CA2021949A1 - Fibrinogen receptor antagonists - Google Patents

Abstract

7652P/ 7/28/89: Fl 17986 TITLE OF THE INVENTION LINEAR FIBRINOGEN RECEPTOR ANTAGONISTS A fibrinogen receptor antagonist compound of the structure: A-B-C-Gly-Asp-D-E (I) wherein A, B, C, D and E are preferably ffollows: A is L-asparagine, D-asparagine or acylated asparagine B is an L-or D-isoer of proline, thioproline, .beta.,.beta.-dimethylthioproline, or N-methylalanine; C is arginine; D is phenylalanine, tryptophan .alpha.-naphthylalanine, .beta.-napthlalanine, arginine or lysine; and E is OH.

Description

7/2~/89: ~1 ~L~
FIBRI~OGEN RECEPTOR ANTAGONISTS

B~KGROUND OF~raE~ EE~IQ~
The invention relates generally to modulating cell adhesion aad to inhibiting the binding o~ fibrino~en and other protein~ to blood -platelet~, and inhibiting the ~ggregati~n o~ blood platelet6 ~pecifically to the IIb/IIIa fibrinoge~
receptor ~ite. FibrinQge~ i8 a ~lycoprotein, pre~ent in blood plasma, which participate8 in platelet aggregation and in f i~rin for~ation. Platelet~ are cell~ e anucleated ~rag~nts, ~ound ~n the ~lo~ of all mammals, which participate ~ blood coa~ulation.
Interaction of fibrino~en ~ith the IIb/IIIa receptor ~te 1~ ~nown to ~e e~ential for normal platelet ~unction.

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7652P/ - 2 17g86 7/28/89: ~1 When a blo~d vessel i~ damaged, platelets adhere ~o the di~rupted ~ubendothelial surface. ~he adherent platelet~ su~sequently release biologically active constituent6 and aggregate. Aggregation i6 initiated by the binding of agoni~t~, such as thrombin, epinephrine, or ADP to ~pecific platelet ~embrane receptors. Stimulation by agonists re~ult~
in e~posure of latent fibrinogen receptors on the platelet surfacc, a~d bindi~ ~f ~ibrin~gen to the glycoprotein IIb/IIla complex.
Attempts have been ~ade to u~e natural pr~duct~ and æynthetic peptide6 to ~tudy the ~echani~m of platelet .ggregation ant adhe~ion.
Rouslahti and Pierschbacher, ~Si~ . 1987,

2~, pp. 491-497, describe adhe~ive proteins cuch as fibronectin, ~itronectin, osteopontin, collagens, thrombo~pondin, fibrinogen, and von Willebrand factor present in extracellular mat~ices and in ~he blood.
The protein~ contain the tripeptide arginine-glycine-a~Rartic acid a~ their cell recognition ~ite. The tripeptides are recognized by at least one ~ember of a family of structurally related receptors, inte~rin~, which are heter~dimeIac protein~ with two membrane-~panning ~u~unit6. The authors ~tate that the c~nformatio~ o~ the tripeptlde equence ln the individual proteins may be cr~tlcal to recognition ~pec~ficity.
Cheresh, ~r~ c~d, Sci~ , 1987, ~4, pp. 6471-6475, de~cribes an A g-Gly-Asp d~sec ed ad~es~on receptor expr~6sed ~y human ~ndothelial cells that ~ structurally simllar to the ~Ib/IIIa complex on ~latelet~ but antigenically and .
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7652P/ - 3 - 17~86 7/28/89: Fl functi~nally distinct. The receptor is directly involved in endothelial cell attachment to fibrinogen, von Willebrand factor, and vitronectin.
Pierschbacher a~d Rouslahti, J. of Biol.
~h~m~, 1987, 2~, 36, pp. 17294-17298 describe 5 ætereochemical i~luence of the ~equence Arg-Gly-Asp-Xaa, where ~aa i~ one of the 20 natural L-amino acid~ other than Me~, Cy8, ~i~, Trp or Gly on ~inding ~peci~icity of peptide~ cont~ining t~e tsipeptide ~equence Arg-Gly-A6p. In ~IQ~ a~ Acad 10 ~, 1984, ~1. pp. 5985-5988, the 6ame authors describe variants of the cell recognition æite of fibronectin that retain attach~ent-promoting activity. The tetrapeptide Arg-Gly-Asp-Ser i6 de~cribed as the minimal BtruCture recognized by cell6 in the lar~e, adhe~ive glycoprotein ~ibronectin.
Peptides having portion6 -Arg-Gly-A~p-Ser- are described in U.S. Patent Nos. 4,589,881 snd 4,614,517. Peptides havin~ p~rtions -Arg-Gly-A6p-R
wherein R i6 selected from Thr or Cy~ or other amino acid ha~ing the ~ame cell-attachment activity as ~ibro~ec~in, are de~cribed in ~.S. Patent NG.
4,57~,079.
Ruggeri et al., Pr~c. Nat'l~_~c~d. ~ci. ~SA, 19B6, ~, pp. 5708 5712, de~cribe~ a series o~
synthetic peptides, design~d i~ l~ngth~ to 16 residues, that contal~ the 8e~uence Arg-Gly-Asp-~al, which lnhiblt ~l~rino~en binding to platelet~.
Wh~l~ it ~B ~nown that the tripeptite ~e~uence Arg-Gly-Asp ic preBent in certain polypeptides whlch can dupllcate or ~nhibit the eell attachment-promoting e~fect6 of ~ibronect~n and 7/28l89: Fl vitronectin, the tripeptide Arg-Gly-A~p ha~ low ~ctivity. There i~ little under~tanding of the ~nfluence on bindin~ ~pecificity of other amino acids in the polypept~de. Applicant~ hav~ prepared small linear pentapeptides which contain the tripeptide 3equence Arg-Gly-A6p which are active platelet aggregation ~nhi~ito~6.
5~9.~.~
The preEent invention i6 a fibrinogen receptor antagoni~t compound of the following structure:
A-B-C-Gly-Asp-D-E (I) wherein:
A i6 any L- or D-isomer of a ~-ami~o acid, an acylated amino acid, a des-~-amino acid, or an N-methyl-~ amino acid;
B i~ an L-i~omer of a 6econdary amino acid 6elected from the group con~i6ting of proline, hydroxyp~oline, thioproline, ~,~-dimethyl-thioproline. dehydr~proline, pipecolic acid, azetidine carboxylic acid and N-methyl amino acid~;
C iB an L-i~omer of arginine, homo-arginine, guanido aminobutyric acid, or guanido ~minopropionic acid;
D i~ an L~omer ~f t~yptophan, phenyl-alanine, leuci~e, ~al~n~ oleuciDe~ napthylalan~ne, methioni~e, tyrosine. or ring ~u~8tltuted derivat~v@s of tryptophan. tyro8ine, phenylala~ine, argin~ne, homo-argin~ne, ornithine, ly81ne or h~ætidine; and O~J N~2, NER, NRlR2, ~herein R 18 an alkyl group haviDg 1 to 4 carbon ato~, ant . . .

' 7/2~/89: Fl RlR2 repre~ent~ an alkyl group having 1 to 4 - carbon at~ms, a ~econdary amino acid, or N
~N ~

~referred co~p~undæ are tho~e where ~ is O~.
~ore preferred compound~ ~ro those ~here:
A i6 L~a~paragi~e, D-a6p~ragi~e or acylated asparagine;
~ i6 an L-i~omer ~f proline, thioproline, dimethylthioproli~e, or N methylalanine;
C iæ arginine;
D is phenylalanine or tryptophan; and E is OH.
More preferred c~mpoundæ are:
i) A~n-Pro-Ar~-Gly-Asp-Phe O~, ii) Asn-ThioPro-Ar~-Gly-A6p-Ph~-O~, ii;) A~n (~,~-dimethylThioPro)-Arg-Gly-Asp-Phe-O~, iv) (D-Asn)-Pro-Ar~-Gly-A~l?-Phe-O~, v) (AeAæn)-Pro-Arg-Gly-Asp-~he-O~, vi) A~a-Pro-Arg-Gly-Asp-Trp-OH, Yi i ~ A~n-ThioPro-Arg-Gly-A~p-Trp-O~, vi~i) A~-(p,~-timethylThioPro)-Arg-Gly-Asp-Trp-O~.
ix~ A~n-(N-methylAla~Arg-Gly-A~p-T~p-O~, s) Ser-Sar-Arg-Gly-A6p-Phe-O~, ~) A~n-Hypro-Arg-Gly-A~p-Ph~O~, ~ii) A~n-dehypro-Arg-~ly-Asp-P~e-O~, ~ill) Asn-(D-Arg)-Arg-Gly-A~p-Phe-O~, xiv) Asn-Hi6-Arg-Gly-Asp-Phe-O~I, xv) A~n-Pro-(D-Arg~-Gly-A~p-Phe-O~, xvl) Asn-Pro-~D-Arg)-Gly-A~p-~he-N~

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7/28/89: Fl --~ vii) A6n-Pro-Arg-Gly-~eAsp-Phe-O~.
sviii) Gly-Pro-Arg-Gly-A6p-Phe-O~, ~ix) Arg-Pro-Arg-Gly-Asp-Phe-OE, ~x) Ala-Pro-Arg-Gly-A~p-Phe-OH, ~xi) Ser-Rro-Arg-Gly-Asp-Phe-O~, æxii) (D-Asn)-Pro-Arg-Gly-Asp-Phe-O~, ~xiii) Ile-Pro-Arg-Gly-A6p-Phe-O~, ~iv) Pro-Pro-Arg-Gly-A~p-Phe-O~, ~V) ~iB-Pr o-Ar~-Gly-A~p-Phe-O~, ~xvi) Gln-P~o-Arg-Gly-Asp-Phe-OH, xxvii) Phe-Pro-Arg-Gly-Asp-Phe-O~, xxviii) Leu-~ro-Arg-Gly-A~p-Phe-O~, ~xix) A~p-Pro-Arg-Gly-A~p-Phe-O~, xxx) Met-Pro-Arg-Gly-Asp-Phe-O~, zxxi) (D-N-methyl Ala)-Pro-Arg-Gly-Asp-Phe-O~, xxxii) (N-methyl-Ala)-Pro-Arg-~ly-A6p-Phe-OB, m ciii) Acetyl-Asn-Pro-Arg-Gly-A~p-Phe-OH, xxxiv) A~n-Pro-Arg-Gly-A~p-Net-O~, ~ xx~) Asn-Pro-Arg-Gly-A~p-Val-O~, xaxvi) A~n-Pro-Arg-Gly-A~p-Trp-O~(For), xxxvii) Asn-Pro-Arg-Gly-A~p-Trp-O~, XXXYiii) A~n-Thio Pro-Asg-GlyAsp Trp-O~, xxxix) Asn-Azt-Arg-Gly-Asp-Trp-OH, xxxx) A~n-~ip-Arg-Gly-Asp-Trp-O~, ~xxx~) Aæn-(D Pip~-Arg-Gly-A6p-Trp-O~, æsxxil) A~-(N-~ethyl-Ala)-Arg-Gly-A~p-Trp-O~, xxxxii1) Asn-~N methyl-~h~)-Ar~-Gly-A~p-Trp-O~, xxxx~v) Asn-~D-Ar~-Arg-Gly-A~p-Trp-O~ and ~a~rv~ As~ -Ar~-Gly-Asp-~rp-O~.
~nless otherwi~e ~d~cated, each ~ino acid iE the

3~ L-~æomer.

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7~52P/ - 7 - . -. 17986 7/28/89: ~1 M~re preferred compounds are:
ii) A6n-ThioPro-Arg-Gly-Asp-Phe-O~, lii) A~n-(~ dimethylThioPro)-Arg-Gly-Asp-Phe-O~, vi) Asn-Pro-Arg~Gly-A~p-Trp-O~, S ~a i ~ Asn-Th~ oPro-Arg-Gly-Asp-Trp-O~, and i~) Asn-(N-methylAla)-Arg-Gly-A~p-Trp-O~.
The ~n~enti~n ~l~o ~nclud~ co~po~ition6.
compri6ing ~ibrinogen receptor a~tB~oni~t peptide~ of the present invention and o~e or ~ore pharma-10 col~ically acceptable carrier~, e.g. ~aline, at aphar~acologically acceptable p~, e.g. 7.4, which are 6uitable for continuous intravenous or oral intra~enou6 b~lus a~mini~tration for promoting inhibition of platelet aggregation.
The invention al60 include~ method~ f~r inhibiting platelet ~ggregation which compri~e atministering to a patient, either by eo~tinuou~
intravenou~ or oral intra~enou~ bolus method, an effective amount of a comp~ition o~ the pre~ent 20 invention-~ETAILE~ ~ES~I TION OF T~E I~ IQ~
Compou~ds of the in~e~tion are fibrinoge~receptor anta~o~ists which in~ib~t f i~ri~ogen ~duc~d 2S platelet ag~regatlon. The~e compound~ are prepared by sol~d pha~e ~ynthe818 which i8 well ~own l~ the art, or by llguld pha8e 8ynthe~i8 whlch i8 well known in the art. The method8 o~ ~ynthe8~s are generally de6cribed ~y Neurath, ~ill and Boeder, Ed~. "The Protel~" 3rd Edit~o~, Volume II, Ac~demic Press 19~6.

7652P/ - 8 - . 17986 7/28/89: Fl The compounds have a relatively 6hor~
duration of activity which makes them desirable for use in therapeutic treatmentB where prevcntion of platelet aggregation over a short period of time iE
de~irable. The compounds are al~o particularly advanta~eous because they do Dot ~ignificantly deplete the platelet count.
An essential ~eature o~ tbe compound~ of the present inven~ion iæ ~he pre~ence of an L-i~omer of a secondary ~mino acid selected from the group lO consisting of proline thioproline, ~,~-dimethyl-thio-proline, dehydroproline, pipecolic acid, azetidine carbo~ylic acid or ~n N-methylamino acid at position B of the compound formula. While applicant~
do nst wi~h to be bound to any particular theory, the 15 pre~ence of a secondary amino acid at po~ition B i~
believed to affect ~he confor~ation ~f the compound such that it iæ readily recognized and accepted by the IIb/lIIa receptor Gite, ther~by enhancing it~
potency.
Common or a-amino acidæ are the twenty with which all protein~ in all ~pecie~, from bacteria to human~ are constructed.

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7/28/89: F1 n Am~R ~cld6 l~c~ A~ L~ ~ ~v~t~on ne ~la L~uc~ne Le~
S /~rtinlne Arg Lyl3~ne L~
A~par~g~ne Asn Methio~ine Met ~spnrtlc Ac~d A6p Phengl~ ne Phe Cy~te~ne Cy6 Proline Pro Glutam~ne Gln Serine Se~
10 Glut~mic Acid Glu Thr~onine Thr Glycine Gly Trypt~phan Trp ~i6tidlrle Bis Tyro~ine Ty~
I601eucine lle Valine Val Compounds of the invention may be prepared using æolid pha~e peptide ~ynthe~is, 6uch a~ that de~cri~ed by Merrifield, ?. A~. Che~_~oc., 85, 2149 (1964), although other equivalent chemical synthese~
know~ in the art can al80 be used, such a8 the 6ynthe~e~ of ~oughten, ~roc. ~atl. ~caL Sci , B2, 5132 (1985). Solid-phase ~ynthesi~ i~ co~enced from the C-terminu of the peptide ~y coupling a protected amino acid to a 6uitable re~ generally ~t forth i~ ~.S. Pate~t No. 4,244,946, ~ued Jan. 21, 1982 to Rivier ~t al., t~e di~clo~ure of whish i8 hereby incorporated by reference. Æx~mples of ~ynthe8~ of th~ gene~al type ~re 6et forth ln ~.S.
Patent Nos. 4,305,87Z and 4, 316, B91. Compound~ of the lnventlon can al~ b~ prepared aecordi~g to liqu~d ~ha~e synthesi~ described by Neurath, ~ill and Boeder, Ed~., "The Protein~" 3rd Edition, Vol, II, Chapter 2 pp. 106-252.

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7652P/ ~ 17986 7/28/89: Fl In ~ynthe6izing these polypeptides, the carboxyl terminal amino acid, ha~ing it~ alpha-amino ~roup sui~ably protected, ~6 coupl~d to a chloromethyl~ted polystyrene resin or the li~e.
After removal of the alpba-~mino protecting group, a~
by using trifluoroacetic acid in methylene chloride, the ~ext step in the synthesi~ i6 ready ~o ~roceed.
Other ~tandard cleaving reagents and condition6 for the removal oP speeific am~o protecting ~roups may be u~ed, a6 de~cribed in the open literature.
~ . The remaining alpha-amino- and side-chain-psotected amino acids are then coupled by conden~ation stepwi~e in the de~ired osder to ~tain an intermediate compound connected to the sesin.
The condensation between two amino acids, or 15 an amino acid and a peptide, or a peptide and a peptide can ~e carried out according to the u6ual condensation method6 ~uch as azide method, mixed acid anhydride method, DCC (dicyclohe~ylcarbodiimide) method, active e~ter method (p-nitrophe~yl ester 20 method, N-hydro~y~uccinic acid imido e~ter ~ethod6, cyanomethyl e6ter method, etc.), Woodward reagent method, carbonyldiimidazol method, oxidation-reduetion ~ethod or BOP b~nzotriazole l-yloxytr~
(timethylamino) pho~phonium hexaf~uoropho~phate) 2s method. In the caæe of elongating the pept~de chai~
in the sol~d phase ~ethod~ the peptide is attached to an ~n~oluble carrier at the C-termi~al amino acid.
For insolu~le carrieræ. tho~e ~hlch react with the carb~y group of the ~-terminal amino acid to ~or~ a bond which iB readily cleaved la~er, ~or e~ample, halomethyl re~in such a~ chloromethyl se in and .
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7652P/ ~ 17986 7/28/89: Fl br~momethyl re~in, hydro~ymethyl re~in, aminomethyl re~in, benzhydryl~mine resin, and t-alkyloxy-carbonylhydrazide re~in can be used.
Common to chemical ~ynthese~ of peptide~ i~
the protection of the reactive 8ide-chain groups of the various ~mino acid moietie~ ~ith ~uitable protecting groups a~ that site until the group i~
ultimately removed after tbe chain ha~ been completely assem~led. ~l~o com~on i~ ~he protection of the alpha-amino group on an amino acid or a 10 fragment while that entity react~ at the carbo2yl group ~ollowed by the selecti~e removal of the alpha-amino-protect~ng group to allow ~ub~equent reaction to take place at that location.
Accordingly, it is common thatl a~ a step in the 15 ~ynthesi~, an intermediate co~pound iB produced which includes each of the amino acid resitue~ located in the de~ired 6equence in the peptide chain with variou~ of theæe re~idues having 6ide-chai~
protecting group~. These protecting groups are the~
2D commonly removed sub~tantially at the 6ame time ~o a~
to produce the de~ired re~ultant product following ~urification.
The applicable protective group~ for protecting ~he alpha-and omega-side cha~n ~ o 2s group~ are exempl~f~ed such as benzylo~ycarbonyl (hereinafter abbrev~ated ~s Z), isonicotlnylo~y-carbonyl (iNOC~, o chlorobenzyloxycarbonyl ~Z(2-Cl)3~
~-nitrobenzyloxycarbonyl ~Z(~2)~' p-methoxybenzylo_ -xycarbonyl tZ(O~e)~ . t-butoxycarbonyl (Boc)~
t-a~yloxycarbonyl (Aoc), ~sobornyloxy~arbonyl~
adamantyloxycarbonyl, 2-(4-biphenyl)-2-propyloxy_ 7/2B/89: Fl carbonyl (Bp~c), g-fluorenylmetho~ycarbonyl (Fmoc), methylsulfonylethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenyl~ulphenyl (NPS~,diphenylpho~phinothioyl (Ppt), dimethylphosph-lnothioyl (Mpt) and the like.
S A6 protective ~roup6 for carb~y group there can be exe~pli~ied, for ~s~mple, benzyl ester (O~zl).

4-nitro~enzyl e~ter (ONb), t-butyl e~ter (OBut), cyclohe~yl (Chg), 4-pyr~dylmethyl e6ter (OPic), a~d the li~e. It i6 desirable that specific a2ino acids lo ~uch as arginine, cysteine, and ~erine p~s~eæsing a functional group other than nmino and carbo~yl groups are protected by a ~uî~able protective group as occa~ion demandæ. For example, the ~uanidino group in arginine may be protected with nitro, p-toluene-15 sulfonyl, benzyloxycarbonyl, atamantyloxycarbonyl,p-methoxybe~zenesulfonyl, 4-methoxy-2, 6-di~ethyl-benzene~ulfonyl (Md ), 1,3,5-trimethylphenyl~ulfonyl (Mts~, and the li~e. The thiol group in cysteine ~ay be protected with ~enzyl, p-meth~ybenzyl/ triphenyl-20 methyl, acetylaminomethyl, ethylcarbamoyl, 4-methyl-ben2yl, 2,4,5-trimethylbenzyl (Tmb) etc., and the hydroxyl group in Eerine ca~ be ~rotected with benzyl, t-butyl, acetyl, tetrahydropyranyl etc.
Stewart and ~oung, "Solid Phase ~ept~de 2~ Synthe~i~", 21erce Che~ical Company, Rockford, ~L
~1984) proY~de~ detailed ~nformatio~ regard~ng procedure~ for preparing ~eptide~. Protectlo~ of a-amino groups 18 de6cribet o~ pa~e~ 14-18, and site-chain ~lockage ~8 described o~ pages 18-2~. A
able of protecting ~roups for am~e, hydroxyl and , .

7/2~/B9: Fl ~ulfhydryl functions i6 provided on pages 149-151.
The~e description~ are hereby incorporated by reference.
A$ter the de~ired ~mino-acid seguence has been completed, the intermetiate peptite i8 removed ~rom the re~in ~upport by treatment with a reagent.
such as liquid EF, which ~t o~ly cleaves the peptide from the r~in, ~ut al80 clea~e~ all the remaining 6ide-chAin protecting group~. The polypeptide can then be purified by gel permeation followed ~y æemi-lO preparative ~PLC, a6 de~cri~ed in Rivier et al.,Peptides: Structure and Biological Func~ion (1979) pp. 125-128.

~.
lS Sy~h~sis of H-Asn-~r~-A~ ly-A~-Phe-n~
Starting with O . 5 mM of Boc-Phe-0-Pam-resin:

0 C~2-Ph o 20 (C~3)3C-0-C-N~-C~ 0-C~2-Ph--C~2C~N~-C~2-~pcr~

wherein "pcr" ~Q a polystyrene cro6~1inked re~iD, the alpha-amino Boc protecting group 25 ~ter~-~utyloxy-car~onyl) ~ rem~ed using trifluoro acet~c acid and methylene chloride, and the deprotscted ph~nylalani~e ~eutralized with diisopropylethyl a~i~e.
Two mM ~oc-protected A~p (benzyl ester~
(A6p-(O~zl)) i~ then coupled to phenylalan~e mediated by 1 mM dicyclohexylcarbodiimide, ~ ~ y ~

7/28/~9: ~1 deprotected usin~ trifluor~acetic acid ant methylene chloride, and ~eutralized with dii~opropylethylamine.
Two mM Boc-protected Gly 1~ the~ coupled to Asp (benzyl ~ster) metia~ed by 1 ~M dicyclohexyl-carbodiimide, deprotected and neutralized as de~cribed above.
Two ~M Boc-protected Arg (4-toluene~ulfonyl) (Arg(To~)) i8 then coupled to glyclne ~ediated by 2 mM dicyeloheæylcarbodii~ite and 2~M l-hydroxy-benzo-triazole, depr~tected and neutr~lized a~ -lo de~cribed above.
Two mM Boc-protected Pro i~ then coupled to Arg (4-toluene6ulfonyl) ~ediated by 1 ~M
dicyclohexylcarbodiimide, deprotec~ed and neutralized a6 described ab~ve.
Two mM Boc-protected Asn is then coupled to Pro mediated by 2 mM dicylohexylear~diimide and 2 mM
l-hydroxybenzotriazole. Trifluor~acetic acid and ~ethylene chl~ride are added to deprotected Asn to form the following ~alt:
To~ Bzl TF~ ~alt ~ A~n-Pro-Arg-Gly-Asp-Phe-0-~am ~

Clea~age of the peptide fro~ the reBin iB achieved 25 using ~F/ani~ole (9~ Iv)), to ~orm EF 6alt ~ Asn-Pr~-Arg-Gly-A~p-Phe-Q~ -Purification iæ conducted u~in~ preparatlve ~PLC in 30 0.1% TFA ~29~C~3CN gradi~nt. The inal TFA salt product 15 converted to ~OAc ~alt by pa~in~ through ion 7652P/ - 15 - 179~6 7/28/B9: Fl exchange column BioRad AG3-X4A (acetate cycle).

Xb~a~e~tic U ~
Reptides of ~he invention may be used for inhibiting i~tegrin protein-comple~ function relating to cell-attachment activity. For example, they may be adminigtered to patient~ where ~nhibition of human or ~a~alian platele~ aggregation or adhesion i~
de~iret.
~olypeptide6 of the inven~ion are eliminated 10 from circulation sapidly and are ~articularly u6eful in inhibiting platelet a~gregation in ~ituations where a ætrong antithr~mbotic of short duration of effectiYeness i~ needed. Thuæ, they may find utility in ~urgery on peripheral arteriex (arterial graft~, 15 carotid endarterectomy) and in cardio~ascular ~urgery where manipulation of arterie~ and ~rgan~, a~d/os the interaction of plateletE with artificial surfaceæ, lead~ to platelet aggregation and co~6umption. The aggregated platelet~ ~ay ~orm thro~ nt thrombo-20 emboli. Polypeptides of the an~entio~ may beadmini~tered to theGe ~urgical patients to prevent the formation of thrombi and thromboemboli.
ExtraeorporQal circulation i6 routi~ely used for cardiovascular ~ur~ery ~ ord~r to oxygenate 25 blood. Plat~lets adh~re to surfaces o~ the extracospcreal circult. Adhe8ion ~8 depe~dent on the ~nteract~o~ between GPIlb/llIa o~ the platelet membrane~ and fibrinoge~ ad~orbet to the ~ur~ace o~
the circuit. ~Glu~z~o et al., 1987, 2~ . pp 61S-621). ~l~telet~ released ~ro~
artificial ~urface~ show impaired hemoætatlc ~ ~s/

7652P/ - 16 - 17~86 7t28/89: Fl function. Polypeptide~ of the invention may be admini~tered to prevent adhe6ion.
Other applications of these polypeptites include prevention of platelet thro~bosi6, thrombo-embolism and r~occlu~ion duri~g and after thrombo-lytic ther2py and prevention of pla~elet thrombosi6,thromboemboli~m and reocclu~ion after angioplasty of coronary and other arteries and after coronary artery ~ypa~6 procedures. Polypeptide~ of the inv~ntion ~ay also be u~ed to prevent myocardial i~farction.
The~e polypeptides ~ay be ad~ini~tered by any convenient ~ean6 which will re~ult in it~
delivery into the blood stream in ~ub~tantial amount including contanuou~ intra~enou~ or bolu~ injection os oral ~ethod~. Compo~ition~ o~ the invention 15 include peptide~ of the invention and pharmacologically acceptable carriers, e.~. ~aline, at a p~ level e.g. 7.4, ~uitable for achieving inhibition of platele~ aggregation. They ~ay be combined with thrombolytic agents such a6 pla~minogen 2~ activators or ~tre~tokina~e in order to inhi~it platelet aggregation. They may also be com~ined with anticoagulant~ such as heparin, aspirin or warfaran.
Intravenous admini~tration i8 pre~ently contemplated ~ the preferred ad~ini~tration route. Th~y are soluble in water, a~d ~ay there~ore ~e effectively adminl~tered ~n solutio~.
In one exemplary application, a ~u~table amount of peptide iB i~travenouBly adminlstergd to a heart attack YiCti~ undergolng angio~la~ty.
Admin~stration OCCUrB duri~g 9r ~everal minutes prior to angioplasty, and i8 in an amount sufficient to `3f 7/28/89: Fl inhibit platelet aggregation, e.g. an am~unt which achieves a ~teady R~ate pla~ma concentration of ~etween about 0.05-30 ~M per kilo, preferably between about 0.3-3 ~M per kilo. When thi~ amount iB achieved, an infusisn of between ~bout 1-100 ~M
per kilo per ~in., preferably between about 10-30 ~M per ~ilo per min. i8 maintained to inhibit platelet aggregation. Should ~he pat~e~t aeet to undergo bypa~ surgery, administr~tion may ~e ~t~pped immediately and will n~t cause c~mp~ications during lo surgery that would be caused by other material6 such as a~pirin or ~onoclonal antibodies, the effect6 of which last hour~ after ces~ation o~ administration.
The pre6ent invention alEo include~ a pharmaceutical compo~iti~n eompri~ing peptides of the 15 present invention and ti~ue-type pla~minogen activator or ~treptokina6e~ The invention al~o include6 a ~ethod ~or promoting thromboly~i~ and preventing re~cclu6i~n in a p~tient whic~ compri6ex admini tering to the patient an e~fective amount of 20 compositions of the invention.
The p~e~ent invention may ~e em~odied iD
other ~pecific f~rms with~ut departing from the fpirit or e~sential attribute~ thereo~. Thu~, the ~pecific examples de~cr~bed above should not ~e interpreted as li~iting the scope of the pre~ent ~nvent~o~.

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Claims (11)

7/28/89: F1 WHAT IS CLAIMED IS:

1. A fibrinogen receptor antagonist compound of the following formula:
A-B-C-Gly-Asp-D-E (I) wherein:
A is any L- or D-isomer of a .alpha.-amino acid, an acylated .alpha.-amino acid, a des-.alpha.-amino acid or an N-methyl-.alpha.amino acid.
B is an L-isomer of a secondary amino acid selected from the group consisting of proline, hydroxyproline, thioproline, .beta.,.beta.-dimethyl-thioproline, dehydroproline, pipecolic acid, azetidine carboxylic acid and N-methyl amino acids;
C is an L-isomer of arginine, homo-arginine, guanido aminobutyric acid, or guanido aminopropionic acid;
D is an L-isomer of tryptophan, phenylalanine, leucine, valine, isoleucine, napthylalanine, methionine, tyrosine, or ring substituted derivatives of tryptophan, tyrosine, phenylalanine, arginine, homo-arginine, ornithine, lysine or histidine; and E is OH, NH2, NHR, NR1R2, wherein R is an alkyl group having 1 to 4 carbon atoms, and R1R2 represents an alkyl group having 1 to 4 carbon atoms, a secondary amino acid, or 7/28/89: Fl

2. A figrinogen receptor antagonist of claim 1 wherein E is OH

3. A fibrinogen receptor antagonist of Claim 2 wherein:.
A is L-asparagine, D-as6paragine or acylated asparagine;
B is an L-isomer of proline, thioproline, .beta.,.beta.-dimethylthioproline, or N-methylalanine;
C is arginine;
D is phenylalanine or tryptophan; and E is OH.

4. A fibrinogen receptor antagonist of Claim 1 which is Asn-Pro-Arg-Gly-Asp-Phe-OH.

5. A fibrinogen receptor antagonist of Claim 1 which is Asn-ThioPro-Arg-Gly-Asp-Phe-OH.

6. A fibrinogen receptor antagonist of Claim 1 which is Asn-(.beta.,.beta.-dimethylThioPro)-Arg Gly-Asp-Phe-OH..

7. A flirinogen receptor antagonist of Claim 1 which is (D-Asn)-Pro-Arg-Gly-Asp-Phe-OH.

8. A fibrinogen receptor antagonist of Claim 1 which is (AcAsn)-Pro-Arg-Gly-Asp-Phe-OH.

7/28/89: Fl

9. A fibrinogen receptor antagonist of Claim 1 which is Asn-Pro-Arg-Gly-Asp-Trp-OH.

10. A composition for inhibiting fibrinogen-induced aggregation in a mammal comprising a peptide of claim 1 and a pharmaceutically acceptable carrier.

11. A method for inhibiting fibrinogen binding to mammalian platelets comprising administering to a patient a composition of claim 10.