WO1993000108A1 - Novel inhibitors of platelet aggregation - Google Patents

Novel inhibitors of platelet aggregation Download PDF

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Publication number
WO1993000108A1
WO1993000108A1 PCT/US1992/005418 US9205418W WO9300108A1 WO 1993000108 A1 WO1993000108 A1 WO 1993000108A1 US 9205418 W US9205418 W US 9205418W WO 9300108 A1 WO9300108 A1 WO 9300108A1
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Prior art keywords
gly
cys
orn
arg
pen
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PCT/US1992/005418
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French (fr)
Inventor
William Charles Ripka
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Corvas International, Inc.
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Publication of WO1993000108A1 publication Critical patent/WO1993000108A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0227—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the (partial) peptide sequence -Phe-His-NH-(X)2-C(=0)-, e.g. Renin-inhibitors with n = 2 - 6; for n > 6 see C07K5/06 - C07K5/10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to novel peptide derivatives having activity as inhibitors of platelet aggregation.
  • platelet aggregation is an essential process in normal blood clotting, and serves as a defense mechanism after injury in the prevention of excessive bleeding, in certain disease states (e.g., myocardial infarction) it can become uncontrolled and lead to restriction or total blood flow blockage. Additionally, fibrinolysis resulting from treatment with thrombolytic agents such as tissue plasminogen activator (tPA) or streptokinase can also result in reocclusion that occurs in part as a result of platelet aggregation.
  • tissue plasminogen activator tPA
  • streptokinase can also result in reocclusion that occurs in part as a result of platelet aggregation.
  • a primary cause of platelet aggre ⁇ gation is the involvement of certain glycoprotein recep- tors known as integrins or cytoadhesins which bind to adhesive proteins such as fibrinogen, fibronectin, von Willebrand factor, and vitronectin. Agents that block this interaction are expected to have therapeutic value particularly in certain disease states and in the reocclu- sion that occurs after angioplasty and fibrinolysis.
  • Certain snake venoms have been reported to possess small proteins that have platelet aggregation inhibitory properties. In certain cases, these proteins are proposed to inhibit platelet aggregation by the antagonism of the gpIIb/IIIa receptor by virtue of the completely conserved Arg-Gly-Asp subsequence present in each of the proteins. (See, Dennis et al., Proc. Natl. Acad Sci. (USA) 87:2471- 2475 (1989); and Seymour et al., J. Biol. Chem. 265:10143- 10147 (1990)).
  • the present invention is directed to the surprising finding that the otherwise invariant aspartic acid in these peptide sequences can be replaced by orni ⁇ thine and yet retain cell adhesion and/or platelet aggre ⁇ gation inhibitory activity.
  • This is particularly surpris ⁇ ing since ornithine is an amino acid residue differing both in size and charge from aspartic acid (the negatively charged carboxyl group of Asp is replaced with an amine which would be protonated and positively charged and the side chain is extended by one methylene) and in view of findings that replacement of aspartic acid with glutamic acid eliminated platelet aggregation activity.
  • This invention is directed to the use of ornithine in any peptide or peptide mimic that derives its biological activity by using an acidic residue such as Asp to bind to an RGD receptor.
  • the present inven ⁇ tion provides novel peptide compounds having activity in inhibiting fibrinogen binding, platelet aggregation and/or glycoprotein Ilb/IIIa binding (collectively referred to as "aggregation inhibiting activity") which comprises a cyclic or rigid peptide including as an aggregation inhib ⁇ iting constituent an amino acid sequence which comprises
  • A is an L or O- isomer of arginine, homoarginine, guanidinoaminobutyric acid, guanidinoaminopropionic acid, N',N"dimethylarginine, N' ,N"-diethylarginine, p-amino- ethyl-phenylalanine, p-amidinophenylalanine, p-guanidino- phenylalanine, a conformationally constrained arginine analog as described by T. R. Webb and C. Eigenbrot, J. Org. Chem.
  • the present invention also includes peptides wherein the Orn of formula (I) is replaced by a 1-methyl Orn.
  • Such peptides typically have an IC 50 ( ⁇ ,M) of no more than about 500 in an assay of thrombin-induced platelet aggregation.
  • Preferred are peptides having less than about 20 amino acid residues, more preferably having from about 6 to about 14 amino acid residues.
  • cyclic peptides that is, peptides wherein a side chain of an amino acid residue on the amino terminal side of -A- is covalently linked to a side chain of an amino acid residue on the carboxy terminal side of -Orn- or cyclic peptides having a cyclic peptide backbone.
  • Covalent linkages between side chains of amino acids may include disulfide linkages between sulfhydryl groups, such as naturally occurring disulfide bridges between cysteine residues, or disulfide bridges between other sulfhydryl- containing amino acids (e.g. penicillamine) or analogs.
  • interamino acid linkages include linkages between (a) alkyl acids and alkyl amines which may form cyclic lactams; (b) alkyl aldehydes or alkylhalides and alkylamines which may condense and be reduced to form an alkylamine bridge; (c) side chains which may be connected to form an alkyl, alkenyl, alkynyl, ether or thioether bond.
  • Preferred alkyl chains include lower alkyl groups having from about 1 to about 6 carbon atoms; or (d) disul ⁇ fide bonds between a thio- containing acyl group on the N-terminus and a cysteine or penicillamine.
  • the present invention is directed to a peptide having the tripeptide sequence of formula I and a rigid, for example, cyclized, structure which may result from a modification in amino acid sequence of a peptide having platelet aggregation inhibitory properties wherein the aspartic acid of an A-Gly-Asp sequence (wherein A is a basic amino acid as described with formula I) is replaced with ornithine to give a peptide having improved inhibitory and/or pharmacokinetic properties.
  • A-Gly-Asp sequence wherein A is a basic amino acid as described with formula I
  • Such pep ⁇ tides which are modified with unnatural amino acids, as well as cyclic versions are considered to fall within the scope of the present invention. Examples of unnatural amino acids are described in T.R. Webb, C. Eigenbrot, J. Org. Chem.
  • Also considered to be within the scope of the present invention are compounds having activity in inhibiting fibrinogen binding or glycoprotein Ilb/IIIa binding in which the aspartic acid of an Arg-Gly-Asp sequence has been replaced by ornithine.
  • polypeptides and smaller cyclic versions of such peptides in which the aspartic acid of the RGD sequence has been replaced with ornithine are considered to fall within the scope of this invention.
  • one such peptide would have the sequence: NH2-ECESGPCCRNCKFLKEGTICKRARG-Om-DMDDYCNGKTCDCPRNPHKGPA T-OH (See EPO 382,538 to Garsky).
  • the invention further provides compositions compris- ing one or more of the foregoing compounds, and methods of using such compounds or compositions in inhibiting fibri ⁇ nogen binding, platelet aggregation, and/or glycoprotein Ilb/IIIa binding.
  • the subject compounds or compositions are also effective in the treatment of certain physiologi- cal conditions, such as thrombosis and/or cancer metas ⁇ tasis, and the present invention is further directed to these uses.
  • the present invention comprehends peptides or small peptides accessible by synthetic routes compris- ing an amino acid sequence corresponding to the amino acid sequence of the variable region of the heavy chain of monoclonal antibody PAC-1, which incorporates the sequence of formula (I) as well as antibodies incorporating that sequence.
  • Pen refers to L-penicillamine or ⁇ , ⁇ -dimethyl cysteine.
  • HCys refers to homocysteine
  • (Me)-Arg refers to N' ,N"-dimethylarginine.
  • Amb refers to aminoisobutyric acid.
  • Ambu refers to aminobutyric acid.
  • Aha refers to 7-aminoheptanoic acid.
  • Boc refers to tert-butyloxycarbonyl.
  • “Bz” refers to benzoyl.
  • DIEA diisopropylethylamine
  • D.I. water refers to deionized water.
  • DMF dimethylformamide
  • (Et 2 )Arg refers to N' ,N"-diethylarginine.
  • Fluorenyl refers to 9-fluorenylmethyloxycarbonyl.
  • HBTU refers to 2-(lH-benzotriazol-l-yl)-l,l,3,3 -tetramethyl uronium hexafluorophosphate.
  • Hys refers to homocysteine.
  • HMP refers tohydroxymethylphenoxymethylpolystyrene resin.
  • HOAc refers to acetic acid
  • HOBT refers to 1-hydroxybenzotriazole.
  • MBHA 4-methylbenzhydralamine resin.
  • Mzl 4-methylbenzyl.
  • MeArg refers to N- ⁇ -methylarginine.
  • (Me 2 )Arg refers to N' ,N"-dimethylarginine.
  • MeOSuc refers to methoxysuccinyl.
  • NMP refers to N-methylpyrrolidine.
  • PBS refers to a buffer of pH of about 7.4 containing 10 mM sodium phosphate and 150 mM sodium chloride.
  • Pen refers to L-penicillamine.
  • PMC refers to 2,2,5,7,8-pentamethylchroman-6- sulfonyl.
  • RINK refers to ((dimethoxyphenyl-Fmoc-aminomethyl)- phenoxy) resin.
  • Tos refers to tosyl or 4-toluenesulfonyl.
  • TFA refers to trifluoroacetic acid.
  • ThioPro refers to L-thioproline.
  • TRT refers to trityl.
  • Tetyrode's Buffer refers to buffer of pH of about 7.4 containing 150 mM sodium chloride, 2.7 mM potassium chloride, 12 mM sodium bicarbonate, 0.4 mM sodium phosphate.
  • ⁇ -R* substituted derivatives of amino acids which may also be denoted as ( ⁇ -R')AA, indicate amino acids which are monosubstituted on the C-alpha of the amino acid by R' wherein R' is alk (alkyl) or benzyl.
  • conformationally constrained arginine analog refers arginine analogs such as those disclosed by Webb et al., “Conformationally Restricted Arginine Ana ⁇ logues", J. Org. Chem. 56:3009-3016 (1991), and, in par ⁇ ticular includes the analogues described therein.
  • alkyl refers to saturated aliphatic groups, including straight, branched and carbocyclic groups.
  • lower alkyl refers to both straight- and branched-chain alkyl groups having a total of from 1 to 6 carbons atoms and includes primary, secondary and tertiary alkyl groups.
  • Typical lower alkyls include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl, and the like.
  • aryl refers to aromatic groups having from about 6 to 14 carbon atoms and includes cyclic aromatic systems such as phenyl and napthyl.
  • aralkyl refers to an alkyl group from about 1 to 4 carbon atoms substituted with an aryl of from 6 to 10 carbon atoms and includes, form example, benzyl, p-chlorobenzyl, p-methylbenzyl and 2-phenylethyl.
  • FIG. 1 depicts a plot of inhibition of HRP-Fibrinogen binding to GPIIb/IIIa by the Elisa-based assay of Example
  • FIG. 2 depicts a plot of inhibition of HRP-Fibrinogen binding to GPIIb/IIIa by the methods of Example A and shows inhibition by the compound of Example 5 in compari- son to a RGD compound.
  • FIG. 3 depicts a plot of inhibition of platelet aggregation by the compound of Example 16 in an assay of
  • Example C at two concentrations, (a) 250 ⁇ M and (b) 500 ⁇ M, the bottom curve showing the inhibited aggregation by the compound of Example 16.
  • the present invention encompasses peptide compounds having the tripeptide sequence depicted in Formula (I) and physiologically acceptable salts thereof.
  • These compounds are characterized by their inhibitory potency, namely their ability to retard or prevent one or more of the following: the binding of the adhesive protein fibrinogen to blood platelets (referred to herein as inhibiting "fibrinogen binding") , the binding of blood platelets to themselves (referred to herein as inhibiting "platelet aggregation”) , and/or the binding of compounds or substances, particularly proteins, to the glycoprotein Ilb/IIIa complex found in blood platelet mem ⁇ branes (referred herein as inhibiting "glycoprotein lib/ Ilia binding”) .
  • the subject compounds are also useful in retarding or preventing the formation of blood clots or thrombi (referred herein as inhibiting "thrombosis”) and/or the spread of cancer cells throughout the body (referred to herein as inhibiting "cancer metastasis”) .
  • inhibiting thrombosis
  • cancer metastasis cancer metastasis
  • the orni ⁇ thine residue in the key tripeptide region, of formula (I) of the present invention is in the L-configuration.
  • the remaining amino acid residues of the peptides of the pres ⁇ ent invention can be present in either the D- or the L- configuration.
  • Amino acid residues may be linked among themselves, or with each other, in branched, cyclic or straight chain form, although cyclic chain linkages are preferred.
  • branched or cyclic chains may be produced by the formation of a peptide bond with amino acid side groups that contain amino or car- boxyl moieties.
  • Amino acids containing such side groups include, for example, glutamic acid (carboxyl group) , aspartic acid (carboxyl group) and lysine (amine group) .
  • Branched or cyclic chains may also be produced through formation of a covalent disulfide bond between amino acid residues having sulfur-containing side groups, such as cysteine, homocysteine and penicillamine.
  • the present invention is directed to novel and very active compounds that contain the tripep ⁇ tide sequence set forth in formula (I) , linked from amino- to carboxy-terminus.
  • the present invention is based upon the surprising finding that, in contrast to previous platelet aggregation inhibitors that require an aspartic acid in the third position of a RYD or RGD sequence, effective and potent inhibitors can be obtained by replacement of the Asp with Orn, an amino acid that differs substantially from aspartic acid in both size and charge.
  • preferred classes of compounds include cyclic peptides having interamino acid peptide linkages of the formula:
  • cyclic peptides having a cyclic peptide back bone of the formula:
  • analogs of the foregoing compounds which include compounds comprising less common or modified amino acids, for example, hydroxyproline, hydroxylysine, cystine, thyroxine, norleucine, pyroglutamic acid or other amino acid derivatives which are capable of incorporation into the peptides of the present invention.
  • Preferred compounds are:
  • the present invention also includes physiologically acceptable salts of the peptides of the present invention.
  • These salts include acid addition salts, for example salts of hydrochloric acid, hydrobromic acid, acetic acid, tri- fluoroacetic acid, citric acid, succinic acid, benzene sulfonic acid or other suitable acid addition salts.
  • acid addition salts for example salts of hydrochloric acid, hydrobromic acid, acetic acid, tri- fluoroacetic acid, citric acid, succinic acid, benzene sulfonic acid or other suitable acid addition salts.
  • the branched, cyclical and straight chain peptides of the present invention can be synthesized using conven ⁇ tional preparative and recovery methods known to those skilled in the art.
  • novel peptides of this invention can be made by conventional methods of peptide synthesis.
  • a "protected" terminal amino group refers to a terminal amino group coupled with any of various amino-terminal protecting groups traditionally employed in peptide synthesis.
  • suitable groups include acyl protecting groups, for example, formyl, acetyl, benzoyl, trifluoroacetyl, succinyl and methoxysuccinyl aromatic urethane protecting groups, for example, benzyloxycarbonyl and aliphatic urethane protect ⁇ ing groups, for example, tert-butyloxycarbony1, adamantyl- oxycarbonyl or fluorenylmethyloxycarbonyl groups.
  • Numer ⁇ ous suitable amino terminal protecting groups are known. See, e.g.. Gross and Mienhofer, eds., The Peptides, Vol. 3, pp 3-88 (Academic Press, New York, 1981) .
  • Other suitable protecting groups are known to those skilled in the art.
  • F oc 9-fluorenymethyloxycarbonyl
  • Ac acetyl
  • Z benzyloxycarbonyl.
  • the amino acid residues of the substituents having side chain amino groups or other reactive groups, for example Lys or Arg, may optionally comprise suitable amino- protecting groups or other functional groups protecting groups attached to the side chains.
  • protected terminal car ⁇ boxyl group refers to a terminal carboxyl group coupled with any of various carboxy-terminal protecting groups.
  • suitable groups include tert-butyl, benzyl or other protecting groups linked to the terminal carboxyl group through an ester or ether bond. Amino acid residues of substituents having acidic or hydroxy side chains may be similarly protected.
  • a preferable synthesis route for the straight-chain peptide intermediates, especially the smaller peptides (of shorter chain length, that is, having from about 3 to about 50 amino acid residues) of the invention is the solid phase method.
  • This method is well known in the art and is described in references such as Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963); Science 150:178-185 (1965); and Science 212:341-347 (1986); Vale et al., Science 211:1394-1397 (1981); and Marke et al., J. Am. Chem. Soc. 101:3178 (1981); the disclosures of which are incorporated herein by reference.
  • Other preparative methods which may be employed include the processes of Houghten, Proc. Natl. Acad. Sci(USA) 82.:5132 (1985).
  • EPO application 382,538 describes the solid phase synthesis of a 49-amino acid cysteine rich peptide. The above disclosures of are also incorporated here
  • Solid phase peptide synthesis is generally commenced from the C-terminus of the peptide by coupling a protected ⁇ -amino acid to a suitable resin, such as a hydroxymethyl- phenoxymethyl polystyrene resin (HMP) or a RINK ([dimeth- oxyphenyl-Fmoc aminomethyl]-phenoxy) resin when synthesiz- ing a peptide amide.
  • HMP hydroxymethyl- phenoxymethyl polystyrene resin
  • RINK [dimeth- oxyphenyl-Fmoc aminomethyl]-phenoxy
  • guanidino group of argi ⁇ nine may be protected by a penta ethyl chroman-6-sulfonyl (PMC) group.
  • PMC penta ethyl chroman-6-sulfonyl
  • TRT trityl
  • the beta carboxyl group of aspartic acid is blocked with a tert-butyl ester.
  • the sulfhydryl groups of cysteine and penicillamine are protected with an acetamidomethyl (ACM) group.
  • Ornithine and lysine are protected on the side chain amine with the t-butoxycarbonyl (BOC) group.
  • Each ⁇ -amino group may be protected with the Fmoc group.
  • the peptide sequence is synthesized by sequential coupling of ⁇ -amino protected amino acids to the a ino- terminal and of the growing peptide chain attached to the solid support. After the desired peptide sequence is complete, the intermediate peptide is cleaved from the resin and the protecting groups are removed with a reagent such as trifluoroacetic acid (TFA) . The peptide is iso ⁇ lated from the TFA solution by techniques such as filtra ⁇ tion, centrifugation or extraction with diethyl ether. The peptide can then be purified by high performance liquid chromatography (HPLC) or other such methods of protein purification.
  • HPLC high performance liquid chromatography
  • the present invention further provides compositions and methods for using these compounds and compositions in inhibiting fibrinogen binding, platelet aggregation, and glycoprotein Ilb/IIIa binding.
  • the ability of the com ⁇ pounds of the present invention to inhibit the foregoing activities makes them useful in inhibiting the physiologi ⁇ cal process of thrombosis.
  • the compounds of the present invention may be employed in inhibiting cancer metastasis (an aberrant physiological phenomenon that is believed to require the adhesion of blood platelets to the cancer cells) .
  • cancer metastasis an aberrant physiological phenomenon that is believed to require the adhesion of blood platelets to the cancer cells.
  • the specific activities of the compounds of pres ⁇ ent invention in carrying out these related functions makes them particularly useful as therapeutic and/or diagnostic agents.
  • the platelet-binding inhibitor activity of the pep ⁇ tide derivatives of this invention may be demonstrated by various assays.
  • the peptides are tested for their inhibition of thrombin-induced plate ⁇ let aggregation in washed human platelets. The percent inhibition is determined for the test peptide by comparing the extent of platelet aggregation in the presence of and absence of the peptide.
  • platelet aggregation is examined in platelet-rich plasma which also is rich in fibrinogen and other plasma proteins.
  • compositions of the present invention comprise an effec ⁇ tive amount of a compound of Formula I and a physiological acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic, diagnostic or other uses are well known in the pharmaceutical art, and are described, for example in Remington's Pharmaceutical Sciences, Gennaro, A. R. , ed. (Mack Publishing Co., Easton PA, 1985) .
  • the co - pounds or compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents.
  • These compounds can be utilized in vivo, ordinarily in a mammal, preferably in a human, or in vitro.
  • the compounds or compo- sitions can be administered to the mammal in a variety of ways, including parenterally, intravenously, subcutane- ously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms.
  • the useful in vivo dosage to be administered and the particu ⁇ lar mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed.
  • the determination of effective dosage levels that is, the dosage levels necessary to achieve the desired result, will be within the ambit of one skilled in the art.
  • applications of com- pound are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved.
  • the compounds of the present invention may be used in conjunction with fibrinolytic therapy for therapeutic indi ⁇ cations such as to prevent reocclusion of a blood vessel following fibrinolytic therapy.
  • fibrinolytic agents such as plasminogen activators including arokinase, pro- urokinase, streptokinase and tissue plasminogen activator as well as derivatives or variants thereof.
  • Antibodies both monoclonal and polyclonal, directed to peptide compounds of the present invention are useful for diagnostic purposes and for the identification of concentration levels of the subject peptides in plasma.
  • any one of a number of conventional techniques which are known in the art can be employed.
  • antibody-containing blood called anti- serum
  • anti- serum antibody-containing blood
  • the compound-specific antibody is isolated from other antibodies in the antiserum by any one of a number of separation techniques (for example, affinity chromatography) .
  • Monoclonal antibodies may be prepared using the technique of Kohler and Milstein, Nature 256:495-497 (1975) and other conven- tional techniques known to those skilled in the art. (See, e.g.. Harlow and Lane, Antibodies a Laboratory Manual (Cold Spring Harbor Laboratory, 1988) .
  • the peptides of this invention were synthesized by the solid phase method using an Applied Biosystems Inc. 431A automated synthesizer. Fmoc amino acids and resins supplied by Calbiochem and Bachem, Inc. were used. - The FASTMOCTM HBTU 2-(lH-benzotriazol-l-yl) 1,1,3,3 tetra ⁇ methyl uronium hexafluorophosphate ⁇ protocol developed by Applied Biosystem Inc was employed as the coupling proto- col (ABI User Bulletin #33 Nov 1990) .
  • novel peptide derivatives of this invention were made by the solid phase synthesis.
  • 0.25 grams of Rink resin containing 0.4 mmoles of amino groups were mixed with 10 equivalents (eq) of F oc-Cys(Acm)-OH , 10 eq of 0.45 M HBTU-HOBT-DMF solution, 1.7 eq of DIEA and 0.8 ml of N-Methylpyrrolidine (NMP) for 9 minutes.
  • the resin was filtered and washed repeatedly with NMP.
  • the Fmoc group was removed by treatment with 50% Piperidine and 50% NMP for 7 minutes and again washed repeatedly with NMP.
  • the resin was then ready for reaction with 10 eq of the next amino acid.
  • the cycle as above described was repeated for each amino acid in the sequence.
  • the resulting peptide was removed from 400 milligrams of resin and deprotected with a cleavage mixture of 10.0 ml TFA; 0.5 ml thioanisole, 0.5 ml D.I. water, 0.25 ml ethanedithiol and 0.75 grams of phenol.
  • the cleavage reac ⁇ tion was done at room temperature for 1.5 hours. In case of cyclic peptides thallium trifluoroaceate was used. After the TFA was evaporated, the peptide was isolated by diethyl ether precipitation and taken up in 10% acetic acid and lyophilized.
  • the peptide product was purified on HPLC on a Waters Delta Pack C-18 reverse phase column using a 0-25% gradient in acetonitrile (0.1%(TFA). Frac- tions containing the product, as ascertained by analytical HPLC, were pooled and lyophilized to yield about 50 milli ⁇ grams of pure heptapeptide from 0.4 grams of resin.
  • the cleaved resin is removed by filtration through sintered glass and the TFA solution concentrated on a rotary evaporator in vacuo. Water was added to the residue and the mixture was extracted twice with ether. The aqueous layer was filtered through a 0.2 ⁇ nylon filter and submitted to preparative high pressure liquid chromatography for purification. A gradient sol ⁇ vent mixture was used which was varied from 0.1% TFA in water to 0.1% TFA in acetonitrile over 50 minutes. The appropriate fraction was detected at 214 nm with an ISCO UA-5 Absorbance detector and was collected. The aceto ⁇ nitrile solvent from the collected fractions was removed in vacuo and the resulting pure cyclic peptide lyophilized to yield a white powder.
  • Fmoc-Gly-O-HMP was coupled by standard deprotection and coupling procedures described above successively with Fmoc-Arg(Tos) , Fmoc-Aha, and t-Boc-Orn(Cbz) .
  • the alpha-amino Fmoc groups are removed by treatment with 50% piperidine and 50% N-methylpyrolli- dine (NMP) .
  • Couplings were effected with dicyclohexyl- carbodiimide, deprotection with piperidine:NMP, and wash- ing the peptide-resin with NMP.
  • Cbz-protected-t-Boc-Orn was used in the final coupling reaction.
  • the protected peptide was cleaved from the resin by the trifluoroacetic acid procedure described above. This procedure ultimately yielded: Cbz Tos
  • linear peptide was then treated with a mixture of N-ethy1-N'-(3-dimethylaminopropyl)carbodiimide, 1-hydroxy- benzotriazole, dimethylformamide andN-methyImorpholine to form:
  • the product cyclized peptide was purified on HPLC using a 0.1% TFA H 2 0-acetonitrile gradient.
  • the TFA salt can be converted to an HOAc salt by passing a solution of the peptide through an ion exchange column BioRad AG3-X4A (acetate cycle) .
  • the alpha-amino Fmoc protecting group was removed (Cys-SH protected with Acm) using 50% piperidine and 50% NMP.
  • Fmoc-protected Orn (Boc protected) was coupled to the cys ⁇ teine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yielded the free amino terminus. Following this stepwise procedure of coupling with dicyclohexylcarbodiimide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Gly, Arg, and Cys residues were coupled in succession. Arg was additionally protected with a PMC group and the final Cys residue was addition- ally protected by an Acm group. The final Cys was acety- lated with acetic anhydride. This yielded the resin coupled peptide:
  • This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid.
  • the cyclized peptide product was purified on HPLC in a 0.1% TFA H20-acetonitrile gradient.
  • the TFA salt produced could be converted to the HOAc salt by pass ⁇ ing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) .
  • the finished peptide was:
  • Fmoc-Cys-O-HMP The alpha-amino Fmoc protecting group was removed (main ⁇ taining protection of Cys with the Acm group) using pipe ⁇ ridine and NMP.
  • Fmoc-protected Orn (Boc) was then coupled to cysteine with dicyclohexylcarbodiimide followed by deprotection again with piperidine and NMP.
  • Fmoc-pro ⁇ tected Gly, Arg(PMC) , Pro, Asn(TRT) , Cys(Acm) residues were coupled in succession.
  • Arg pentamethyl chroman-6-sulfonyl
  • Asn(TRT) Asn with trityl
  • Cys(Acm) the final Cysteine with an Acm group
  • the final Cys was acety- lated with acetic anhydride.
  • Mass spectral analysis using FAB showed a mass peak of Mw. 802.5 (calculated 802.3).
  • the alpha-amino Fmoc protecting group was removed (Cys SH protected with Acm) using 50% piperidine and 50% NMP.
  • Fmoc-protected Ser(tBu) was coupled to the cysteine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yields the free amino terminus. Following this stepwise procedure of coupling with dicyclohexylcarbodiimide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Orn (Boc), Gly, MeArg (PMC), and Cys(Acm) residues are coupled in succession.
  • MeArg was additionally protected with a PMC group and the final Cys residue was addition ⁇ ally protected by an Acm group.
  • the final Cys is acety- lated with acetic anhydride. This yields the resin coupled peptide: Acm PMC Boc tBu Acm
  • This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid.
  • the cyclized peptide product is purified on HPLC in a 0.1% TFA H 2 0-acetonitrile gradient.
  • the result ⁇ ing TFA salt can be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) .
  • the finished peptide is:
  • the alpha-amino Fmoc protecting group was removed (Pen-SH protected with Acm) using 50% piperidine and 50% NMP.
  • Fmoc-protected Orn was coupled to the penicillamine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yielded the free amino terminus. Following this stepwise procedure of coupling with dicyclohexylcarbodi ⁇ imide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Gly, Arg, and Pen residues were coupled in succession.
  • This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid.
  • the cyclized peptide product was purified on HPLC in a 0.1% TFA H20-acetonitrile gradient.
  • the TFA salt produced could be converted to the HOAc salt by pass- ing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) .
  • the finished pep ⁇ tide was:
  • the protected peptide-resin intermediate N°-Ac-Cys (SEt)-MeArg(Tos)-Gly-Orn(Cbz)-Cys(4-MBzl)-MBHA, is syn ⁇ thesized by solid phase methods.
  • the amino acids, pro ⁇ tected as the t-Boc derivatives, are coupled successively using N,N-dicyclohexylcarbodiimide/ HOBt and deprotection of the N-terminal protecting groups in each step with TFA. After the last amino acid is coupled the peptide is acety- lated with acetic anhydride and diisopropylethylamine in DMF.
  • the peptide is cleaved from the resin with deprotec- tion of the side chain protecting groups using anhydrous HF in the presence of anisole at 0°. After evaporation of HF in vacuo, the residue is washed with anhydrous ether, and the crude peptide is extracted with 50% acetic acid, then diluted with deionized water. The pH of the aqueous solution is adjusted to 7.5 with concentrated ammonium hydroxide. The basic conditions causes the free thiol generated by removal of the 4-MBzl group from the cysteine to displace the mercaptoethyl protecting group of the second cysteine to cause cyclization of the peptide.
  • the cyclized peptide product is purified on HPLC in a 0.1% TFA H 2 0-acetonitrile gradient.
  • the TFA salt which is produced can be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) .
  • the finished pep ⁇ tide is:
  • the protected peptide-resin intermediate N ⁇ -Ac-Pen (4-MBzl)-MeArg(Tos)-Gly-Orn(Cbz)-Pen(4-MBzl)-MBHA is prepared, cleaved and isolated in the same manner as described in Example 10 above.
  • the peptide is cyclized using a 0.01% K 3 Fe(CN) 5 solution.
  • the cyclized peptide product is purified on HPLC in a 0.1% TFA H 2 0-acetonitrile gradient.
  • the TFA salt produced can be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) .
  • the finished peptide is:
  • the alpha-amino Fmoc protecting group is removed (Cys-SH protected with Acm) using 50% piperidine and 50% NMP.
  • Fmoc-protected Orn (Boc protected) is coupled to the cys ⁇ teine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yielded the free amino terminus. Following this stepwise procedure of coupling with dicyclohexylcarbodiimide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Gly, Arg, Pro and Cys residues were coupled in succession. Arg is additionally protected with a PMC group and the final Cys residue is additionally pro ⁇ tected by an Acm group. The final Cys is acetylated with acetic anhydride. This yields the resin coupled peptide:
  • This peptide is cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacete, anisole, and trifluor- acetic acid.
  • the cyclized peptide product was purified on HPLC in a 0.1% TFA H 2 0- acetonitrile gradient.
  • the TFA salt produced could be converted to the HOAc salt by pass ⁇ ing a solution of the peptide through an ion exchange column BioRad Ag3-X4-X4A (acetate cycle) .
  • the finished peptide is:
  • the alpha-amino Fmoc protecting group is removed (Pen-SH protected with Acm) using 50% piperidine and 50% NMP.
  • Fmoc-protected Orn (Boc protected) is coupled to the penicillamine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yielded the free amino terminus. Following this stepwise procedure of coupling with dicyclohexylcarbodii ⁇ mide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Gly, Arg(PMC) , and Cys(Acm) resi- dues were coupled in succession. Arg is additionally pro ⁇ tected by a PMC group and the Cys with an Acm group. The final Cys is acetylated with acetic anhydride. This yields the resin coupled peptide:
  • This peptide is cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid.
  • the cyclized peptide product was purified on HPLC in a 0.1% TFA H 2 0- acetonitrile gradient.
  • the TFA salt produced could be converted to the HOAc salt by pass ⁇ ing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) .
  • the finished pep- tide is: Acetyl-Cys-Arg-Gly-Orn-Pen-NH 2
  • the alpha-amino Fmoc protecting group was removed (Cys-SH 15 protected with Acm) using 50% piperidine and 50% NMP.
  • Fmoc-protected Orn was coupled to the cysteine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yielded the free amino terminus. Following this stepwise 20 procedure of coupling with dicyclohexylcarbodiimide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Gly, Arg(PMC), Cys(Acm), Gly, and Tyr(tBu) residues were coupled in succession.
  • This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoracetate, anisole, and trifluoro- 35 acetic acid.
  • the cyclized peptide product was purified on HPLC in a 0.1% TFA H 2 0-acetonitrile gradient.
  • the TFA salt produced could be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4a (acetate cycle) .
  • the finished peptide is:
  • This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoracetate, anisole, and trifluoro- 35 acetic acid.
  • the cyclized peptide product was purified on HPLC in a 0.12% TFA H 2 0-acetonitrile gradient.
  • the TFA salt is produced by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) .
  • the finished peptide is:
  • Fmoc-Cys-NH-RINK The alpha-amino Fmoc protecting group was removed (main ⁇ taining protection of Cys with the Acm group) using pipe ⁇ ridine and NMP.
  • Fmoc-protected Pro was then coupled to cysteine with dicyclohexylcarbodiimide followed by depro ⁇ tection again with piperidine and NMP.
  • deprotection with piperidine/NMP Fmoc-protected Ser(t-Bu), Orn(t-Boc), Gly, Arg(PMC) , Gly, Pen(Acm) , and Gly residues were coupled in succession.
  • the Arg was additionally protected by penta ethyl chroman-6-sulfonyl (Arg(PMC)), the Ser with t-butyl, the Orn with t-Boc, and the penicillamine with an Acm group.
  • the following pep ⁇ tide resin was formed:
  • This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid.
  • the cyclized peptide product was purified on HPLC in a 0.1% TFA H 2 0-acetonitrile gradient.
  • the TFA salt produced could be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) .
  • the finished peptide was:
  • the alpha-amino Fmoc protecting group was removed using piperidine and NMP.
  • Fmoc-protected Cys(Acm) was then coupled to alanine with dicyclohexylcarbodiimide followed by deprotection again with piperidine and NMP.
  • deprotection with piperidine/NMP Fmoc-pro- tected Arg(PMC) , Leu, Orn(t-Boc) , Gly, Arg(PMC) , His(TRT), Gly, Pen(Acm) , and Gly residues were coupled in succession ⁇ sion.
  • the Arg was additionally protected by pentamethyl chroman-6-sulfonyl, the His with trityl, the Orn with t-Boc, and the penicillamine with an Acm group.
  • the fol- lowing peptide resin was formed:
  • This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid.
  • the cyclized peptide product was purified on HPLC in a 0.1% TFA H 2 0-acetonitrile gradient.
  • the TFA salt produced could be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) .
  • the finished peptide was:
  • the alpha-amino Fmoc protecting group was removed (main ⁇ taining protection of Cys with the TPM group) using pipe- ridine and NMP.
  • Fmoc-protected Orn(Boc) was then coupled to cysteine with dicyclohexylcarbodiimide followed by deprotection again with piperidine and NMP.
  • deprotection with piperidine/NMP deprotection with piperidine/NMP, Gly, Arg (PMC) , and Gly residues were coupled in succession.
  • the Arg was additionally protected by pentamethyl chroman-6- sulfonyl (Arg(PMC)).
  • the final Gly was acetylated with bromoacetic acid by activation of the carboxyl group with DCC followed by coupling to the N-terminal amino group. Following acetylation the following peptide resin was formed:
  • the platelet integrin gpIIb/IIIa was purified from platelets by affinity chromatography using immobilized concanavalin A (Con A) .
  • Con A immobilized concanavalin A
  • Five units of platelets obtained from a local blood bank were washed using differential centrifugation.
  • GPIIb/IIIa was extracted from the mem ⁇ brane with 100 mM- octylthioglucopyranoside at 4° C for 20 min. The extract was run through a 9 ml Con A Sepha- rose 4B column and retained glycoprotein (including GPIIb/ Ilia) was eluted with 150 mM mannopyranoside.
  • This glyco- protein-containing fraction is composed of 50-60% GPIIb/ Ilia as judged by SDS-polyacrylamide gel electrophoresis.
  • Figure 2 depicts inhibition of HRP-fibrinogen binding to GPIIb/IIIa and compares the compound of Example 5 to the i 1 cyclic peptide Cys-Asn-Pro-Arg-Gly-Asp-Cys.
  • Poly L lysine was coated on dynatech immullon II microtiter plates at 10 ug/ml in 0.1 M bicarbonate buffer pH 9.5, 2 hours at 37°C. The plate was washed three times with distilled water and dried for 30 minutes at 37°C. Thrombin stimulated platelets at 6.7 x 107 cells/ml in PBS pH 7.2 with 1 mM calcium chloride were added at 50 uL per well. Incubation was carried out for 18 hours at 4°C. The plate was washed three times with PBS, lmM calcium chloride, 0.02% Tween 20 (PBS/Tween) and blocked with PBS/0.5% BSA O.N. at 4°C.
  • the blocking buffer was removed from the above prepa ⁇ ration and the inhibitory peptide, diluted in PBS. lmM calcium chloride was added (50 uL per well) followed by incubation for 1 hour at 37°C.
  • Peroxidase labeled fibri ⁇ nogen 125 ng/ml
  • TBS platelet diluent buffer
  • 50 mM Tris 50 mM Tris, 140 mM NaCl, pH 7.4, 0.1% BSA, 0.02% Tween 20
  • TBS platelet diluent buffer
  • the plate was washed five times with PBS/ Tween then tetramethybenzidine (100 uL per well) was added and the plate was incubated for 10 minutes. The color reaction was stopped with 100 uL per well of 1 N sulfuric acid. Optical density was read at 450-650 nm and test compounds were compared with controls.
  • Figure 1 depicts inhibition of HRP-fibrinogen binding to GPIIb/IIIa by the following: (a) the compound of Exam ⁇ ple 16; (b) the compound of Example 17; (c) the compound of Example 5; (d) the peptide Gly-Arg-Gly-Asp-Ser; (e) the 17-amino acid peptide: Cys-Gly-Gly-Ser-Thr-Ser-Tyr-Asn- Arg-Gly-Asp-Ser-Thr-Phe-Glu-Ser-Lys-COOH.
  • Arg-Gly-Orn peptides of the pres- ent invention were examined. Platelet aggregation was performed with platelet-rich plasma (PRP) . PRP was stirred at 37°C in an aggregometer (Sienco Model 247, Morrison, CO) and aggregation was initiated by the addition of 12.5 ⁇ M of ADP.
  • the control compound used was the peptide Gly-Arg-Gly-Asp-Ser, syn ⁇ thesized at Corvas, International. Aggregation was moni ⁇ tored as a change in light transmittance, and is expressed as the initial rate of aggregation.
  • Figures 3(a) and 3(b) depicts inhibition of platelet aggregation over time by the compound of Example 16 at two concentrations (a) 250 ⁇ M and (b) 500 ⁇ M.
  • the top curve of each figure depicts uninhibited aggregation and the bottom curve inhibition of aggregation due to the compound of Example 16.

Abstract

Provided are novel peptides with platelet aggregation inhibitory activity which include the tripeptide sequence A-Gly-Orn wherein A is a basic amino acid. These peptides are preferably cyclic.

Description

NOVEL INHIBITORS OF PLATELET AGGREGATION
Background and Introduction to the Invention
This invention relates to novel peptide derivatives having activity as inhibitors of platelet aggregation.
While platelet aggregation is an essential process in normal blood clotting, and serves as a defense mechanism after injury in the prevention of excessive bleeding, in certain disease states (e.g., myocardial infarction) it can become uncontrolled and lead to restriction or total blood flow blockage. Additionally, fibrinolysis resulting from treatment with thrombolytic agents such as tissue plasminogen activator (tPA) or streptokinase can also result in reocclusion that occurs in part as a result of platelet aggregation. A primary cause of platelet aggre¬ gation is the involvement of certain glycoprotein recep- tors known as integrins or cytoadhesins which bind to adhesive proteins such as fibrinogen, fibronectin, von Willebrand factor, and vitronectin. Agents that block this interaction are expected to have therapeutic value particularly in certain disease states and in the reocclu- sion that occurs after angioplasty and fibrinolysis.
When blood vessels are injured, platelets are acti¬ vated by several released stimuli and become competent to bind fibrinogen (Fg) . (Bennett et al., J. Clin. Invest. .64:1393-1400 (1979); Marguerie et al. J. Biol. Chem. 254; 5357-5363 (1979).) This activated platelet then initiates aggregation and thrombus formation. The interaction of fibrinogen with platelets occurs through a membrane bound glycoprotein receptor, known as gpIIb/IIIa. (Phillips et al.. Blood 21:831-843 (1988)). This receptor has been reported to recognize a specific amino acid sequence pres¬ ent in several of the adhesive proteins. (Hynes, Cell 48:549-554 (1987); Ruoslahti et al., Science 23.8:491-497 (1987)) . Specifically, it has been reported that the Arg- Gly-Asp-Ser sequence at positions 572-575 of the fibri¬ nogen αA-chain plays a major role in the binding of this protein to the gpIIb/IIIa receptor, although other regions, particularly one in the α-chain C-terminus of fibrinogen, also appear to be involved. (Gartner et al. J. Biol. Chem. 260:11891-11894 (1985); Ginsberg et al. J. Biol. Chem. 260:3931-3936 (1985); Haverstick et al., Blood «S6_:946-952 (1985); Pytela et al. , Science 231: 1159-1162 (1986); Kloczewiak et al.. Thrombosis Res. 29:249-255 (1983); Kloczewiak et al. , Biochemistry 23:1767-1774 (1984)). In addition to occurring in fibrinogen this important tripeptide sequence, Arg- Gly-Asp, has been reported to occur in other adhesive proteins including fibronectin, von Willebrand factor and vitronectin. (Hynes, Cell 4J:549-554 (1987); Ruoslahti et al., Science 238:491-497 (1987)). One of the few naturally occurring modifications of this tripeptide sequence has been found in von Willebrand factor which has been reported to contain an Arg-Tyr-Asp-Ala sequence as well as the Arg-Gly-Asp sequence. (Titani et al.. Bio¬ chemistry 2_:3171-3184 (1986)). The replacement of Tyr for Gly in the middle position of this receptor tripeptide ligand has also been reported in the urine PAC-1 antibody which also binds to the activated gpIIb/IIIa receptor. (Shattil et al.. Blood 68..1224-1231 (1986)).
Several patents and patent applications disclose peptides containing the Arg-Gly-Asp sequence which are said to inhibit cellular adhesion in general, and platelet aggregation specifically. Without exception, the aspartic acid in this tripeptide sequence has been completely con¬ served. See e.g., EPO 319,506; U.S. Patent No. 4,857,508; U.S. Patent No. 4,792,525; U.S. Patent No. 4,683,291; EPO 410,539; EPO 341,915; EPO 410,767; EPO 411,833; U.S. Patent No. 4,952,562; U.S. Patent 4,879,313; and EPO 410,537. These peptides are also said to be effective in inhibiting the formation of blood clots, other synthetic peptides which contain the Arg-Gly-Asp sequence and their use as inhibitors of fibrinogen binding to platelets have been reported. See, e.g.. Kloczewiak et al.. Biochemistry 22:1767-1774 (1984); Plow et al. Proc. Natl. Acad. Sci. (USA) 81:8057-8061 (1985); Ruggeri et al., Proc. Nat. Acad. Sci. (USA) 81:5708-5712 (1986); Ginsberg et al., J. Biol. Chem. 260(7) :3931-3936 (1985); and Haverstick et al.. Blood 66(4) :946-952 (1985). It is important to note that while some of these peptides have replaced the arginine with another amino acid and in some cases the glycine of the Arg-Gly-Asp sequence has been replaced with another amino acid, without exception the aspartic acid is conserved. PAC-1, an IgM murine monoclonal antibody that, like fibrinogen, gpIIb/IIIa receptor on activated plate- lets, is also inhibited by such peptides. Synthetic pep¬ tides which contain the sequence Arg-Tyr-Asp have been claimed as platelet aggregation inhibitors. (See EPO 368,486) .
Certain snake venoms have been reported to possess small proteins that have platelet aggregation inhibitory properties. In certain cases, these proteins are proposed to inhibit platelet aggregation by the antagonism of the gpIIb/IIIa receptor by virtue of the completely conserved Arg-Gly-Asp subsequence present in each of the proteins. (See, Dennis et al., Proc. Natl. Acad Sci. (USA) 87:2471- 2475 (1989); and Seymour et al., J. Biol. Chem. 265:10143- 10147 (1990)). One of these venom proteins has been reported to increase the rate and extent of thrombolysis with reduced doses of recombinant tissue plasminogen activator and prevents reocclusion. (Yashuda et al.. Circulation J53.:1038-1047 (1991)). Such small proteins have been prepared by total peptide synthesis. (See, EPO 382,538.)
Since the original discovery of the Arg-Gly-Asp bind- ing sequence in fibrinogen, this tripeptide sequence has been proposed to be a primary recognition sequence for several of the adhesive proteins. It has been proposed that the Fg-α fragment Arg-Gly-Asp and the Fg-λ-fragment Ala-Lys-Gln-Ala-Gly-Asp-Val present the same pharmacophore to the gpIIb/IIIa receptor, i.e.. a cationic group (K in Fg-λ and R in Fg-α) and an invariant aspartic acid in both fragments. (Shabuski et al. , Thrombosis and Hemostasis ■51:183-188 (1989)). The importance of conserving the aspartic acid residue for activity is emphasized by Shebuski et al. who point out:
"..the antiaggregatory action of RGDS appears to be specific, since the analog RGES, in which [the] critical aspartic acid has been replaced with the homologous glutamic acid (E) , is inactive as an antiaggregatory agent". Shebuski et al.. Thrombosis and Hemostasis .61, 183-188 (1989), well as by D'Souza, Ginsburg, and Plow:
"Within the RGD tripeptide sequence, conserva¬ tive amino acid substitutions profoundly influence function....Particularly profound is the effect of a glutamic acid substitution for the aspartic acid; this conservative substitu¬ tion diminishes function to an immeasurable level." S.E. D'Souza, M.H. Ginsburg, E.F. Plow, TIBS, in press, (1991) . It has also been reported that RGD peptides can inhibit tumor cell invasion during the metastatic process. (Gehlsen et al., J. Cell Biology 106:925-930 (1988). Studies have implicated platelet aggregation in the pro¬ cess of metastasis formation; inhibitors of this process may have therapeutic value in the treatment of cancer. (Gasic, Cancer Metastasis Reviews 3.:99-116 (1984)).
Additional peptides or peptides having improved activity and properties which meet the important goals of inhibiting fibrinogen binding to activated platelets and preventing blood clot formation are needed. The present invention describes a completely novel class of compounds that meet these needs. Summary of the Invention
Most platelet aggregation inhibitors, to date, have been patterned after the natural Arg-Gly-Asp (RGD) sequence found in several of the adhesive proteins and naturally occurring platelet inhibitory snake venom proteins. While synthetically some substitutions have been made, for example, substituting Arg mimics for Arg, or substituting Tyr in place of Gly (in the PAC-1 anti¬ body) , the Asp residue has been invariant and considered to be critical for activity. Indeed, the replacement of the Asp even by the closely related Glu group (differing from Asp by only one methylene) has been found to elimi¬ nate platelet aggregation inhibitory activity altogether. (Shabuski et al. Thrombosis and Hemostasis 61:183-188 (1989); Plow et al., Proc. Natl. Acad Sci (USA) .31:8057- 8061 (1985); Gartner et al., Blood 66 (SUPPI. 1) :305a (1987)) .
Accordingly, the present invention is directed to the surprising finding that the otherwise invariant aspartic acid in these peptide sequences can be replaced by orni¬ thine and yet retain cell adhesion and/or platelet aggre¬ gation inhibitory activity. This is particularly surpris¬ ing since ornithine is an amino acid residue differing both in size and charge from aspartic acid (the negatively charged carboxyl group of Asp is replaced with an amine which would be protonated and positively charged and the side chain is extended by one methylene) and in view of findings that replacement of aspartic acid with glutamic acid eliminated platelet aggregation activity. The sub- stitution of ornithine for aspartic acid in RGD sequences gives the resulting novel peptide compounds of this inven¬ tion which show potent platelet aggregation inhibitory properties. As noted above, this finding is particularly surprising in view of all previous observations that the aspartic acid had to be conserved for activity and even the insertion of one methylene into the aspartic acid of RGD-containing analogs to give glutamic acid (D —> E) resulted in inactive peptides.
This invention is directed to the use of ornithine in any peptide or peptide mimic that derives its biological activity by using an acidic residue such as Asp to bind to an RGD receptor.
According to one preferred aspect, the present inven¬ tion provides novel peptide compounds having activity in inhibiting fibrinogen binding, platelet aggregation and/or glycoprotein Ilb/IIIa binding (collectively referred to as "aggregation inhibiting activity") which comprises a cyclic or rigid peptide including as an aggregation inhib¬ iting constituent an amino acid sequence which comprises
- A - Gly - Orn - (I) wherein A is an L or O- isomer of arginine, homoarginine, guanidinoaminobutyric acid, guanidinoaminopropionic acid, N',N"dimethylarginine, N' ,N"-diethylarginine, p-amino- ethyl-phenylalanine, p-amidinophenylalanine, p-guanidino- phenylalanine, a conformationally constrained arginine analog as described by T. R. Webb and C. Eigenbrot, J. Org. Chem. 56.: 3009 (1991) , lysine, or an α-R' substi¬ tuted derivative thereof wherein R' is lower alkyl, such as methyl, said peptide having aggregation inhibiting activity. The present invention also includes peptides wherein the Orn of formula (I) is replaced by a 1-methyl Orn. Such peptides typically have an IC50 (μ,M) of no more than about 500 in an assay of thrombin-induced platelet aggregation. Preferred are peptides having less than about 20 amino acid residues, more preferably having from about 6 to about 14 amino acid residues. Especially pre¬ ferred are cyclic peptides, that is, peptides wherein a side chain of an amino acid residue on the amino terminal side of -A- is covalently linked to a side chain of an amino acid residue on the carboxy terminal side of -Orn- or cyclic peptides having a cyclic peptide backbone. Covalent linkages between side chains of amino acids may include disulfide linkages between sulfhydryl groups, such as naturally occurring disulfide bridges between cysteine residues, or disulfide bridges between other sulfhydryl- containing amino acids (e.g. penicillamine) or analogs. Other suitable interamino acid linkages include linkages between (a) alkyl acids and alkyl amines which may form cyclic lactams; (b) alkyl aldehydes or alkylhalides and alkylamines which may condense and be reduced to form an alkylamine bridge; (c) side chains which may be connected to form an alkyl, alkenyl, alkynyl, ether or thioether bond. Preferred alkyl chains include lower alkyl groups having from about 1 to about 6 carbon atoms; or (d) disul¬ fide bonds between a thio- containing acyl group on the N-terminus and a cysteine or penicillamine.
In particular, the present invention is directed to a peptide having the tripeptide sequence of formula I and a rigid, for example, cyclized, structure which may result from a modification in amino acid sequence of a peptide having platelet aggregation inhibitory properties wherein the aspartic acid of an A-Gly-Asp sequence (wherein A is a basic amino acid as described with formula I) is replaced with ornithine to give a peptide having improved inhibitory and/or pharmacokinetic properties. Such pep¬ tides which are modified with unnatural amino acids, as well as cyclic versions are considered to fall within the scope of the present invention. Examples of unnatural amino acids are described in T.R. Webb, C. Eigenbrot, J. Org. Chem. 5j5: 3009 (1991); J. C. Kauer et al., J. Biol. Chem. 261: 10695 (1986), R. S. Glass et al., J. Org. Chem. 55: 3797 (1990), R. T. Shuman et al., in Peptides, Proceedings of the Eleventh American Peptide Symposium, July 9-14, 1989, Eds. J.E. Rivier and G.R. Marshall, ESCOM Science Publishers, 1990, p. 944, G. Chassaing et al., ibid, p. 935, R. Dharanipragada et al., ibid., p 937, S. E. deLaszlo et al., ibid., p. 409, and D.J. Kempf et al., ibid., p. 393.
Also considered to be within the scope of the present invention are compounds having activity in inhibiting fibrinogen binding or glycoprotein Ilb/IIIa binding in which the aspartic acid of an Arg-Gly-Asp sequence has been replaced by ornithine.
In addition, it has been reported that a number of snake venom cysteine-rich polypeptides contain RGD sequences that are said to be responsible for inhibition of platelet aggregation (See, e.g. f WO 9015072 to Lazarus and Dennis; WO 90/08772 to Maraganore, Jakubowski and Chao; EP 382538 to Garsky, EP 382451 to Friedman, Jacobs, Gould, Polokoff, Gan, Niewiarows, Holt, and Rucinski; and EP 338634 to Friedman, Polokoff, Gould, Bencen, Jacobs, Garsky; and Gan) . Accordingly, polypeptides and smaller cyclic versions of such peptides in which the aspartic acid of the RGD sequence has been replaced with ornithine are considered to fall within the scope of this invention. For example, one such peptide would have the sequence: NH2-ECESGPCCRNCKFLKEGTICKRARG-Om-DMDDYCNGKTCDCPRNPHKGPA T-OH (See EPO 382,538 to Garsky).
The invention further provides compositions compris- ing one or more of the foregoing compounds, and methods of using such compounds or compositions in inhibiting fibri¬ nogen binding, platelet aggregation, and/or glycoprotein Ilb/IIIa binding. The subject compounds or compositions are also effective in the treatment of certain physiologi- cal conditions, such as thrombosis and/or cancer metas¬ tasis, and the present invention is further directed to these uses.
Finally, the present invention comprehends peptides or small peptides accessible by synthetic routes compris- ing an amino acid sequence corresponding to the amino acid sequence of the variable region of the heavy chain of monoclonal antibody PAC-1, which incorporates the sequence of formula (I) as well as antibodies incorporating that sequence. Definitions
As used herein, the following terms have the follow¬ ing meanings unless expressly stated to the contrary:
"Pen" refers to L-penicillamine or β,β-dimethyl cysteine.
"HCys" refers to homocysteine.
"(Me)-Arg" refers to N' ,N"-dimethylarginine.
("Et2)Arg" refers to N' ,N"-diethylarginine.
"Aib" refers to aminoisobutyric acid. "Abu" refers to aminobutyric acid.
"Ac" refers to acetyl.
"Acm" refers to acetamidomethyl.
"Aha" refers to 7-aminoheptanoic acid.
"Boc" refers to tert-butyloxycarbonyl. " "Bz" refers to benzoyl.
"Cbz" refers to benzyloxycarbonyl.
"DIEA" refers to diisopropylethylamine.
"D.I. water" refers to deionized water.
"DMF" refers to dimethylformamide. "(Et2)Arg" refers to N' ,N"-diethylarginine.
"Fmoc" refers to 9-fluorenylmethyloxycarbonyl.
"HBTU" refers to 2-(lH-benzotriazol-l-yl)-l,l,3,3 -tetramethyl uronium hexafluorophosphate.
"HCys" refers to homocysteine. "HMP" refers tohydroxymethylphenoxymethylpolystyrene resin.
"HOAc" refers to acetic acid.
"HOBT" refers to 1-hydroxybenzotriazole.
"MBHA" refers to 4-methylbenzhydralamine resin. "MBzl" refers to 4-methylbenzyl.
"MeArg" refers to N-α-methylarginine.
"(Me2)Arg" refers to N' ,N"-dimethylarginine.
"MeOSuc" refers to methoxysuccinyl.
"NMP" refers to N-methylpyrrolidine. "PBS" refers to a buffer of pH of about 7.4 containing 10 mM sodium phosphate and 150 mM sodium chloride. "Pen" refers to L-penicillamine.
"PMC" refers to 2,2,5,7,8-pentamethylchroman-6- sulfonyl.
"RINK" refers to ((dimethoxyphenyl-Fmoc-aminomethyl)- phenoxy) resin.
"Tos" refers to tosyl or 4-toluenesulfonyl. "TFA" refers to trifluoroacetic acid. "ThioPro" refers to L-thioproline. "TRT" refers to trityl. "Tyrode's Buffer" refers to buffer of pH of about 7.4 containing 150 mM sodium chloride, 2.7 mM potassium chloride, 12 mM sodium bicarbonate, 0.4 mM sodium phosphate.
The "α-R* substituted" derivatives of amino acids, which may also be denoted as (α-R')AA, indicate amino acids which are monosubstituted on the C-alpha of the amino acid by R' wherein R' is alk (alkyl) or benzyl.
The term "conformationally constrained arginine analog" refers arginine analogs such as those disclosed by Webb et al., "Conformationally Restricted Arginine Ana¬ logues", J. Org. Chem. 56:3009-3016 (1991), and, in par¬ ticular includes the analogues described therein.
The term "alkyl" refers to saturated aliphatic groups, including straight, branched and carbocyclic groups. The term "lower alkyl" refers to both straight- and branched-chain alkyl groups having a total of from 1 to 6 carbons atoms and includes primary, secondary and tertiary alkyl groups. Typical lower alkyls include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl, and the like.
The term "aryl" refers to aromatic groups having from about 6 to 14 carbon atoms and includes cyclic aromatic systems such as phenyl and napthyl.
The term "aralkyl" refers to an alkyl group from about 1 to 4 carbon atoms substituted with an aryl of from 6 to 10 carbon atoms and includes, form example, benzyl, p-chlorobenzyl, p-methylbenzyl and 2-phenylethyl. Brief Description of the Drawings
FIG. 1 depicts a plot of inhibition of HRP-Fibrinogen binding to GPIIb/IIIa by the Elisa-based assay of Example
B, showing effective inhibition of fibrinogen binding by the compounds of Examples 5, 16 and 17 along with two RGD peptides.
FIG. 2 depicts a plot of inhibition of HRP-Fibrinogen binding to GPIIb/IIIa by the methods of Example A and shows inhibition by the compound of Example 5 in compari- son to a RGD compound.
FIG. 3 depicts a plot of inhibition of platelet aggregation by the compound of Example 16 in an assay of
Example C at two concentrations, (a) 250 μM and (b) 500 μM, the bottom curve showing the inhibited aggregation by the compound of Example 16.
Detailed Description of the Invention
More specifically, the present invention encompasses peptide compounds having the tripeptide sequence depicted in Formula (I) and physiologically acceptable salts thereof. These compounds are characterized by their inhibitory potency, namely their ability to retard or prevent one or more of the following: the binding of the adhesive protein fibrinogen to blood platelets (referred to herein as inhibiting "fibrinogen binding") , the binding of blood platelets to themselves (referred to herein as inhibiting "platelet aggregation") , and/or the binding of compounds or substances, particularly proteins, to the glycoprotein Ilb/IIIa complex found in blood platelet mem¬ branes (referred herein as inhibiting "glycoprotein lib/ Ilia binding") . The subject compounds are also useful in retarding or preventing the formation of blood clots or thrombi (referred herein as inhibiting "thrombosis") and/or the spread of cancer cells throughout the body (referred to herein as inhibiting "cancer metastasis") . The selectivity of these compounds in carrying out the foregoing related activities makes them particularly useful as therapeutic and/or diagnostic agents.
For maximal activity, it is preferred that the orni¬ thine residue in the key tripeptide region, of formula (I) of the present invention is in the L-configuration. The remaining amino acid residues of the peptides of the pres¬ ent invention can be present in either the D- or the L- configuration.
Amino acid residues may be linked among themselves, or with each other, in branched, cyclic or straight chain form, although cyclic chain linkages are preferred. As one skilled in the art would recognize, branched or cyclic chains may be produced by the formation of a peptide bond with amino acid side groups that contain amino or car- boxyl moieties. Amino acids containing such side groups include, for example, glutamic acid (carboxyl group) , aspartic acid (carboxyl group) and lysine (amine group) . Branched or cyclic chains may also be produced through formation of a covalent disulfide bond between amino acid residues having sulfur-containing side groups, such as cysteine, homocysteine and penicillamine.
In one aspect, the present invention is directed to novel and very active compounds that contain the tripep¬ tide sequence set forth in formula (I) , linked from amino- to carboxy-terminus. The present invention is based upon the surprising finding that, in contrast to previous platelet aggregation inhibitors that require an aspartic acid in the third position of a RYD or RGD sequence, effective and potent inhibitors can be obtained by replacement of the Asp with Orn, an amino acid that differs substantially from aspartic acid in both size and charge.
According to' one aspect of the present invention, preferred classes of compounds include cyclic peptides having interamino acid peptide linkages of the formula:
L-(K) -G-(E)0-(B)m-A-Gly-0m-(D)n-F-( ) -M (II) wherein m is O or l; n, o, p and q are independently selected integers from 0 to 2; A is a D- or L-isomer of arginine, homoarginine, guanidinoaminobutyric acid, guanidinoaminopropionic acid, (Me2)Arg, (Et2)Arg, p-aminomethyl phenylalanine, p-amidinophenylalanine, p-guanidinophenylalanine, a conformationally constrained arginine analog as described by T. R. Webb and C. Eigenbrot, J. Org. Chem. 56_:3009 (1991), Lys or an α-R' substituted derivative thereof wherein R' is lower alkyl such as methyl; B is a D- or L- isomer of proline, β-methylproline, β,β-dimethylproline; hydroxyproline, anhydroproline, thioproline, β-methylthioproline, β,β- dimethylthioproline, pipecolic acid, azetidine carboxylic acid, N-methylarginine, N-methylhistidine, N-methylaspara- gine, N-methy1 Aib, Abu, Ala, Gly, (Me2)Arg, (Et2)Arg, Arg, His, Asn, homoarginine, guanidinoaminobutyric acid, guani¬ dinoaminopropionic acid, sarcosine or an α-R' substituted derivative thereof wherein R' is lower alkyl such as methyl; D is an independently selected D- or L-isomer of Tyr, (alk)Tyr, Phe, Trp, His, Ser, (alk)Ser, Thr, (alk) Thr, Ala, Val, Norvaline, Met, Leu, lie, Nle, or an α-R» substituted derivative thereof wherein R1 is a lower alkyl, such as methyl and alk is lower alkyl of 1 to 6 carbon atoms; E is an independently selected D- or L-amino acid; F and G are independently selected (L-) amino acids having side chains which are chemically bonded to each other; J and K are independently selected amino acids; L is hydrogen, aryl, aminoacyl, or N-methylaminoacyl; and M is -OH, -NH2 -NHRj, -N(R3)2, or the C-terminal carboxylic acid group of the peptide is esterified with -OR4 wherein R2 and Rj are independently selected alkyl of 1 to 4 carbon atoms and R4 is alkyl of 1 to 6 carbon atoms or aralkyl of 7 to 9 carbon atoms. According to one preferred aspect, F and G may be cysteine and penicillamine in any combina- tion as well as groups having side chain linkages where F is -NHCH(X)C(=0)- and G is -NHCH(Y)C(=0)- and where F is cyclized to G as depicted in formula (III) below: I
CZ, cz, -NH-CH-C (=0) - -NH-CH-C (=0) - (III) where Z may be H or lower alkyl such as methyl and X,Y may be -S-S-, -CH2-S-, -S-CH2-, -(CH2)3-, -(CH2)4-, -CH2-S-S-CH2-, ■~CΪ-[ ,'™S"~S~* i "■"S*~S'~'CΓI-5~*•
Additionally, when F is Cys or Pen and q=0, then G may be CH2C(=0)- to form the cyclic thioether wherein M is as defined below:
S CZ2
I I
CH2-C(=0)-(E)0-(B)0-A-Gly-Om-(D)n-NH-CH-C(=0)-( )p-M (IV) Also preferred are cyclic peptides having a cyclic peptide back bone of the formula:
J-(E)s-(B)m-A-Gly-0m-(D)p-K (V) wherein m is 0 or 1; r and s are independently selected integers from 0 to 3; A is an L-or D-isomer of arginine, homoarginine, guanidinoaminobutyric acid, guanidinoamino- propionic acid, (Me2)Arg, (Et2)Arg, p-aminomethyl-phenyl- alanine, p-amidinophenylalanine, p-guanidinophenylalanine, a conformationally constrained arginine analog as described by T. R. Webb and C. Eigenbrot, J. Org. Chem. 56.:3009 (1991), Lys or an α-R' substituted derivative thereof wherein Rf is lower alkyl, preferably methyl; B is a D- or L- isomer of proline, β-methylproline, β,β- dimethylproline; hydroxyproline, anhydroproline, thiopro- line, β-methylthioproline, β,β-dimethylthioproline, pipe- colic acid, azetidine carboxylic acid, N-methylarginine, N-methylhistidine, N-methylasparagine, N-methy1 Aib, Abu, Ala, Gly, (Me2)Arg, (Et2)Arg, Arg, His, Asn, homoarginine, guanidinoaminobutyric acid, guanidinoaminopropionic acid, sarcosine or an α-R' substituted derivative thereof wherein R» is lower alkyl, preferably methyl; D is an independently selected D- or L-isomer of Tyr, (alk)Tyr, Phe, Trp, His, Ser, (alk)Ser, Thr, (alk)Thr, Ala, Val, Norvaline, Met, Leu, lie, Nle, or an α-R' substituted derivative thereof wherein R' is lower alkyl preferably methyl, and alk is lower alkyl of 1 to 6 carbon atoms; E is an independently selected D- or L- amino acid; and J and K are independently selected D- or L- amino acids and are linked by a peptide linkage.
Also included within the scope of the present inven¬ tion are analogs of the foregoing compounds which include compounds comprising less common or modified amino acids, for example, hydroxyproline, hydroxylysine, cystine, thyroxine, norleucine, pyroglutamic acid or other amino acid derivatives which are capable of incorporation into the peptides of the present invention. Preferred compounds are:
Ac-Cys-(D-Arg)-Arg-Gly-Orn-Cys-OH
Ac-Cys-ThioPro-Arg-Gly-Orn-Cys-OH
Ac-Cys-Pro-Arg-Gly-Orn-Cys-OH
Ac-Cys-Asn-Pro-Arg-Gly-Orn-Cys-NH2
Ac-Cys-Asn-Pro-Arg-Gly-Orn-Cys-OH
Ac-Cys-Asn-ThioPro-Arg-Gly-Orn-Cys-OH
Ac-Cys-Arg-Gly-Orn-Cys-NH2 Ac- (D-Cys) -Arg-Gly-Orn-Cys-NH2
Ac-Pen-Arg-Gly-Orn-Cys-NH2
Ac- (homoCys) -Arg-Gly-Orn-Cys-NH2
Ac-Cys-Arg-Gly-Orn-Pen-NH2
Ac-Cys-Arg-Gly-Orn-Cys-OH
Ac-Pen-Arg-Gly-Orn-Cys-OH
Ac-Cys-Arg-Gly-Orn-Cys-Pro-NH2
Figure imgf000018_0001
Cys-Arg-Gly-Orn-Cys-OH
Ac- (N-Methy ICys ) -Arg-Gly-Orn-Cys-OH
Figure imgf000018_0002
-NH2 Ac-Cys-Arg-Gly-Orn-Pen-OH
Ac-Pen-Arg-Gly-Orn-Pen-NH2
Ac- (D-Pen) -Arg-Gly-Orn-Pen-OH
Ac-Cys-Arg-Gly-Orn- (homoCys) -NH2
Ac-Cys- (D-Arg) -Gly-Orn-Cys-NH2
Ac-Cys-MeArg-Gly-Orn-Ser-Cys-NH2
Ac-Cys-Arg-Gly-Orn-Ser-Cys-NH2
Ac-Cys-MeArg-Gly-Orn- (Me) Ser-Cys-NH,
Ac-Cys-MeArg-Gly-Orn-Cys-NH2
Ac-Cys-Arg-Gly-Orn-Pen-NH2
Ac-Cys-Arg-Gly-Orn- (D-Pen) -NH2 Ac-Cy s-Ly s -Gly-Orn-Pen-NH2
Ac-Cys-MeArg-Gly-Orn-Pen-NH2
Ac-Cys-Arg-Gly-Orn-Pen-NHEt
Ac-Cys-DArg-Gly-Orn-Pen-NH,
Ac-Cys-MeArg-Gly-Orn-Tyr-Cys-NH2
Ac-Pen-MeArg-Gly-Orn-Pen-NH2
Ac-Pen-Arg-Gly-Orn-Cys-NH2
Ac-Cys-DArg-Gly-Orn-Ser-Cys-NH2
Ac-Cys-Sar-Arg-Gly-Orn-Pen-NH2
Ac-Cys -Arg-Gly-Or n-Cy s-NH2
Figure imgf000020_0001
Ac-Cys-DMeArg-Gly-Orn-Pen-NH2
Arg-Gly-Orn-Arg-Gly
Arg-Gly-Orn-Ser-Pro-Ala-Gly
Arg-Gly-Orn-Ser-Pro-Ala-Val-Thr-Gly
Arg-Gly-Orn-Ser-Pro-Ala-Ala-Val-Thr-Gly
Arg-Gly-Orn-Asn-Pro-Ala-Gly
Gly-Cys-Gly-Lys-Gly-Orn-Trp-Pro-Cys-Ala-NH2
Acetyl-Tyr-Gly-Cys-Arg-Gly-θrn-Cys-NH2
Acetyl-Cys-Asn-Pro-Arg-Gly-MeOrn-Cys-OH
Gly-Pen-Gly-Arg-Gly-Orn-Ser-Pro-Cys-NH2
Gly-Pen-Gly-His-Arg-Gly-Orn-Leu-Arg-Cys-Ala-NH, cyclo-S-acetyl-Gly-Arg-Gly-Orn-Cys-OH
The present invention also includes physiologically acceptable salts of the peptides of the present invention. These salts include acid addition salts, for example salts of hydrochloric acid, hydrobromic acid, acetic acid, tri- fluoroacetic acid, citric acid, succinic acid, benzene sulfonic acid or other suitable acid addition salts. The branched, cyclical and straight chain peptides of the present invention can be synthesized using conven¬ tional preparative and recovery methods known to those skilled in the art.
The novel peptides of this invention can be made by conventional methods of peptide synthesis.
As used herein, a "protected" terminal amino group, refers to a terminal amino group coupled with any of various amino-terminal protecting groups traditionally employed in peptide synthesis. Examples of suitable groups include acyl protecting groups, for example, formyl, acetyl, benzoyl, trifluoroacetyl, succinyl and methoxysuccinyl aromatic urethane protecting groups, for example, benzyloxycarbonyl and aliphatic urethane protect¬ ing groups, for example, tert-butyloxycarbony1, adamantyl- oxycarbonyl or fluorenylmethyloxycarbonyl groups. Numer¬ ous suitable amino terminal protecting groups are known. See, e.g.. Gross and Mienhofer, eds., The Peptides, Vol. 3, pp 3-88 (Academic Press, New York, 1981) . Other suitable protecting groups are known to those skilled in the art.
The following represent preferred amino terminal protecting groups:
Boc = tert-butyloxycarbonyl; Bz = benzyl; MeOSuc = methoxysuccinyl;
F oc = 9-fluorenymethyloxycarbonyl; Ac = acetyl, and Z = benzyloxycarbonyl. The amino acid residues of the substituents having side chain amino groups or other reactive groups, for example Lys or Arg, may optionally comprise suitable amino- protecting groups or other functional groups protecting groups attached to the side chains.
As used herein, the term "protected" terminal car¬ boxyl group, refers to a terminal carboxyl group coupled with any of various carboxy-terminal protecting groups. As will be readily apparent to one skilled in the art, suitable groups include tert-butyl, benzyl or other protecting groups linked to the terminal carboxyl group through an ester or ether bond. Amino acid residues of substituents having acidic or hydroxy side chains may be similarly protected.
A preferable synthesis route for the straight-chain peptide intermediates, especially the smaller peptides (of shorter chain length, that is, having from about 3 to about 50 amino acid residues) of the invention is the solid phase method. This method is well known in the art and is described in references such as Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963); Science 150:178-185 (1965); and Science 212:341-347 (1986); Vale et al., Science 211:1394-1397 (1981); and Marke et al., J. Am. Chem. Soc. 101:3178 (1981); the disclosures of which are incorporated herein by reference. Other preparative methods which may be employed include the processes of Houghten, Proc. Natl. Acad. Sci(USA) 82.:5132 (1985). On particular EPO application 382,538 describes the solid phase synthesis of a 49-amino acid cysteine rich peptide. The above disclosures of are also incorporated herein by reference.
Solid phase peptide synthesis is generally commenced from the C-terminus of the peptide by coupling a protected α-amino acid to a suitable resin, such as a hydroxymethyl- phenoxymethyl polystyrene resin (HMP) or a RINK ([dimeth- oxyphenyl-Fmoc aminomethyl]-phenoxy) resin when synthesiz- ing a peptide amide. The RINK resin is a modified benzhy- drylamine resin that contains ortho and para electron- donating methoxy groups.
During synthesis, suitable protecting groups are used to prevent side reactions with functional groups on amino acid side chains as needed. The guanidino group of argi¬ nine may be protected by a penta ethyl chroman-6-sulfonyl (PMC) group. The side chain amides of asparagine and glu- tamine are protected with the trityl (TRT) group. The beta carboxyl group of aspartic acid is blocked with a tert-butyl ester. The sulfhydryl groups of cysteine and penicillamine are protected with an acetamidomethyl (ACM) group. Ornithine and lysine are protected on the side chain amine with the t-butoxycarbonyl (BOC) group. Each α-amino group may be protected with the Fmoc group.
The peptide sequence is synthesized by sequential coupling of α-amino protected amino acids to the a ino- terminal and of the growing peptide chain attached to the solid support. After the desired peptide sequence is complete, the intermediate peptide is cleaved from the resin and the protecting groups are removed with a reagent such as trifluoroacetic acid (TFA) . The peptide is iso¬ lated from the TFA solution by techniques such as filtra¬ tion, centrifugation or extraction with diethyl ether. The peptide can then be purified by high performance liquid chromatography (HPLC) or other such methods of protein purification.
Background information on established solid phase synthesis procedures which can be used for the preparation of the peptides described herein is set forth in treatise by Stewart and Young, "Solid Phase Peptide Synthesis", W.H. Freeman & Co., San Francisco, 1969; in the review chapter by Merrifield in Advances in Enzymology 32:221- 296, F. F. Nold, Ed., Interscience Publishers, New York, 1969; and in Erickson and Merrifield, The Proteins, Vol. 2, page 255 et seg. (ed. Neurath and Hill) , Academic Press, New York, 1976; the disclosures of which are incorporated herein by reference.
Preferred synthesis procedures, particularly for the smaller branched or cyclic chain peptides, as a skilled artisan would recognize, would include conventional liquid phase processes. The liquid phase processes, as well as other synthesis methods, are described in Principles of Peptide Synthesis. M. Bodansky, ed. , (Springer-Verlag, 1984), the disclosures of which are incorporated herein by reference.
Suitable recovery methods for the synthesized pep¬ tides are described in the foregoing references. Other recovery methods which may be employed include those described in Rivier et al., Peptides: The Structure and Biological Function, pages 125-128 (1979) , the disclosures of which are incorporated herein by reference.
The present invention further provides compositions and methods for using these compounds and compositions in inhibiting fibrinogen binding, platelet aggregation, and glycoprotein Ilb/IIIa binding. The ability of the com¬ pounds of the present invention to inhibit the foregoing activities makes them useful in inhibiting the physiologi¬ cal process of thrombosis. In addition, in light of their demonstrated activities, the compounds of the present invention may be employed in inhibiting cancer metastasis (an aberrant physiological phenomenon that is believed to require the adhesion of blood platelets to the cancer cells) . The specific activities of the compounds of pres¬ ent invention in carrying out these related functions makes them particularly useful as therapeutic and/or diagnostic agents.
The platelet-binding inhibitor activity of the pep¬ tide derivatives of this invention may be demonstrated by various assays. According to one such assay, the peptides are tested for their inhibition of thrombin-induced plate¬ let aggregation in washed human platelets. The percent inhibition is determined for the test peptide by comparing the extent of platelet aggregation in the presence of and absence of the peptide.
In another assay, platelet aggregation is examined in platelet-rich plasma which also is rich in fibrinogen and other plasma proteins.
In certain of these assays, the results using com¬ pounds of the present invention were then compared with the activity of the known active inhibitor tetrapeptide, Arg-Gly-Asp-Ser and the corresponding Arg-Gly-Asp analog. According to one aspect of the present invention, the compositions of the present invention comprise an effec¬ tive amount of a compound of Formula I and a physiological acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic, diagnostic or other uses are well known in the pharmaceutical art, and are described, for example in Remington's Pharmaceutical Sciences, Gennaro, A. R. , ed. (Mack Publishing Co., Easton PA, 1985) .
In practicing the methods of the invention, the co - pounds or compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These compounds can be utilized in vivo, ordinarily in a mammal, preferably in a human, or in vitro. In employing them in vivo, the compounds or compo- sitions can be administered to the mammal in a variety of ways, including parenterally, intravenously, subcutane- ously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms. As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particu¬ lar mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is, the dosage levels necessary to achieve the desired result, will be within the ambit of one skilled in the art. Typically, applications of com- pound are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved.
The compounds of the present invention may be used in conjunction with fibrinolytic therapy for therapeutic indi¬ cations such as to prevent reocclusion of a blood vessel following fibrinolytic therapy. Thus, these compounds may be administered in conjunction with fibrinolytic agents such as plasminogen activators including arokinase, pro- urokinase, streptokinase and tissue plasminogen activator as well as derivatives or variants thereof.
Antibodies, both monoclonal and polyclonal, directed to peptide compounds of the present invention are useful for diagnostic purposes and for the identification of concentration levels of the subject peptides in plasma. To prepare the subject antibodies, any one of a number of conventional techniques which are known in the art can be employed. In one such technique, polyclonal antibodes synthesized by injecting an animal (for example a rabbit) with one or more compounds of the invention. After injec¬ tion, the animal naturally produces antibodies to these compounds. When the antibody level (or titer) reaches a sufficient level, antibody-containing blood, called anti- serum, is then drawn from the animal, and the compound- specific antibody is isolated from other antibodies in the antiserum by any one of a number of separation techniques (for example, affinity chromatography) . Monoclonal antibodies may be prepared using the technique of Kohler and Milstein, Nature 256:495-497 (1975) and other conven- tional techniques known to those skilled in the art. (See, e.g.. Harlow and Lane, Antibodies a Laboratory Manual (Cold Spring Harbor Laboratory, 1988) .
To assist in understanding the present invention, the following Examples are included which describe the results of a series of experiments. The following examples relat¬ ing to this invention should not, of course, be construed in specifically limiting the invention and such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are considered to fall within the scope of the invention as described herein and hereinafter claimed.
Examples Example 1 General Methods of Peptide Synthesis and Analysis
The peptides of this invention were synthesized by the solid phase method using an Applied Biosystems Inc. 431A automated synthesizer. Fmoc amino acids and resins supplied by Calbiochem and Bachem, Inc. were used. - The FASTMOC™ HBTU 2-(lH-benzotriazol-l-yl) 1,1,3,3 tetra¬ methyl uronium hexafluorophosphate} protocol developed by Applied Biosystem Inc was employed as the coupling proto- col (ABI User Bulletin #33 Nov 1990) .
The peptides were cleaved from the solid support by the TFA (trifluoroacetic acid) Cleavage Procedure described in the ABI Manual, Introduction to Cleavage
Techniques, pp.6-19 (1990). The peptides were purified by reverse phase HPLC
(Waters Delta Pack C-18, lOoA 19mm X 30cm column, eluting using a gradient of 0-25% B in 60 minutes at a flow rate of 12 ml/min. ; A=0.1% TFA-H20 B=0.1% TFA-CH3CN) . Products were characterized by amino acid analyses after acid hydrolysis and fast atom bombardment mass spectrometry
(FABMS) . Analyses were consistent with the expected structures, with product purities in excess of 95%.
The novel peptide derivatives of this invention were made by the solid phase synthesis. For example, 0.25 grams of Rink resin containing 0.4 mmoles of amino groups were mixed with 10 equivalents (eq) of F oc-Cys(Acm)-OH , 10 eq of 0.45 M HBTU-HOBT-DMF solution, 1.7 eq of DIEA and 0.8 ml of N-Methylpyrrolidine (NMP) for 9 minutes. The resin was filtered and washed repeatedly with NMP. The Fmoc group was removed by treatment with 50% Piperidine and 50% NMP for 7 minutes and again washed repeatedly with NMP. The resin was then ready for reaction with 10 eq of the next amino acid. The cycle as above described was repeated for each amino acid in the sequence.
The resulting peptide was removed from 400 milligrams of resin and deprotected with a cleavage mixture of 10.0 ml TFA; 0.5 ml thioanisole, 0.5 ml D.I. water, 0.25 ml ethanedithiol and 0.75 grams of phenol. The cleavage reac¬ tion was done at room temperature for 1.5 hours. In case of cyclic peptides thallium trifluoroaceate was used. After the TFA was evaporated, the peptide was isolated by diethyl ether precipitation and taken up in 10% acetic acid and lyophilized. The peptide product was purified on HPLC on a Waters Delta Pack C-18 reverse phase column using a 0-25% gradient in acetonitrile (0.1%(TFA). Frac- tions containing the product, as ascertained by analytical HPLC, were pooled and lyophilized to yield about 50 milli¬ grams of pure heptapeptide from 0.4 grams of resin.
Substantially similar synthesis procedures were used for the solid phase synthesis of other derivatives of this invention by substituting equivalent amounts of other Fmoc-amino acids.
Example 2
General Method for Cyclic Cys Peptides
The procedure to produce cyclized Cys-Cys peptides was based on a modified version of the method by Fujii et al. (J. Chem. Commun. 1987, 163). Thus, 0.1 mmole of linear peptide on RINK resin (used to produce C-terminal amides) or HMP (used to produce C-terminal acids) is cooled to 4°C and 1 equivalent of Thallium tristrifluoro- acetate and 2 equivalents of anisole per peptide blocking group is added followed by 50 ml of trifluoroacetic acid (TFA) . The solution is stirred for one hour at 0βC fol¬ lowed by one hour at 25°C. The cleaved resin is removed by filtration through sintered glass and the TFA solution concentrated on a rotary evaporator in vacuo. Water was added to the residue and the mixture was extracted twice with ether. The aqueous layer was filtered through a 0.2 μ nylon filter and submitted to preparative high pressure liquid chromatography for purification. A gradient sol¬ vent mixture was used which was varied from 0.1% TFA in water to 0.1% TFA in acetonitrile over 50 minutes. The appropriate fraction was detected at 214 nm with an ISCO UA-5 Absorbance detector and was collected. The aceto¬ nitrile solvent from the collected fractions was removed in vacuo and the resulting pure cyclic peptide lyophilized to yield a white powder.
Example 3
Synthesis of Cvclo(Aha-Arg-Gly-Orn)
Commercially available Fmoc-Gly-O-HMP was coupled by standard deprotection and coupling procedures described above successively with Fmoc-Arg(Tos) , Fmoc-Aha, and t-Boc-Orn(Cbz) . The alpha-amino Fmoc groups are removed by treatment with 50% piperidine and 50% N-methylpyrolli- dine (NMP) . Couplings were effected with dicyclohexyl- carbodiimide, deprotection with piperidine:NMP, and wash- ing the peptide-resin with NMP. Cbz-protected-t-Boc-Orn was used in the final coupling reaction. The protected peptide was cleaved from the resin by the trifluoroacetic acid procedure described above. This procedure ultimately yielded: Cbz Tos
I I
NH2-Orn-Aha-Arg-Gly-OH
The linear peptide was then treated with a mixture of N-ethy1-N'-(3-dimethylaminopropyl)carbodiimide, 1-hydroxy- benzotriazole, dimethylformamide andN-methyImorpholine to form:
Cbz Tos
I I
I I cyclo (Orn-Aha-Arg-Gly) Final deprotection was done with Zn/HOAc, H2/Pd, or with HF/anisole (9:1 (v:v)) to form:
Figure imgf000031_0001
The product cyclized peptide was purified on HPLC using a 0.1% TFA H20-acetonitrile gradient. The TFA salt can be converted to an HOAc salt by passing a solution of the peptide through an ion exchange column BioRad AG3-X4A (acetate cycle) .
EXAMPLE 4 Synthesis of
Acetyl-Cys-Arg-Gly-Orn-Cys-NH2
Fmoc and Acm protected cysteine was combined with RINK resin to form:
Acm
I
Fmoc-Cys-NH-RINK
The alpha-amino Fmoc protecting group was removed (Cys-SH protected with Acm) using 50% piperidine and 50% NMP. Fmoc-protected Orn (Boc protected) was coupled to the cys¬ teine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yielded the free amino terminus. Following this stepwise procedure of coupling with dicyclohexylcarbodiimide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Gly, Arg, and Cys residues were coupled in succession. Arg was additionally protected with a PMC group and the final Cys residue was addition- ally protected by an Acm group. The final Cys was acety- lated with acetic anhydride. This yielded the resin coupled peptide:
Acm PMC Boc Acm
I I I I Acety1-Cys-Arg-Gly-Orn-Cys-NH-RINK
This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid. The cyclized peptide product was purified on HPLC in a 0.1% TFA H20-acetonitrile gradient. The TFA salt produced could be converted to the HOAc salt by pass¬ ing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished peptide was:
Acetyl-Cys-Arg-Gly-Orn-Cys-NH2
Example 5 Synthesis of
Ac-Cys-Asn-Pro-Arg-Gly-Orn-Cys-OH
The synthesis was started with Acm
I I
Fmoc-Cys-O-HMP The alpha-amino Fmoc protecting group was removed (main¬ taining protection of Cys with the Acm group) using pipe¬ ridine and NMP. Fmoc-protected Orn (Boc) was then coupled to cysteine with dicyclohexylcarbodiimide followed by deprotection again with piperidine and NMP. Following this stepwise procedure of coupling with dicyclohexyl¬ carbodiimide, deprotection with piperidine/NMP, Fmoc-pro¬ tected Gly, Arg(PMC) , Pro, Asn(TRT) , Cys(Acm) residues were coupled in succession. The Arg was additionally protected by pentamethyl chroman-6-sulfonyl (Arg(PMC)), the Asn with trityl (Asn(TRT)), and the final Cysteine with an Acm group (Cys(Acm)). The final Cys was acety- lated with acetic anhydride.
Following acetylation the following peptide resin was formed: Acm TRT PMC Boc Acm
I I 1 I I
I I I I I
Acetyl-Cys-Asn-Pro-Arg-Gly-Orn-Cys-O-HMP This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid. The cyclized peptide product was purified on HPLC in a 0.1% TFA H20-acetonitrile gradient. The TFA salt produced could be converted to the HOAc salt by pass¬ ing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished peptide was:
Acetyl-Cys-Asn-Pro-Arg-Gly-Orn-Cys-OH
Mass spectral analysis using FAB (fast atom bombardment) showed a mass peak of Mw. 802.5 (calculated 802.3).
Example 6
Synthesis of
Gly-Pen-Gly-His-Arg-Gly-Orn-Leu-Arg-Cys-Ala-NH2 The synthesis was started with
Fmoc-Ala-NH-RINK
The alpha-amino Fmoc protecting group was removed using piperidine and NMP. Fmoc-protected Cys(Acm) was then coupled to Alanine with dicyclohexylcarbodiimide followed by deprotection again with piperidine and NMP. Following this stepwise procedure of coupling with dicyclohexyl¬ carbodiimide, deprotection with piperidine/NMP, Fmoc- protected Arg(PMC) , Leu, Orn(Boc) , Gly, Arg(PMC) , His, Gly, Pen(Acm) , and Gly residues were coupled in succes- sion. The Orn was additionally protected with t-butyloxy- carbonyl and the Pen and Cys with an Acm group and the Arg with a PMC group. The following peptide resin was formed: Acm PMC Boc PMC Acm
I I I I I
I I I I I Ac-Gly-Pen-Gly-His-Arg-Gly-Orn-Leu-Arg-Cys-Ala-NH-RINK This peptide is cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid. The cyclized peptide product is purified on HPLC in a 0.1% TFA H20-acetonitrile gradient. The TFA salt produced could be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished peptide was:
Gly-Pen-Gly-His-Arg-Gly-Orn-Leu-Arg-Cys-Ala-NH2
Example 7
Synthesis of Acetyl-Cys-ThioPro-Arg-Gly-Orn-Cys-NH2
Fmoc and Acm protected cysteine was combined with RINK resin to form:
Acm
I I Fmoc-Cys-NH-RINK
The alpha-amino Fmoc protecting group was removed (Cys-SH protected with Acm) using 50% piperidine and 50% NMP. Fmoc-protected Orn (Boc protected) was coupled to the cysteine with dicyclohexylcarbodimide. Deprotection with piperidme and NMP, followed by thorough washing with NMP, yielded the free amino terminus. Following this stepwise procedure of coupling with dicyclohexylcarbodiimide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Gly, Arg(PMC) , ThioPro and Cys resi- dues were coupled in succession. Arg was additionally protected with a PMC group and the final Cys residue was additionally protected by an Acm group. The final Cys was acetylated with acetic anhydride. This yielded the resin coupled peptide: Acm PMC Boc Acm
I I I I
I 1 1 1
Acetyl-Cys-ThioPro-Arg-Gly-Orn-Cys-NH-RINK This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid. The cyclized peptide product was purified on HPLC in a 0.1% TFA H20-acetonitrile gradient. The TFA salt produced could be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished peptide was:
Acetyl-Cys-ThioPro-Arg-Gly-Orn-Cys-NH2
Example 8 Synthesis of
Acetyl-Cys-MeArg-Gly-Orn-Ser-Cys-NH2
Fmoc and ACM protected cysteine is combined with RINK resin to form:
Acm
I
Fmoc-Cys-NH-RINK
The alpha-amino Fmoc protecting group was removed (Cys SH protected with Acm) using 50% piperidine and 50% NMP. Fmoc-protected Ser(tBu) was coupled to the cysteine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yields the free amino terminus. Following this stepwise procedure of coupling with dicyclohexylcarbodiimide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Orn (Boc), Gly, MeArg (PMC), and Cys(Acm) residues are coupled in succession. MeArg was additionally protected with a PMC group and the final Cys residue was addition¬ ally protected by an Acm group. The final Cys is acety- lated with acetic anhydride. This yields the resin coupled peptide: Acm PMC Boc tBu Acm
I I I I I
I I I I I
Acetyl-Cys-MeArg-Gly-Orn-Ser-Cys-NH-RINK
This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid. The cyclized peptide product is purified on HPLC in a 0.1% TFA H20-acetonitrile gradient. The result¬ ing TFA salt can be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished peptide is:
Acetyl-Cys-MeArg-Gly-Orn-Ser-Cys-NH2
Example 9
Acetyl-Pen-Arg-Gly-Orn-Pen-NH2
Fmoc and Acm protected penicillamine was combined with RINK resin to form:
Acm
I
Fmoc-Pen-NH-RINK
The alpha-amino Fmoc protecting group was removed (Pen-SH protected with Acm) using 50% piperidine and 50% NMP. Fmoc-protected Orn (Boc protected) was coupled to the penicillamine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yielded the free amino terminus. Following this stepwise procedure of coupling with dicyclohexylcarbodi¬ imide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Gly, Arg, and Pen residues were coupled in succession. Arg was additionally protected with a PMC group and the final Pen residue was addition- ally protected by an Acm group. The final Pen is acety- lated with acetic anhydride. This yielded the resin coupled peptide: Acm PMC Boc Acm
I I I I
I I I I
Acetyl-Pen-Arg-Gly-Orn-Pen-NH-RINK
This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid. The cyclized peptide product was purified on HPLC in a 0.1% TFA H20-acetonitrile gradient. The TFA salt produced could be converted to the HOAc salt by pass- ing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished pep¬ tide was:
Acetyl-Pen-Arg-Gly-Orn-Pen-NH2
Example 10 Synthesis of
Ac-Cys-MeArg-Gly-Orn-Cys-NH2
The protected peptide-resin intermediate, N°-Ac-Cys (SEt)-MeArg(Tos)-Gly-Orn(Cbz)-Cys(4-MBzl)-MBHA, is syn¬ thesized by solid phase methods. The amino acids, pro¬ tected as the t-Boc derivatives, are coupled successively using N,N-dicyclohexylcarbodiimide/ HOBt and deprotection of the N-terminal protecting groups in each step with TFA. After the last amino acid is coupled the peptide is acety- lated with acetic anhydride and diisopropylethylamine in DMF. The peptide is cleaved from the resin with deprotec- tion of the side chain protecting groups using anhydrous HF in the presence of anisole at 0°. After evaporation of HF in vacuo, the residue is washed with anhydrous ether, and the crude peptide is extracted with 50% acetic acid, then diluted with deionized water. The pH of the aqueous solution is adjusted to 7.5 with concentrated ammonium hydroxide. The basic conditions causes the free thiol generated by removal of the 4-MBzl group from the cysteine to displace the mercaptoethyl protecting group of the second cysteine to cause cyclization of the peptide. Purging the solution with nitrogen removes excess ethyl mercaptan. The cyclized peptide product is purified on HPLC in a 0.1% TFA H20-acetonitrile gradient. The TFA salt which is produced can be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished pep¬ tide is:
Ac-Cys-MeArg-Gly-Orn-Cys-NH2
Example 11 Synthesis of
Acetyl-Pen-MeArg-Gly-Orn-Pen-NH2
The protected peptide-resin intermediate Nβ-Ac-Pen (4-MBzl)-MeArg(Tos)-Gly-Orn(Cbz)-Pen(4-MBzl)-MBHA, is prepared, cleaved and isolated in the same manner as described in Example 10 above. The peptide is cyclized using a 0.01% K3Fe(CN)5 solution. The cyclized peptide product is purified on HPLC in a 0.1% TFA H20-acetonitrile gradient. The TFA salt produced can be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished peptide is:
Ac-Pen-MeArg-Gly-Orn-Pen-NH2
Example 12 Synthesis of
Figure imgf000038_0001
Acety -Cys-Pro-Arg-Gly-Orn-Cys-NH2 Fmoc and Acm protected cysteine is combined with RINK resin to form:
Acm
I Fmoc-Cys-NH-RINK
The alpha-amino Fmoc protecting group is removed (Cys-SH protected with Acm) using 50% piperidine and 50% NMP. Fmoc-protected Orn (Boc protected) is coupled to the cys¬ teine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yielded the free amino terminus. Following this stepwise procedure of coupling with dicyclohexylcarbodiimide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Gly, Arg, Pro and Cys residues were coupled in succession. Arg is additionally protected with a PMC group and the final Cys residue is additionally pro¬ tected by an Acm group. The final Cys is acetylated with acetic anhydride. This yields the resin coupled peptide:
Acm PMC Boc Acm
I I I I
I I I I
Acetyl-Cys-Pro-Arg-Gly-Orn-Cys-NH-RINK
This peptide is cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacete, anisole, and trifluor- acetic acid. The cyclized peptide product was purified on HPLC in a 0.1% TFA H20- acetonitrile gradient. The TFA salt produced could be converted to the HOAc salt by pass¬ ing a solution of the peptide through an ion exchange column BioRad Ag3-X4-X4A (acetate cycle) . The finished peptide is:
Acetyl-Cys-Pro-Arg-Gly-Orn-Cys-NH2 Example 13 Synthesis of
Acetyl-Cys-Arg-Gly-Orn-Pen-NH2
Fmoc and ACM protected penicillamine is combined with RINK resin to form:
Acm
I I Fmoc-Pen-NH-RINK
The alpha-amino Fmoc protecting group is removed (Pen-SH protected with Acm) using 50% piperidine and 50% NMP. Fmoc-protected Orn (Boc protected) is coupled to the penicillamine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yielded the free amino terminus. Following this stepwise procedure of coupling with dicyclohexylcarbodii¬ mide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Gly, Arg(PMC) , and Cys(Acm) resi- dues were coupled in succession. Arg is additionally pro¬ tected by a PMC group and the Cys with an Acm group. The final Cys is acetylated with acetic anhydride. This yields the resin coupled peptide:
Acm PMC Boc Acm
I I I I
I I I I
Acetyl-Cys-Arg-Gly-Orn-Pen-NH-RINK
This peptide is cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid. The cyclized peptide product was purified on HPLC in a 0.1% TFA H20- acetonitrile gradient. The TFA salt produced could be converted to the HOAc salt by pass¬ ing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished pep- tide is: Acetyl-Cys-Arg-Gly-Orn-Pen-NH2
EXAMPLE 14 5 Synthesis of
Acetyl-Tyr-Gly-Cys-Arg-Gly-Orn-Cys-NH2 Fmoc and ACM protected cysteine was combined with
10 RINK resin to form:
Acm
I
Fmoc-Cys-NH-RINK
The alpha-amino Fmoc protecting group was removed (Cys-SH 15 protected with Acm) using 50% piperidine and 50% NMP. Fmoc-protected Orn (Boc protected) was coupled to the cysteine with dicyclohexylcarbodimide. Deprotection with piperidine and NMP, followed by thorough washing with NMP, yielded the free amino terminus. Following this stepwise 20 procedure of coupling with dicyclohexylcarbodiimide, deprotection with piperidine and NMP, and washing with NMP, Fmoc-protected Gly, Arg(PMC), Cys(Acm), Gly, and Tyr(tBu) residues were coupled in succession. Arg was additionally protected with a PMC group, the second Cys 25 residue was additionally protected by an Acm group and Tyr was protected with a t-butyl group. The final Tyrosine was acetylated with acetic anhydride. This yielded the resin coupled peptide: tBu Acm PMC Boc Acm
Figure imgf000041_0001
s-NH-RINK
This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoracetate, anisole, and trifluoro- 35 acetic acid. The cyclized peptide product was purified on HPLC in a 0.1% TFA H20-acetonitrile gradient. The TFA salt produced could be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4a (acetate cycle) . The finished peptide is:
Acetyl-Tyr-Gly-Cys-Arg-Gly-Orn-Cys-NH2
Example 15 Synthesis of
10 Acetyl-Cys-Asn-Pro-Arg-Gly-MeOrn-Cys-OH
The synthesis was started with
Acm
I
Fmoc-Cys-O-HMP
15 The alpha-amino Fmoc protecting group was removed (main¬ taining protection of Cys with the Acm group.) using pipe¬ ridine and NMP. Fmoc-protected DL-1-Me-Orn(Boc) was then coupled to cysteine with dicyclohexylcarbodiimide followed by deprotection again with piperidine and NMP. Following
20 this stepwise procedure of coupling with dicyclohexylcar¬ bodiimide, deprotection with piperidine/NMP, Fmoc-pro¬ tected Gly, Arg(PMC) , Pro, Asn(TRT) , Cys(Acm) residues were coupled in succession. The Arg was additionally pro¬ tected by pentamethyl chroman-6-sulfonyl (Arg(PMC)), the
25 Asn with trityl (Asn(TRT)) , and the final Cys with an Acm group (Cys(Acm)). The final Cys was acetylated with ace¬ tic anhydride. Following acetylation, the following pep¬ tide resin as formed:
Acm TRT PMC Boc Acm
-if* I I I I I
*-5U 1 1 1 I I
Acetyl-Cys-Asn-Pro-Arg-Gly-MeOrn-Cys-O-HMP
This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoracetate, anisole, and trifluoro- 35 acetic acid. The cyclized peptide product was purified on HPLC in a 0.12% TFA H20-acetonitrile gradient. The TFA salt is produced by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished peptide is:
Acetyl-Cys-Asn-Pro-Arg-Gly-MeOrn-Cys-OH
Example 16 Synthesis of
Gly-Pen-Gly-Arg-Gly-Orn-Ser-Pro-Cys-NH2
The synthesis was started with
Acm
I
Fmoc-Cys-NH-RINK The alpha-amino Fmoc protecting group was removed (main¬ taining protection of Cys with the Acm group) using pipe¬ ridine and NMP. Fmoc-protected Pro was then coupled to cysteine with dicyclohexylcarbodiimide followed by depro¬ tection again with piperidine and NMP. Following this stepwise procedure of coupling with dicyclohexylcarbodi¬ imide, deprotection with piperidine/NMP, Fmoc-protected Ser(t-Bu), Orn(t-Boc), Gly, Arg(PMC) , Gly, Pen(Acm) , and Gly residues were coupled in succession. The Arg was additionally protected by penta ethyl chroman-6-sulfonyl (Arg(PMC)), the Ser with t-butyl, the Orn with t-Boc, and the penicillamine with an Acm group. The following pep¬ tide resin was formed:
Acm PMC Boc t-Bu Acm
I I I I I
I I I I I Gly-Pen-Gly-Arg-Gly-Orn-Ser-Pro-Cys-NH-RINK
This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid. The cyclized peptide product was purified on HPLC in a 0.1% TFA H20-acetonitrile gradient. The TFA salt produced could be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished peptide was:
Gly-Pen-Gly-Arg-Gly-Orn-Ser-Pro-Cys-NH2
Example 17 Synthesis of
Figure imgf000044_0001
Gly-Pen-Gly-His-Arg-Gly-Orn-Leu-Arg-Cys-Ala-NH2
The synthesis was started with
Fmoc-Ala-NH-RINK
The alpha-amino Fmoc protecting group was removed using piperidine and NMP. Fmoc-protected Cys(Acm) was then coupled to alanine with dicyclohexylcarbodiimide followed by deprotection again with piperidine and NMP. Following this stepwise procedure of coupling with dicyclohexylcar¬ bodiimide, deprotection with piperidine/NMP, Fmoc-pro- tected Arg(PMC) , Leu, Orn(t-Boc) , Gly, Arg(PMC) , His(TRT), Gly, Pen(Acm) , and Gly residues were coupled in succes¬ sion. The Arg was additionally protected by pentamethyl chroman-6-sulfonyl, the His with trityl, the Orn with t-Boc, and the penicillamine with an Acm group. The fol- lowing peptide resin was formed:
Acm TRT PMC Boc PMC Acm
I I I I I I
I I I I I I
Gly-Pen-Gly-His-Arg-Gly-Orn-Leu-Arg-Cys-Ala-NH-RINK
This peptide was cleaved from the resin, deprotected, and cyclized by treatment of the peptide-resin with a mixture of thallium tristrifluoroacetate, anisole, and trifluoro- acetic acid. The cyclized peptide product was purified on HPLC in a 0.1% TFA H20-acetonitrile gradient. The TFA salt produced could be converted to the HOAc salt by passing a solution of the peptide through an ion exchange column BioRad Ag3-X4A (acetate cycle) . The finished peptide was:
Gly-Pen-Gly-His-Arg-Gly-Orn-Leu-Arg-Cys-Ala-NH2
Example 18 Synthesis of cyclo-S-acetyl-Gly-Arg-Gly-Orn-Cys-OH The synthesis was started with TPM
I I
Fmoc-Cys-O-HMP
The alpha-amino Fmoc protecting group was removed (main¬ taining protection of Cys with the TPM group) using pipe- ridine and NMP. Fmoc-protected Orn(Boc) was then coupled to cysteine with dicyclohexylcarbodiimide followed by deprotection again with piperidine and NMP. Following this stepwise procedure of coupling with dicyclohexylcar¬ bodiimide, deprotection with piperidine/NMP, Gly, Arg (PMC) , and Gly residues were coupled in succession. The Arg was additionally protected by pentamethyl chroman-6- sulfonyl (Arg(PMC)). The final Gly was acetylated with bromoacetic acid by activation of the carboxyl group with DCC followed by coupling to the N-terminal amino group. Following acetylation the following peptide resin was formed:
PMC Boc TPM
I I I
I I I
Bromo-acetyl-Gly-Arg-Gly-Orn-Cys-O-HMP Repeated treatment of the resin bound peptide with a dilute solution of trifluoroacetic acid in dichloromethane results in the cleavage of the S-triphenylmethyl group as evidenced by a bright yellow color of the solution. Treat¬ ment is continued until the yellow color disappeared. After complete cleavage of the S-triphenylmethyl group (TPM) the resin bound peptide is washed several times with a 5% solution of N-methyImorpholine in N,N-dimethylace- ta ide and then shaken in pure N,N-dimethylacetamide for 12 hr to complete the cyclization. Treatment of the cyclized resin bound peptide with TFA containing (v/v) 1% phenol, 1% anisole and 1% ethanedithiol completed cleavage of the protective groups and cleaved the desired product from the resin. The finished product is:
cyclo-S-acetyl-Gly-Arg-Gly-Orn-Cys-OH
Example A
(i) Receptor GPIIb/IIIa preparation
The platelet integrin gpIIb/IIIa was purified from platelets by affinity chromatography using immobilized concanavalin A (Con A) . Five units of platelets obtained from a local blood bank were washed using differential centrifugation. GPIIb/IIIa was extracted from the mem¬ brane with 100 mM- octylthioglucopyranoside at 4° C for 20 min. The extract was run through a 9 ml Con A Sepha- rose 4B column and retained glycoprotein (including GPIIb/ Ilia) was eluted with 150 mM mannopyranoside. This glyco- protein-containing fraction is composed of 50-60% GPIIb/ Ilia as judged by SDS-polyacrylamide gel electrophoresis.
(ii) Compound Testing The inhibition of fibrinogen binding to GPIIb/IIIa by different compounds was ascertained using an assay in which fibrinogen-GPIIb/IIIa complexes are captured by an anti-GPIIb/IIIa monoclonal antibody (10D8) immobilized in the wells of a microtiter plate. HRP-fibrinogen (67 ng/ml) was incubated with GPIIb/IIIa-containing platelet glycoprotein fraction (4.5 ug/ml) in the presence or absence of sample compound at 37°C for 1 hour. Fifty microliters of the mixture was then transferred to a microtiter plate previously coated with 10 ug/ml MAb 10D8 and incubated at 37°C for 1 hr. The plate was washed 3 times with Tyrode's buffer containing 1 mM CaCl2 and 0.05% (w/v) Tween 20 before adding chromogenic substrate, tetra- methylbenzidine. Color reaction was stopped with 1 N H2SOA. A fifty molar excess of fibrinogen was used in this assay to determine non-specific binding control. Figure 2 depicts inhibition of HRP-fibrinogen binding to GPIIb/IIIa and compares the compound of Example 5 to the i 1 cyclic peptide Cys-Asn-Pro-Arg-Gly-Asp-Cys.
Example B
(i) ELISA-based Assay for Fibrinogen Binding to Platelets
Poly L lysine was coated on dynatech immullon II microtiter plates at 10 ug/ml in 0.1 M bicarbonate buffer pH 9.5, 2 hours at 37°C. The plate was washed three times with distilled water and dried for 30 minutes at 37°C. Thrombin stimulated platelets at 6.7 x 107 cells/ml in PBS pH 7.2 with 1 mM calcium chloride were added at 50 uL per well. Incubation was carried out for 18 hours at 4°C. The plate was washed three times with PBS, lmM calcium chloride, 0.02% Tween 20 (PBS/Tween) and blocked with PBS/0.5% BSA O.N. at 4°C.
(ii) Compound Testing
The blocking buffer was removed from the above prepa¬ ration and the inhibitory peptide, diluted in PBS. lmM calcium chloride was added (50 uL per well) followed by incubation for 1 hour at 37°C. Peroxidase labeled fibri¬ nogen (125 ng/ml) , diluted in platelet diluent buffer (TBS; 50 mM Tris, 140 mM NaCl, pH 7.4, 0.1% BSA, 0.02% Tween 20) was added (50 uL per well) and incubated for 1 hour at 25°C. The plate was washed five times with PBS/ Tween then tetramethybenzidine (100 uL per well) was added and the plate was incubated for 10 minutes. The color reaction was stopped with 100 uL per well of 1 N sulfuric acid. Optical density was read at 450-650 nm and test compounds were compared with controls. Figure 1 depicts inhibition of HRP-fibrinogen binding to GPIIb/IIIa by the following: (a) the compound of Exam¬ ple 16; (b) the compound of Example 17; (c) the compound of Example 5; (d) the peptide Gly-Arg-Gly-Asp-Ser; (e) the 17-amino acid peptide: Cys-Gly-Gly-Ser-Thr-Ser-Tyr-Asn- Arg-Gly-Asp-Ser-Thr-Phe-Glu-Ser-Lys-COOH.
Example C
Platelet Aggregation Studies
The effects of the Arg-Gly-Orn peptides of the pres- ent invention on blood platelet aggregation were examined. Platelet aggregation was performed with platelet-rich plasma (PRP) . PRP was stirred at 37°C in an aggregometer (Sienco Model 247, Morrison, CO) and aggregation was initiated by the addition of 12.5 μM of ADP. The control compound used was the peptide Gly-Arg-Gly-Asp-Ser, syn¬ thesized at Corvas, International. Aggregation was moni¬ tored as a change in light transmittance, and is expressed as the initial rate of aggregation.
Figures 3(a) and 3(b) depicts inhibition of platelet aggregation over time by the compound of Example 16 at two concentrations (a) 250 μM and (b) 500 μM. The top curve of each figure depicts uninhibited aggregation and the bottom curve inhibition of aggregation due to the compound of Example 16.

Claims

Claims
1. A peptide containing the tripeptide sequence:
-A-Gly-Orn- wherein A is an L- or D-isomer of arginine, homoarginine, guanidinoaminobutyric acid, guanidinoaminopropionic acid, N1, N"-dimethylarginine, N' ,N"-diethylarginine, p-amino- methylpheny1-alaninine, p-amidinophenylalanine, p-guani- dinophenylalanine, D-argine, a conformationally con¬ strained arginine analog, lysine or an α-R'-substituted derivative thereof wherein R* is lower alkyl wherein said peptide has an IC50 (μM) of no more than about 500 in an assay of thrombin-induced platelet aggregation.
2. A peptide according to claim 1 which has a rigid structure.
3. A peptide according to claim 1 which is cyclic.
4. A cyclic peptide according to claim 3 wherein the cycle comprises a covalent linkage through an amino acid side chain.
5. A cyclic peptide according to claim 4 wherein the cycle comprises a peptide linkage of the peptide backbone between amino acids.
6. A cyclic peptide according to any of claims 3, 4 or 5 wherein the cyclic peptide has from about 5 to about 12 amino acids in the ring.
7. A cyclic peptide containing the tripeptide sequence:
A-Gly-Orn wherein A is an L- or D-isomer of arginine, homoarginine, guanidinoaminobutyric acid, guanidinopropionic acid, N', N"-dimethylarginine, N' ,N"-diethylarginine, p-amino- methylphenylalanine, p-amidinophenylalanine, or p-guani- dinophenylalanine, a conformationally constrained arginine analog, lysine or an α-R'-substituted derivative thereof wherein R' is lower alkyl.
8. A cyclic peptide according to claim 7 wherein the cycle comprises a covalent linkage through an amino acid side chain.
9. A cyclic peptide according to claim 7 wherein the cycle comprises a peptide linkage of the peptide backbone between amino acids.
10. A cyclic peptide according to any of claims 7, 8 or 9 wherein the cyclic peptide has from about 5 to about 12 amino acids.
11. A cyclic peptide according to claim 7 which is selected from:
Ac-Cys-(D-Arg)-Arg-Gly-Orn-Cys-OH
Ac-Cys-ThioPro-Arg-Gly-Orn-Cys-OH
Ac-Cys-Pro-Arg-Gly-Orn-Cys-OH
Ac-Cys-Asn-Pro-Arg-Gly-Orn-Cys-NH2
Ac-Cys-Asn-Pro-Arg-Gly-Orn-Cys-OH Ac-Cys-Asn-ThioPro-Arg-Gly-Orn-Cys-OH
Ac-Cys-Arg-Gly-Orn-Cys-NH2
Ac- (D-Cys) -Arg-Gly-Orn-Cys-NH2
Ac-Pen-Arg-Gly-Orn-Cys-NH2
Ac- (homoCys) -Arg-Gly-Orn-Cys-NH2
Ac-Cys-Arg-Gly-Orn-Pen-NH2
Ac-Cys-Arg-Gly-Orn-Cys-OH
Ac-Pen-Arg-Gly-Orn-Cys-OH
Ac-Cys-Arg-Gly-Orn-Cys-Pro-NH2
(CH2 ) 6
C (=0) -Arg-Gly-Orn-NH
Figure imgf000051_0001
Ac- (N-MethylCys) -Arg-Gly-Orn-Cys-OH
Ac-Cys-Arg-Gly-Orn- (D-Cys) -NH2
Ac-Cys-Arg-Gly-Orn-Pen-OH
Ac-Pen-Arg-Gly-Orn-Pen-NH.
Ac- (D-Pen) -Arg-Gly-Orn-Pen-OH
Ac-Cys-Arg-Gly-Orn- (homoCys) -NH2
Ac-Cys- (D-Arg) -Gly-Orn-Cys-NH2
Ac-Cys-MeArg-Gly-Orn-Ser-Cys-NH2
Ac-Cys-Arg-Gly-Orn-Ser-Cys-NH,
Ac-Cys-MeArg-Gly-Orn- (Me) Ser-Cys-NH2
Ac-Cys-MeArg-Gly-Orn-Cys-NH2 Ac-Cys-Arg-Gly-Orn-Pen-NH2
Ac-Cys-Arg-Gly-Orn- (D-Pen) -NH2
Ac-Cys-Lys-Gly-Orn-Pen-NH2
Ac-Cys-MeArg-Gly-Orn-Pen-NH2
Ac-Cys-Arg-Gly-Orn-Pen-NHEt
Ac-Cys-DArg-Gly-Orn-Pen-NH2
Ac-Cys-MeArg-Gly-Orn-Tyr-Cys-NH-
Ac-Pen-MeArg-Gly-Orn-Pen-NH2
Ac-Pen-Arg-Gly-Orn-Cys-NH2
Ac-Cys-DArg-Gly-Orn-Ser-Cys-NH2
Ac-Cys-Sar-Arg-Gly-Orn-Pen-NH2 Ac-Cys-Arg-Gly-Orn-Cys-NH2
Ac-Cys-Arg(Et) 2-Gly-Orn-Pen-NH2
Ac-Cys-DMeArg-Gly-Orn-Pen-NH2
Arg-Gly-Orn-Arg-Gly
Arg-Gly-Orn-Ser-Pro-Ala-Gly
Arg-Gly-Orn-Ser-Pro-Ala-Val-Thr-Gly
Arg-Gly-Orn-Ser-Pro-Ala-Ala-Val-Thr-Gly
Arg-Gly-Orn-Asn-Pro-Ala-Gly
Gly-Cys-Gly-Lys-Gly-Orn-Trp-Pro-Cys-Ala-NH2
Acetyl-Tyr-Gly-Cys-Arg-Gly-Orn-Cys-NH2
Acetyl-Cys-Asn-Pro-Arg-Gly-MeOrn-Cys-OH Gly-Pen-Gly-Arg-Gly-Orn-Ser-Pro-Cys-NH2
Gly-Pen-Gly-His-Arg-Gly-Orn-Leu-Arg-Cys-Ala-NH2
cyclo-S-acetyl-Gly-Arg-Gly-Orn-Cys-OH
12. A cyclic peptide according to claim 7 of the formula:
L-(K)q-G-(E)0-(B)m-A-Gly-Orn-(D)n-F-(J)p-M wherein m is O or 1; n, o, p, and q are independently 0, 1 or 2; B is a D- or L-isomer of proline, β-methylproline, β,β-dimethylproline; hydroxyproline, anhydroproline, thio- proline, β-methylthioproline, β,β-dimethylthioproline, pipecolic acid, azetidine carboxylic acid, N-methylargi- nine, N-methylhistidine, N-methylasparagine, N-methy1-Aib, Abu, alanine, glycine, N' ,N"-dimethylarginine, arginine, histidine, asparagine, homoarginine, guanidinoaminobutyric acid, guanidinoaminopropionic acid, sarcosine or an α-R1 derivative thereof wherein R' is lower alkyl; D is an amino acid independently selected from a D- or L-isomer of Tyr, (alk)Tyr, Phe, Trp, His, Ser, (alk)Ser, Thr, (alk) Thr, Ala, Val, norvaline, Met, Leu, lie, Nle or an α-R' derivative thereof and alk is lower alkyl; E is an independently selected D- or L-amino acid; F and G are independently selected amino acids having side chains chemically bonded to each other; J and K are independently selected amino acids; L is hydrogen, aryl, aminoacyl, or N-methylaminoacy1; and M is -OH, -NH2, -NHR3, -N ^ or the C-terminal carboxylic acid is esterified with -OR4 wherein R2 and R3 are independently selected alkyl groups of 1 to 4 carbon atoms and R4 is alkyl of 1 to 6 carbon atoms or aralkyl of 7 to 9 carbon atoms.
13. A cyclic peptide according to claim 12 wherein F and G are selected from: (a) cysteine and penicillamine in any combination; (b) F is -NHCH(X)C(=0)- and G is -NHCH (Y)C(=0)-; (c) F and G taken together are cyclized to give
Figure imgf000056_0001
wherein each Z is independently hydrogen or lower alkyl and -X-Y- are selected from -S-S-, -CH2-S-, -S-CH2-, (CH2)3-, (CH2)4-, -CH2-S-S-CH2-, -CH2-S-S, and -S-S-CH2~; (or) (d) when q is 0, F is cysteine or penicillamine* and G is -(CH2C(=0)- S CZ2
I I
CH2-C(=0)-(E)0-(B)m-A-Gly-Om-(D)n-NH-CH-C(=0)-(J)p-M
14. A cyclic peptide according to claim 7 of the formula:
J-(E)s-("•B)'m-A-Gly•*-Or—n-("»D) • r-K wherein m is 0 or 1; r and s are independently 0, 1, 2 or 3; B is a D- or L-isomer of proline, β-methylproline, β,β- dimethylproline, hydroxyproline, anhydroproline, thiopro- line, β-methylthioproline, β,β-dimethylproline, pipecolic acid, azetidine carboxylic acid, N-methylarginine, N-methylhistidine, N-methylasparagine, N-methy1-Aib, Abu, alanine, glycine, N* ,N"-dimethylarginine, arginine, histi- dine, asparagine, homoarginine, guanidinoaminobutyric acid, guanidinoaminopropionic acid, sarcasine or an α-R' derivative thereof wherein R' is lower alkyl; D is an amino acid independently selected from a D- or L-isomer of Tyr, (alk)Tyr, Phe, Trp, His, Ser, (alk)Ser, Thr, (alk) Thr, Ala, Val, norvaline. Met, Leu, lie, Nle or an α-R* derivative thereof and alk is lower alkyl; and J and K are independently selected D- or L-amino acids and are linked by a peptide linkage.
15. A method for reducing platelet aggregation in a mammal which comprises administering a pharmaceutically effective amount of a peptide according to any of claims 1, 7, 12 or 14 to said mammal.
16. A method for treating a mammal in order to diminish the propensity for blood clotting which comprises administering a pharmaceutically amount of a peptide according to any of claims 1, 7, 12 or 14 to said mammal.
17. A method for inhibiting fibrinogen binding to platelets in a mammal which comprises administering a pharmaceutically effective amount of a peptide according to any of claims 1, 7, 12 or 14.
18. A composition having aggregation inhibiting activity which comprises an aggregation inhibiting effec- tive amount of a compound according to any of claims 1, 7, 12 or 14 and a pharmaceutically acceptable carrier.
19. A peptide according to any of claim 1, 7, 12 or 14 wherein Orn is replaced by 1-methyl Orn.
PCT/US1992/005418 1991-06-28 1992-06-24 Novel inhibitors of platelet aggregation WO1993000108A1 (en)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011943A1 (en) * 1994-10-14 1996-04-25 Astra Aktiebolag New peptides with immunomodulatory effects
WO1997011718A1 (en) * 1995-09-27 1997-04-03 The Victoria University Of Manchester Inhibitors of integrin receptors and their therapeutical uses
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6228373B1 (en) 1995-03-24 2001-05-08 Astra Aktiebolag Peptides with immunomodulatory effects
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US9988434B2 (en) * 2014-09-12 2018-06-05 Wisconsin Alumni Research Foundation Optimal interstrand bridge for collagen mimics

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4857508A (en) * 1987-12-03 1989-08-15 Monsanto Company Novel platelet-aggregation inhibitor peptide derivatives
EP0341915A2 (en) * 1988-05-09 1989-11-15 Smithkline Beecham Corporation Anti-aggregatory peptides
US4952562A (en) * 1989-09-29 1990-08-28 Rorer Pharmaceutical Corporation Anti-thrombotic peptides and pseudopeptides
EP0410540A1 (en) * 1989-07-28 1991-01-30 Merck & Co. Inc. Fibrinogen receptor antagonists
EP0411833A1 (en) * 1989-07-28 1991-02-06 Merck & Co. Inc. Fibrinogen receptor antagonists

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4857508A (en) * 1987-12-03 1989-08-15 Monsanto Company Novel platelet-aggregation inhibitor peptide derivatives
EP0341915A2 (en) * 1988-05-09 1989-11-15 Smithkline Beecham Corporation Anti-aggregatory peptides
EP0410540A1 (en) * 1989-07-28 1991-01-30 Merck & Co. Inc. Fibrinogen receptor antagonists
EP0411833A1 (en) * 1989-07-28 1991-02-06 Merck & Co. Inc. Fibrinogen receptor antagonists
US4952562A (en) * 1989-09-29 1990-08-28 Rorer Pharmaceutical Corporation Anti-thrombotic peptides and pseudopeptides

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PEPTIDE HORMONES, issued June 1976, J. RUDINGER, "Characteristics of the Amino Acids as Components of a Peptide Hormone Sequence", pages 1-7. *
PEPTIDES CHEMISTRY, STRUCTURE AND BIOLOGY, PROCEEDINGS OF THE ELEVENTH AMERICAN PEPTIDE SYMPOSIUM, issued 09-14 July 1989, F.E.F. ALI et al., "Structure-Activity Studies Toward the Improvement of Antiaggregatory Activity of Arg-Gly-Asp-Ser (RGDS)", pages 94-96. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, Volume 3, issued August 1986, Z.M. RUGGERI et al., "Inhibition of Platelet Function with Synthetic Peptides Designed to be High-Affinity Antagonists of Fibrogen Binding to Platelets", pages 5708-5712. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
WO1996011943A1 (en) * 1994-10-14 1996-04-25 Astra Aktiebolag New peptides with immunomodulatory effects
AU705364B2 (en) * 1994-10-14 1999-05-20 Astra Aktiebolag New peptides with immunomodulatory effects
US6228374B1 (en) 1994-10-14 2001-05-08 Astra Aktiebolag Peptides with immunomodulatory effects
US6228373B1 (en) 1995-03-24 2001-05-08 Astra Aktiebolag Peptides with immunomodulatory effects
WO1997011718A1 (en) * 1995-09-27 1997-04-03 The Victoria University Of Manchester Inhibitors of integrin receptors and their therapeutical uses
US9988434B2 (en) * 2014-09-12 2018-06-05 Wisconsin Alumni Research Foundation Optimal interstrand bridge for collagen mimics

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