cyclohexylester ditrifluoroacetate, Cal'd: 570, Found: 570 11 -Aminoundecanoyl-L-aspartyl-L-valine 0.58 1.7 0.98 trifluoroac-etate, Cal'd: 416, Found: 416 N-(9-guanidinononanoyl-L-aspartyl)-(R)-4- 54 >25 13.0 amino-4-isobutyl butyric acid ditrif luo ro acetate, Cal'd: 472, Found: 472 4-[N-(9-guanidinononanoyl-L-aspartyl)amino]- 10.52.940n 4-isobutyl butyl guanidine ditrifluoroacetate, IV.S., CaI'd: 479, Found: 479

C,

TABLE I (cont'd) Inhibition of Fibrinogen Inhibition of 25 1-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at 8-Aminooctanoyl-sarcosyl-L-aspartyl-L-valine 0.23 2.6 96 trifluoroacetate, CaI'd: 445, Found: 445 6-Guanidinohexanoyl-sarcosyl-L-aspartyl-L- valine methylester trifluoroacetate, Cal'd: 473, Found: 473 5-(5'-Guanidinopentyl-1 H-tetrazo le-1 -acetyl- 2.2 >25 40.0 acetyl-L-aspartyl-L-valine trifluoroacetate salt, CaI'd: 470, Found: 470 9-Guanidinononanoy-L-asparty-13-homo-L- >200 >25 38 isoleucyl psi(CH 2 NH)-L-arginine tritrifluoroacetate, Cal'dl: 586, Found: 586 9-Guanidinononanoyl-L-aspartyl-L-isoleucyl-D- 15.0 2-0o 97.0 arginine ditrifluoroacetate, MS., CaI'd: 600, Found: 600 c- 9-Guaniciinononanoyl-L-aspartyl-(S)-cz-benzyl-L- 20 13.3 69 4 arginine methylester ditrif luoroacetate, CaI'd: 591, Found: 591 TABLE I (cont'd) Inhibition of Fibrinogen Inhibition of 125 1-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at J~ilw M-Q{ 25 M N-(9-Guanidino non anoyl-L-aspartyl)-3-amino- 4.3 0.45 89.0 2-sec-butyl propionic acid L-arginine- amide ditriftuoroacetate, CaI'd: 614, Found: 614 9-Giaanidinononanoyl-L-aspartyl-D-homoisoleucyl- 4.7 5.8 98 L-arg-;i~e diirifuoroacetate,0~ ZG'ai'd: 614, Found: 614 9-Guaridinononanoyl-L-aspartyl-L-phenylaanine- 6.0 0.32 98.0 L-arginina dlitrifluoroacetate, Cal'd: 634, Found: 634 9-Guanidinornonanoyl-L-aspartyl-L-arginine 1.7 91.0 methylester 'Jitrifluoroacetate, CaI'd: 501, Found: 501 9-Guanidinononanoyl-L-aspartyl-L-isoleucyl- >25 45.0 L-isoleucine trifluoroacetate, Cal'd: 557, Found: 557 c3 9-Guanidinononanoy-L-aspartyl-L-alany-L- 0.54 89.0 arginine ditrifluoroacetate, CaI'd: 558, Found: 558 TABLE I (cont'd) Inhibition of Fibrinogen Inhibition -of 125 1-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at MUL" lQuum1 25 LM 9-Guanidinononanoy-L-aspartyl-L-t-butylg lycyl- 1.5 96.0 L-arginine ditritluoroacetate.

Cal'd: 600, Found: 600 9-Guanidinononanoy-L-asparty-[(S)-4-amiflo- 11.2 76.0 4-sec-butyl-butyljguanidine dlitrifluoroacetate, Cal'd: 499, Found: 499 9-Guanidinononanoyi-L-aspartyl-2-armilo-- 0.36 90.0 butanoyl-L-arginine ditrifluoroacetate, Cal'd: 572, Found: 572 9-Guanidinonanoyl-L artyl-D-J-homolso- 3.3 94.0 leucyl-(des-carboxy-L-arginine ditrifluoroacetate, Cal'd: 570, Found: 570 9-Guanidinononanoy-L-aspartyl-(S)-(x-iSObutyl-- >25 0.0 arginine methyzster ditritluoroacetate, Cal'd: 557, Found: 557 c 9.,Guanidinonolaloyl-L-aspatyl-L-valifle-L---0.290 arginine ditrifluoroacetate, Cal'd: 586, Found: 586 TABLE I (cont'd) Inhibition of Fibrinogen Inhibition of 125 1-Fibrinogen Mediated Platelet Aggregation Binding to Platelets inhibition at 25 Lim 9-Guanidinononanoyl-L-aspartyl-cyclohexyl- alanyl-L-arginine ditrifluoroacetate, CaI'd: 640, Found: 640 9-Guanidinononanoyl-L-aspartyl-L-leucyl-L- 0.52 96 0 arginine ditrifluoroacetate, CaI'd: 600, Found: 600 9-Guanidinono'nanoyl-L-aspartyl-(R)-6-amino-6sec-butyl hexanoyl-L arginine ditrifluoroacetat,, Cal'd: 656, Found: 656 6-Guanidi riohexanoyl-N-ethylglycyl-L-aspartyl-[(R)- 4-amino-4-isobutyl-butyl]guanidine acetate, CaI'd: 542, Found: 542 9-guanidinononanoyt-L-aspartyl-[(R)-6-amino-6sec-butyl-hbx1yl]guanidine ditrifluoroacetate, M.S, 527, Found: 527 9-G uan Idin on onan oyl-L-asparty, no rva ly l-L- arginine ditrifluoroacetate, Cal'd: 585, Found: 585 TABLE I (cont'd) Inhibition of Fibrinogen Inhibition of 125 1-Fibrinoge1 Mediated Platelet Aggregation Binding to Platelets inihibition at IQ5QI"25

W

N-(9-Gu an Win ononanoyl-L-aspartyl)-3- butyi-3-ami'iobutyryl-L-argi nine dlitrifluoroacetate, CaI'd: 628, Found: 623 6-Guanidinohexanoyl-sarcosyl-L-aspartyl4-- 4.5 90.0 valine-butylester trifluoroacetate, MRS., Cal'd: 515, Found: 515 6- Guanidino hexanoyl-sarcosyl-L-aspartyl- L- 4.5 90.0 valine-butylester trifluoroacetate, CaI'd: 515, Found: 515 9-Guanidinononanoyl-L-aspartyl-D-homonot'Ieucyl-L-arginine ditrifluoroacetate, CaI'd: 614, Round: 614 8-Guanidinooctanoyl L-aspartic acid 8-guanidino--- 1 -aminooctan e-a-amide ditrifluoroacetate, CaI'd: 485, Found: 485 6-Guanid>r--nexanoyI-sarcosy-L-aspartyl- 1- >25 18.0 butylester-L-vatine trifluoroacetate, Cal'd: 515, Found: 515 TABLE I (1cont'd) Inhibition of 125 1-Fibrinogen Binding to Platelets 404m1 Inhibition of Fibrinogen Mediated Platelet Aggregation inhib.ition -at lc~l~w25

M

6-Guanidinohexanoyl-sarcosyl-L-aspartyl- 1butylester-L-valine-butyl ester trifluoroacetate, Cal'd: 571, Found: 571 6-Guanidinohexanoyl-N-ethylglycl-L-a-spartyl- 2,2-diethyl glycine, Cal'd: 464, Found: 464 11.4 73.0 WO 92/17196 PCT/US92/O263r 69 One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compounds, compositions, and methods described herein are presented as representative of the preferred embodiments, or intended to be exemplary and not intended as limitations on the scope of the present invention. Changes therein and other uses will occur to those of skill in the art which are encompassed within the spirit of the invention or defined by the scope of the appended claims.

Claims (16)

1. A compound of the formula 0 I I OO A (0H2)i(C)hi (B)k IC )h2- (CH 2 )m 2 -D -CH Z I I I R2 R4 (OH 2 )n COOQH wherein: A is N ~NR N 11 -C -R 6 or (CO)x" 13 5 R 5 R B and D are independently -CH 2 -CH 2 -CH 2 0 R 9 0 111 1 It -C-NR 9 -(CH 2 -CH-CH 2 or I I I i R 1 0 R 7 Y -CH-0CH 2 0- Y 1 B may also be 5-tetLrazol-1 -yl or -CR 7 =CR 8 Z is O01a, a D- or L-isomer of an ae-amino acid bonded at the (x-nitrogen, a dipeptide bonded at the N-terminal a-amino acid, or -NRaRx, where Rx is H or I~I (CH V C- Rt where V is 0 0 C -NRg C H- -OH 2 -OH 2 NH-, -CH 2 or -CH 2 S-; Re and Rf are independently 1H, alkyl, cycloalkyl, cycloalkylmethyl, or -(0H2)s-Rz where Rz is;-CO0Rn, -0Rn, -SRn, -N Rn, R 0 NHl Nil NHl-C- NH 23 NH 2 1 1- 10 and Rn are independently H, alkyl, cyoloalkyl, cycloal kyl methyl, aryl, substituted aryl, aralkyl or substituted aralkyl; Ra, Rg, and R 0 are independently H, alkyl, cycloalkyl, cycloalkylmethyl; Rt is -COOH, -COORk, carbamoyl, or 0 11 C -NRkRm where Rk and Rm are independently H, alkyl, cycloalkyl, cyclo lkylmethyl; Yj is H, amino or 0 11 -NH -C-R where Rp is H, alkyl, cycloalkyl, cycloal'.yl methyl, aryl, substituted aryl, aralkyl or substituted aralkyl; x, and are independently 0 or 1; ml and M2 are independently 0 to 9; 11 1 h2, and k are independently 0 or 1; n is i to 3; q is i to 5; and p and s are independently 0 to 6; provided that when ;21 0 I I oC A (CH2) m1(Chil k {C)h. 2 (CH 2 m 2 -D O H R 2 R4(OH 2 )n COOH is arginyl-glycyl-aspartyl, then Z is other than a naturally occurring amino arid or a dipeptide composed of two naturally occurring amino acids; and provided that Z is other than serine or a pharmaceutically acceptable salt thereof.

2. A compound of claim 1 wherein: B and D are independently -H 2 -CH 2 0 R 9 0 -C-NR 9 -(CH 2 -OH-0H 2 or Rt 1 0 Rt 7 Ft 7 -CH-CH 2 Y 1 B may also be -CR 7 and Rz is -C00Rn, -ORn, -SRr,~ NRn'R 0 NH NH -NH-C-NH 2 NH 2

3. A compound of claim 2 wherein: Z is -ORa, a D- or L-,."omer of an a-amino acid bonded at the ax-nitrogen, a dipeptide bonded at the N-terminal a-amino acid, or -NRaRx, where Rx is H or R e CH-V C-Rt I I Re R t

4. A compound of claim 1 of the formula 0 II 0 C II A (CH 2 )ml (B)k (CH 2 2 C- NH CH Z CH 2 COOH wherein: A is guanidino or N -N Rs mi is 1 to 9; m 2 is 0 or 1; and B is -CH=CH- or 0 II C- NR 9 (CH 2 x"' A compound of claim 4 wherein A is guanidino.

6. A compound of claim 5 wherein Z is a D- or L-isomer of an a-amino acid bonded at the a-nitrogen, or Z is a dipeptide bonded at the N-terminal a-amino acid.

7. A compound of claim 6 wherein Z is a D- or L-isomer of an a-amino acid bonded at the a-nitrogen.

8. A compound of claim 7 wherein the cx-amino acid is selected from the group consisting of glycine alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, proline, hydroxyproline, aspartic acid, aspargine, glutamine, glutamic acid, histidine, arginine, ornithine, and lysine.

9. A compound of claim 8 wherein the a-amino acid is selected from the group consistinig of valine, leticine, isoleucine, and arginine. A compound of claim 1 which is: o r th e trifluoroacetate salt thereon 6-guenidinohexanoy-N-(ethyl)-glycyl-L-aspartyl-L-leucine or the trifluoroacetate salt thereof; 6-guanidinohexanoyl-,N-(ethyl)-glycyl-L-aspartyl-L-isoleuclne or the trifluoro- acetate salt thereof; 6-guanidinohexanoyl-sarcosyl-L-aspartyl-L-leucine or the trifluoroacetate salt thereof; or a pharamaceutically acceptable salt thereof,

11. A compound of claim 1 which is: 6-guanidinoiiexanoyk-N-(ethyl)-glycyl-L-aspartyl-L-valine or the trifluoroacetate salt thereof; 6-guanidinohexanoyl-sarcosyl-L-e, partyl-L-valine or the trifluoroacetate salt thereof; :5-guanidinovaleroyl-sarcosyl-L-aspartyl-L-valine or the trifluoroacetate salt thereof; or a pharmaceutically acceptable salt thereof.

12. A compound of claim 1 which is aspartyl-L.,arginine or the ditrifluoroacetate salt thereof or a pharmaceutically accepablesalt thereof. 12. A compound of claim 1 which is aspartyl-L-arginine or the ditrifluoroacetate salt thereof or a pharmaceutically acceptable salt thereof.

13. A compound of claim 1 which is 8-guankIinooct-2-enoyl-L-aspartyl-L- valine or the trifluoroacetate salt thereof or a pharmaceutically acceptable salt thereof.

14. A compound of claim 1 which is 9-guanidinononanoy-L-aspartyl-L- !sole uci ne-4-g uan idi nobutyl amide or the ditrifluoroacetate salt thereof or a pharmaceutically acceptable salt thereof. A compound of claim 1 which is 9 guanidinononanoyl-L-aspartyl-L- !eucine or the trifluoroacetate salt thereof or a pharmaceutically acceptable salt thereof.

16. A compound of claim 1 which is 9-guanidinononanoylkL-aspartyl-L- arginine or the difluoroacetate salt thereof or a pharmaceutically -acceptable salt thereof. 7. A compound of claim 1 which is 9-guanidinononanoyl-L-aspartyl-L- arginine-isobutyl ester or the ditrifluoroacetate salt thereof or a pharmaceutically acceptable salt thereof.

18. A compound of claim 1 which is-, 9-guanldlnononanoyl-L-aspartyl-L-aspartyl-L-leucyl-arglnine or the ditrifluoro- 'acetate salt thereof; 9-guanidinononanoyl-L-aspartyl-L-valyl-arginine or the ditrlluoro-acetate salt thereof; N-[N-(9-guanldinononanoyl-L-aspartyl)-2-aminobutanoyl]-L-arginine or the ditrifluoroacetate salt thereof; 9 -guanidinononanoyl-L-aspartyl-L-alanyl-arginine or the ditrifluoroacetate salt thereof; 9-guanidinononanoyl-L-aspartyl-L-norleucyl-arginine or the ditrifluoroacetate salt thereof; 9-guanidinononanoyl-L-aspartyl-D-homoisoleucyl-L-arginine or the ditrifluoro- acetate salt thereof; 9-guanidinonon,.noyl-L-aspartyl-L-phenylalanyl-L-arginine or the ditrifluoro- acetate salt thereof; N-(9-guanidinoionanoyl-L-aspartyl)-3 amino-2-secbutylpropionyl-L-arginine of the ditrifluoroacetate salt thereof; or a pharmaceu,ically acceptable salt thereof.

19. A pharmaceutical composition for the prevention or treatment of abnormal thrombus formation in a mammal comprising a pharmaceutically acceptable carrier and an antithrombotic effective amount of a compound of claim 1. A method for the prevention r treatment of abnormal thrombus formation in a mammal comprising the administration of a therapeutically effective amount of a compound of claim 1.

21. A method for the prevention or treatment of abnormal thrombus formation in a mammal comprising the administration of a therapeutically effective amount of the composition of claim 19. o DATED this 16th day of December, 1994. RH6NE-POULENC RORER INTERNATIONAL (HOLDINGS) INC. WATERMARK PATENT TRADEMARK ATTORNEYS 290 BURWOOD ROAD -AWTHORN VICTORIA 3122 UJSTRALIA DBM:SI:JL VAX doc 040 AU2014392.WPC INT'ERNATIONAL SEARCH REPORT FIneiiational application No. PCT/US92/02637 I, A. CLASSIFICATION OF SUBJECT MATT'ER :A61K 37/0-2; C07K 5106, 5/08, 5/10, 7/06 US CL :514/18, 19, 20; 530/330, 331; 562/560, 571 According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHIED Minimum document'AtioV' searched (classification system followed by classification symbols) U.S. 514/18, 19, 20-.5301330, 331,,5621560, 571 Documentation searched other than minimum documentation to the extent that such documents are included in the fields searchted Electronic data base consulted during the interniational search (nrtam of data base and. where practicable, search terms used) APS search terms: thrombo?, peptide?, guanidin?, arginyl C. DOCUMENTS CONSIDERED TO BE RELEVANT Category's Citation of document, with iilication, where appropriate, of the relevant passages Relevant to claim No, X US, A, 4,629,736 (Tsukarnoto et at) 16 December 1986, column 2 lines 3-35 and column 1-3, 2 1-23 12 lines 24-41. X US, A. 4,879,313 (Tjoeng et al.) 07 November 1989, 1-3,6,7,21-23 column 2,lines 13-68 Y 4, 5, 8-20 column 10,llne 59 to column I11)ie 9. X US, A, 4,992,463 (Tjoeng et al). 12 February 1991, t-3,6,7,21-23 column 2,l1ines 6-59 Y 4, 5, 8-20 column l~ines 18-33, X US, A, 5,037,808 (Tjoeng et al) 06 August 1991, 1.3,6,7.21-23 column 2,line 20 to column 3, line Y 4, 5, 8-20 column 17, lines 21-46, X US, A, 5,053,393 (Tjoeng et al) 01 October 1991, 1-3,6-10,21-23 -'Pcolumn 2,lines 23-50, column lies 12-31 4, colum 10, line 63 to column 11, line 19. LI Further documents are listed in the continuation of Box f See patent family annex. Special categories of cited documents: dnwnteaderning the general stat-e of the inl which is not consWerel to be pant of particular rceviince eE arlier document published on or after the international filing date L document which may throw doubua on priority claim(s) or which is cited to establish the publication date of snother citation or other special reason (as specified) .0 document referring to an oral disclosure. use, exhibition or other means Tp* document published prior to the international riling date but later than the priority date claimed Date of the actual completion of thc international search Date of mailnh.of the international search report June 1992 1 "JUL 139 Name and mailing address of the ISAJ Authorized officer Commissioner of Patent& and Trademarks p w 4 IFacsimile No. NOT APPLICABLE jTelerghoneNo._ (703) 308-2957 Form PCT/ISA/210 (second sheet)(July 199,1)*