WO1994015913A1 - Chemical compounds - Google Patents

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Publication number
WO1994015913A1
WO1994015913A1 PCT/US1993/012531 US9312531W WO9415913A1 WO 1994015913 A1 WO1994015913 A1 WO 1994015913A1 US 9312531 W US9312531 W US 9312531W WO 9415913 A1 WO9415913 A1 WO 9415913A1
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WO
WIPO (PCT)
Prior art keywords
compound according
benzoyl
4alkyl
phenoxy
pyrrolidinyl
Prior art date
Application number
PCT/US1993/012531
Other languages
French (fr)
Inventor
James Samanen
Original Assignee
Smithkline Beecham Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to EP94905503A priority Critical patent/EP0677041A1/en
Priority to JP6516048A priority patent/JPH08505391A/en
Publication of WO1994015913A1 publication Critical patent/WO1994015913A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • This invention relates to novel compounds which inhibit platelet aggregation, pharmaceutical compositions containing the compounds and methods of using the compounds.
  • GPIIb-IIIa platelet receptor complex which is a member of a family of adhesion receptors referred to as integrins. It has been found that frequently the natural ligands of integrin receptors are proteins which contain an Arg-Gly-Asp sequence (RGD in single letter amino acid code). Von Willebrand factor and fibrinogen, which are considered to be natural ligands for the GPIIb-IIIa receptor, possess an RGD sequence in their primary structure. Functionally, these proteins are able to bind and crosslink GPIIb-IIIa receptors on adjacent platelets and thereby effect aggregation of platelets.
  • RGD Arg-Gly-Asp sequence
  • Fibronectin, vitronectin and thrombospondin are RGD-containing proteins which have also been demonstrated to bind to GPIIb-IIIa.
  • Fibronectin is found in plasma and as a structural protein in the intracellular matrix. Binding between the structural proteins and GPIIb-IIIa may function to cause platelets to adhere to damaged vessel walls.
  • Linear and cyclic peptides which bind to vitronectin and contain an RGD sequence are disclosed in WO 89/05150 (PCT US88/04403).
  • EP 0 275 748 discloses linear tetra- to hexapeptides and cyclic hexa- to octapeptides which bind to the GPIIb-IIIa receptor and inhibit platelet aggregation.
  • Other linear and cyclic peptides are reported in EP-A 0 341 915.
  • the peptide like structures of such inhibitors often pose problems, such as in drug delivery, metabolic stability and selectivity.
  • Inhibitors of the fibrinogen receptor which are not constructed of natural amino acid sequences are disclosed in EP-A 0372,486, EP-A 0 381 033 and EP-A 0478 363.
  • WO 92/07568 discloses fibrinogen receptor antagonists which mimic a conformational ⁇ -turn in the RGD sequence by forming a monocyclic seven-membered ring structure.
  • novel fibrinogen receptor antagonists e.g. inhibitors of the GPIIb-IIIa protein
  • the present invention discloses novel compounds that inhibit binding to the GPIIb-IIIa receptor and inhibit platelet aggregation.
  • this invention is a compound comprising a five- or six- membered ring as described hereinafter in formula (I).
  • This invention is also a pharmaceutical composition for inhibiting platelet aggregation or clot formation, which comprises a compound of formula (I) and a pharmaceutically acceptable carrier.
  • This invention is further a method for inhibiting platelet aggregation in a mammal in need thereof, which comprises internally administering an effective amount of a compound of formula (I).
  • this invention provides a method for inhibiting reocclusion of an artery or vein in a mammal following fibrinolytic therapy, which comprises internally administering an effective amount of a fibrinolytic agent and a compound of formula (I).
  • This invention is also a method for treating stroke, transient ischemia attacks, myocardial infarction, or atherosclerosis.
  • This invention discloses novel compounds which inhibit platelet aggregation.
  • the novel compounds comprise a five- or six-membered ring.
  • the five- or six- membered ring may contain heteroatoms, such as nitrogen, oxygen and sulfur.
  • the five- or six-membered ring system is believed to interact favorably with the GPIIb- IIIa receptor and to orient the substituent sidechains on the five- or six-membered rings so that they may also interact favorably with the receptor.
  • the compounds of this invention are compounds of formula (I):
  • A is -(CH2)r > Het, Ar, or C3_7cycloalkyl
  • Qi to Q" form any accessible substituted five- or six-membered ring, which may be saturated or unsaturated, optionally containing up to two heteroatoms chosen from the group of O, S, and N wherein S and N may be optionally oxidized;
  • Y is -CO-, -CR'(OH)-, -SO 2 -, -CR'2-, -CONR'-, -NR'CO-, SO 2 NR', or -NR'SO 2 -;
  • M is Het, Ar, or C3_7cycloalkyl, each of which is unsubstituted or substituted by R 3 ;
  • each R' independently is hydrogen, C ⁇ _4alkyl, C3.7cycloalkyl-C ⁇ -4alkyl, or
  • each R" independently is R', -C(O)R', or -C(O)OR 15 ;
  • R 2 is absent or present as C ⁇ alkyl, CO2 ', Z-CO2R', C ⁇ alkoxy, hydroxy, C ⁇ alkylthio, CONR'R', CN, CF 3 , halo, or NR'R';
  • R 3 is C ⁇ _4al yl, C ⁇ alkoxy, Cj ⁇ alkylthio, hydroxy, halo, CO2R', Z-CO 2 R', NR'R', CN CONR'R', CF3, or Ar-Co-4alkyl;
  • each R*5 independently is Cj.galkyl, C3.7cycloalkyl-C ⁇ -4alkyl, or Ar-Co-4alkyl;
  • J is absent or present as S or O;
  • n 1 or 2;
  • each n independently is 0 or 1;
  • each t independently is 0 to 2;
  • Prodrugs are considered to be any covalently bonded carriers which release the active parent drug according to formula (I) in vivo.
  • this invention includes each unique nonracemic compound which may be synthesized and resolved by conventional techniques.
  • compounds may have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention.
  • compounds may exist in tautomeric forms, such as keto-enol tautomers, such as and tautomers of guanidine-type groups, such as NR" NR' 2
  • R-R'N I NR'-X- and R . R . N NR'-X-, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or locked in one form by appropriate substitution with R'.
  • the meaning of any substituent at any one occurrence is independent of its meaning, or any other substituent's meaning, at any other occurrence, unless specified otherwise.
  • Q ⁇ to Q" optionally substituted by R 2 are any accessible combination of the following:
  • Q 1 is CH, C, or N
  • Q 2 is CH, C, or N
  • Q3 is CH 2 , CHR 2 , C(R 2 ) 2 , CH, CR 2 , NH, NR 2 , N, O, or S;
  • Q 4 is CH 2 , CHR 2 , C(R 2 ) 2 , CH, CR 2 , NH, NR 2 , N, O, or S;
  • Q 5 is absent or present as CH2, CHR 2 , C(R 2 )2, CH, CR 2 , NH, NR 2 , N, O, or S;
  • Q 6 is CH 2 , CHR 2 , C(R 2 ) 2 , CH, CR 2 , NH, NR 2 , N, O, or S.
  • Q 1 to Q ⁇ optionally substituted by R 2 is
  • A is phenyl;
  • X is -CO-, -CH(OH)-, -SO 2 -, -CONR'- or -NR'CO-;
  • Y is -CO-, -CH(OH)-, -SO2-, -CONR'- or -NR'CO-;
  • M is phenyl;
  • R 2 is absent; t is O to 1; each n independently is 0; and Q 1 to Q 6 is
  • Particular compounds of the invention include, but are not limited to, the following:
  • the most prefered compound of this invention is N-[4- aminoiminomethyl)benzoyl]-4-oxyacetic acid-(R)-prolylbenzene or a pharmaceutically acceptable salt thereof.
  • C ⁇ _4alkyl as applied herein means carbon chains which are branched or unbranched and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t- butyl.
  • Ci- ⁇ -alkyl additionally includes pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof.
  • Co-4alkyl additionally indicates that no alkyl group need be present (e.g. that a covalent bond is present).
  • Ar, or aryl as applied herein, means phenyl or naphthyl, or phenyl or naphthyl substituted by one to three moieties R 2 .
  • R 2 may be Ci ⁇ alkyl, Ci ⁇ alkoxy, C ⁇ alkythio, CF3, OH, CI, Br, I, F, C ⁇ 2R', or Z-CO2R', wherein Z and R' are as defined in formula (I).
  • Het, or heterocycle indicates an optionally substituted five or six membered monocyclic ring, or a nine or ten-membered bicyclic ring containing one to three heteroatoms chosen from the group of nitrogen, oxygen and sulfur, which are stable and available by conventional chemical synthesis.
  • Illustrative heterocycles are imidazole, benzimidazole, pyrrole, indole, pyridine, pyrimidine, pyrazine, quinoline, benzofuran, furan, benzopyran, benzothiophene, thiophene, thiazole, benzothiazole, indoline, moipholine, piperidine, piperazine, pyrrolidine, isoquinoline, and tetra- and perhydro- quinoline and isoquinoline. Any accessible combination of up to three substituents, such as chosen from R 2 , on the Het ring that is available by chemical synthesis and is stable is within the scope of this invention.
  • C3_.7cycloalkyl refers to an optionally substituted carbocyclic system of three to seven carbon atoms, which may contain up to two unsaturated carbon-carbon bonds.
  • Typical of C3-7cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl and cycloheptyl. Any combination of up to three substituents, such as chosen from R 2 , on the cycloalkyl ring that is available by conventional chemical synthesis and is stable, is within the scope of this invention.
  • Any accessible substituted five- or six-membered ring as referred to herein is any saturated or unsaturated five- or six-membered ring which (i) is optionally substituted by R 2 , wherein the substituents may be present on any atom or heteroatom that results in a stable structure, and (ii) contains up to two heteroatoms selected from the group of N, O and S, wherein S and N may optionally be oxidized, and (iii) is stable and may be synthesized by one skilled in the chemical arts.
  • Typical of accessible five-membered rings are the common saturated and unsaturated rings of cyclopentane, cyclopentene, furan, thiophene, imidazole, pyrrole, thiazole, oxazole, pyrrolidine, pyrroline, and pyrazole.
  • Typical of accessible six-membered rings are the common saturated and unsaturated rings of cyclohexane, cyclohexene, benzene, pyridine, pyrimidine, pyridazine, pyrazine, piperidine, piperazine, morpholine, and dihydropyridine. ⁇ .
  • a nitrogen heterocycle which may be a saturated or an unsaturated stable five-, six- or seven -membered monocyclic ring, or a seven- to ten membered bicyclic ring, containing up to three nitrogen atoms or containing one nitrogen atom and a heteroatom chosen from oxygen and sulfur, and which may be substituted on any atom that results in a stable structure, and wherein the nitrogen heteroatom may be optionally quaternized.
  • _ are pyrroline, pyrrolidine, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, piperidine, piperazine, morpholine, pyridine, tetrahydropyridine, tetrahydro- and hexahydro-azepine.
  • _ may be pyrolidinyl, piperidinyl, tetrahydropyridinyl, or piperidinyl.
  • t-Bu refers to the tertiary butyl radical
  • Boc refers to the t-butyloxycarbonyl radical
  • Fmoc refers to the fluorenylmethoxycarbonyl radical
  • Ph refers to the phenyl radical
  • Cbz refers to the benzyloxycarbonyl radical
  • BrZ refers to the o-bromobenzyloxycarbonyl radical
  • C1Z refers to the o-chlorobenzyloxycarbonyl radical
  • Bzl refers to the benzyl radical
  • 4-MBzl refers to the 4-methyl benzyl radical
  • Me refers to methyl
  • Et refers to ethyl
  • Ac refers to acetyl
  • Alk refers to C ⁇ _4alkyl
  • Nph refers to 1- or 2-naphthyl
  • cHex refers to cyclohexyl.
  • DCC refers to dicyclohexylcarbodiimide
  • DMAP refers to dimethylaminopyridine
  • DIEA refers to diisopropylethyl amine
  • EDC refers to N- ethyl-N'(dimethylaminopropyl)carbodiimide.
  • HOBt refers to
  • THF tetrahydrofuran
  • DIEA diisopropylethylamine
  • DMF dimethyl formamide
  • NBS N- bromosuccinimide
  • Pd/C a palladium on carbon catalyst
  • PPA 1- propanephosphonic acid cyclic anhydride
  • DPPA diphenylphosphoryl azide
  • BOP refers to benzotriazol-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate
  • HF hydrofluoric acid
  • TEA triethylamine
  • TFA trifluoroacetic acid
  • PCC pyridinium chlorochromate.
  • the compounds of formula (I) are generally prepared by reacting a compound :
  • L 1 and L 2 are groups which are capable of reacting to form the linkage -X-, wherein X is as defined in formula (I); or L* is hydrogen, Ql is nitrogen, and L 2 is a functional group which is capable of reacting with the Q* nitrogen to from certain X linkages; and thereafter removing any protecting groups, and optionally forming a pharmaceutically acceptable salt. It will be apparent that the precise identity of L 1 and L 2 will be dependent upon the site of the linkage being formed. For instance, if X is CONH, L 1 may be -NH2 L 2 and may be COOH or COC1. When L 2 is COOH, a coupling agent is used. Similarly, if X is NHCO, L 1 may be -CO2H or COC1 and L 2 may be -NH2.
  • L 1 may be H and Q 1 is nitrogen and L 2 may be COOH or COC1.
  • L 1 may be SO2CI and L 2 may be -NH2.
  • L 1 may be -NH2 and L 2 may be SO2CI.
  • Coupling reagents as used herein denote reagents which may be used to form peptide bonds. Typical coupling methods employ carbodiimides, activated anhydrides and esters and acyl halides. Reagents such as EDC, DCC, DPPA, PPA, BOP reagent, HOBt, N-hydroxysuccinimide and oxalyl chloride are typical.
  • Coupling methods to form peptide bonds are generally well known to the art.
  • the methods of peptide synthesis generally set forth by Bodansky et al., THE PRACTICE OF PEPTIDE SYNTHESIS, Springer- Verlag, Berlin, 1984, Ali et al. in J. Med. Chem., 29, 984 (1986) and J. Med. Chem., 30, 2291 (1987) are generally illustrative of the technique and are incorporated herein by reference.
  • Solution synthesis for the formation of amide or peptide bonds is accomplished using conventional methods used to form amide bonds.
  • the amine or aniline is coupled via its free amino group to an appropriate carboxylic acids substrate using a suitable carbodiimide coupling agent, such as N,N' dicyclohexyl carbodiimide (DCC), optionally in the presence of catalysts such as 1- hydroxybenzotriazole (HOBt) and dimethylaminopyridine (DMAP).
  • DCC N,N' dicyclohexyl carbodiimide
  • catalysts such as 1- hydroxybenzotriazole (HOBt) and dimethylaminopyridine (DMAP).
  • Other methods such as the formation of activated esters, anhydrides or acid halides, of the free carboxyl of a suitably protected acid substrate, and subsequent reaction with the free amine of a suitably protected amine, optionally in the presence of a base, are also suitable.
  • a protected Boc-amino acid or Cbz-amidino benzoic acid is treated in an anhydrous solvent, such as methylene chloride or tetrahydrofuran(THF), in the presence of a base, such as N-methylmorpholine, DMAP or a trialkylamine, with isobutyl chloroformate to form the "activated anhydride", which is subsequently reacted with the free amine of a second protected amino acid or aniline.
  • anhydrous solvent such as methylene chloride or tetrahydrofuran(THF)
  • a base such as N-methylmorpholine, DMAP or a trialkylamine
  • D is generally a basic functional group attached to A, optionally via an alkyl chain, and is protected during the synthesis, where necessary.
  • the reactive functional groups of the sidechains of each synthetic fragment are suitably protected as known in the art. Suitable protective groups are disclosed in Greene, PROTECTIVE GROUPS IN ORGANIC CHEMISTRY, John Wiley and Sons, New York, 1981.
  • the Boc, Cbz, phthaloyl orFmoc group may be used for protection of an amino or amidino group.
  • the Boc group is generally preferred for protection of an ⁇ -amino group.
  • a t-Bu, cHex or benzyl ester may be used for the protection of the side chain carboxyl.
  • a benzyl group or suitably substituted benzyl group eg.
  • 4-methoxy-benzyl or 2,4-dimethoxy-benzyl) is used to protect the mercapto group or the hydroxyl group.
  • the tosyl group may be used for protection of the imidazolyl group and tosyl or nitro group for protection of the guanidino group.
  • a suitably substituted carbobenzyloxy group or benzyl group may be also be used for the hydroxyl group or amino group.
  • Suitable substitution of the carbobenzyloxy or benzyl protecting groups is ortho and/or para substitution with chloro, bromo, nitro or methyl, and is used to modify the reactivity of the protective group.
  • the protective groups for the amino moiety are, most conveniently, those which are not removed by mild acid treatment. These protective groups are removed by such methods as catalytic hydrogenation, sodium in liquid ammonia or HF treatment, as known in the art.
  • Modification of amino groups may be accomplished by alkylation, sulfonylation, cyanation or acylation as is generally known in the art.
  • Scheme I provides a method of preparing compounds wherein Q ⁇ is absent, trogen, Q 2 is CH, Q 3 , OA and Q ⁇ are CH2, and A and M are each phenyl.
  • me I, R 2 ' and R 3 ', D', and R* indicate R 2 , R 3 , D, and R, respectively, or a suitable precursor thereof, wherein any functional groups are protected as known in the art.
  • Y' represents -CH(OH)- and -CO- groups.
  • a suitably protected prolylhalide of formula (I), such as N-trifluoroacetyl-D-prolylchloride is reacted with an aromatic compound of formula (2), such as anisole, in the presence of a Lewis acid catalyst, such as aluminum chloride, aluminum bromide, titanium tetrachloride or boron trifluoride, particularly aluminum chloride, in a suitable solvent, such as methylene chloride.
  • a Lewis acid catalyst such as aluminum chloride, aluminum bromide, titanium tetrachloride or boron trifluoride, particularly aluminum chloride
  • a suitable solvent such as methylene chloride
  • prolyl compounds are coupled with an appropriately substituted D'-(CH2)t-A-carboxylic acid compound, such as 4-N-Cbz- amidinobenzoic acid, in the presence of a suitable amide coupling agent, such as N- ethyl-N'(dimethylaminopropyl)carbodiimide, and optionally in the presence of a catalyst, such as 1-hydroxybenzotriazole, in a suitable solvent, such as dimethylfor amide.
  • a suitable amide coupling agent such as N- ethyl-N'(dimethylaminopropyl)carbodiimide
  • a catalyst such as 1-hydroxybenzotriazole
  • the formula (5) compounds may then be deprotected using conventional chemical techniques, for example, using hydrogen in the presence of a suitable catalyst, such as palladium on carbon, when a Cbz-protecting group is present, or using trifluoroacetic acid in a suitable solvent, such as methylene chloride, when a t-butyl ester is present.
  • a suitable catalyst such as palladium on carbon
  • trifluoroacetic acid in a suitable solvent, such as methylene chloride, when a t-butyl ester is present.
  • Acid addition salts of the compounds of formula (I) are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic or methanesulfonic.
  • the acetate salt form is especially useful.
  • Certain of the compounds form inner salts or zwitterions which may be acceptable.
  • Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine.
  • Cations such as Li+, Na+, K+, Ca++, Mg++ and NH4+ are specific examples of cations present in pharmaceutically acceptable salts.
  • This invention provides a pharmaceutical composition which comprises a compound according to formula (I) and a pharmaceutically acceptable carrier. Accordingly, the compounds of formula (I) may be used in the manufacture of a medicament.
  • Pharmaceutical compositions of the compounds of formula (I) prepared as hereinbefore described may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use.
  • the liquid formulation may be a buffered, isotonic, aqueous solution.
  • Suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution.
  • Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate. Alternately, these compounds may be encapsulated, tableted or prepared in a emulsion or syrup for oral administration.
  • Solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition.
  • Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
  • Liquid carriers include syrup, peanut oil, olive oil, saline and water.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit.
  • the pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms.
  • a liquid carrier When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension.
  • Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
  • the compounds of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
  • This invention also provides a method of inhibiting platelet aggregation and clot formation in a mammal, especially a human, which comprises the internal administration of a peptide of formula (I) and a pharmaceutically acceptable carrier.
  • Indications for such therapy include acute myocardial infarction (AMI), deep vein thrombosis, pulmonary embolism, dissecting aneurysm, transient ischemia attack (TIA), stroke and other infarct-related disorders, and unstable angina.
  • DIC disseminated intravascular coagulation
  • septicemia surgical or infectious shock
  • post-operative and post-partum trauma cardiopulmonary bypass surgery
  • incompatible blood transfusion abruptio placenta
  • thrombotic thrombocytopenic purpura TTP
  • snake venom and immune diseases
  • the compounds of this invention may be useful in a method for the prevention of metastatic conditions, the prevention or treatment of fungal or bacterial infection, inducing immunostimulation, and the prevention or treatment of diseases in which bone resorption is a factor.
  • the compounds of mis invention are administered either orally or parenterally to the patient, in a manner such that the concentration of drug in the plasma is sufficient to inhibit platelet aggregation, or other such indication.
  • the pharmaceutical compositions containing the compounds of this invention are administered at a dose between about 0.2 to about 50 mg kg in a manner consistent with the condition of the patient.
  • parenteral administration is preferred.
  • an intravenous infusion of the compound in 5% dextrose in water or normal saline is most effective, although an intramuscular bolus injection may be sufficient.
  • oral administration of a capsule or tablet, or a bolus intramuscular injection is suitable.
  • the compounds of this invention are administered one to four times daily at a level of about 0.4 to about 50 mg/kg to achieve a total daily dose of about 0.4 to about 200 mg/kg/day.
  • This invention further provides a method for inhibiting the reocclusion of an artery or vein following fibrinolytic therapy, which comprises internal administration of a compound of formula (I) and a fibrinolytic agent. It has been found that administration of certain compounds in fibrinolytic therapy either prevents reocclusion completely or prolongs the time to reocclusion.
  • fibrinolytic agent is intended to mean any compound, whether a natural or synthetic product, which directly or indirectly causes the lysis of a fibrin clot.
  • Plasminogen activators are a well known group of fibrinolytic agents.
  • Useful plasminogen activators include, for example, anistreplase, urokinase (UK), pro-urokinase (pUK), streptokinase (SK), tissue plasminogen activator (tPA) and mutants, or variants, thereof, which retain plasminogen activator activity, such as variants which have been chemically modified or in which one or more amino acids have been added, deleted or substituted or in which one or more or functional domains have been added, deleted or altered such as by combining the active site of one plasminogen activator with the fibrin binding domain of another plasminogen activator or fibrin binding molecule.
  • tPA molecules in which one or more glycosylation sites have been altered.
  • Preferred among plasminogen activators are variants of tPA in which the primary amino acid sequence has been altered in the growth factor domain so as to increase the serum half-life of the plasminogen activator.
  • tPA Growth factor variants are disclosed, e.g., by Robinson et al, EP-A 0 297 589 and Browne et al., EP-A 0 240 334.
  • Other variants include hybrid proteins, such as those disclosed in EP 0 028 489, EP 0 155 387 and EP 0 297 882, all of which are incorporated herein by reference.
  • Anistreplase is a preferred hybrid protein for use in this invention.
  • Fibrinolytic agents may be isolated from natural sources, but are commonly produced by traditional methods of genetic engineering. Useful formulations of tPA, SK, UK and pUK are disclosed, for example, in EP-A 0 211 592, EP-A 0092 182 and U.S. Patent 4,568,543, all of which are incorporated herein by reference.
  • the fibrinolytic agent may be formulated in an aqueous, buffered, isotonic solution, such as sodium or ammonium acetate or adipate buffered at pH 3.5 to 5.5. Additional excipients such as polyvinyl pyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene, glycol, mannitol and sodium chloride may also be added. Such a composition can be lyophilized.
  • the pharmaceutical composition may be formulated with both the compound of formula (I) and fibrinolytic in the same container, but formulation in different containers is preferred.
  • both agents are provided in solution form they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement.
  • Indications for such therapy include myocardial infarction, deep vein thrombosis, pulmonary embolism, stroke and other infarct-related disorders.
  • the compound of this invention is administered just prior to, at the same time as, or just after parenteral administration of tPA or other fibrinolytic agent. It may prove desirable to continue treatment with the claimed compounds for a period of time well after reperfusion has been established to maximally inhibit post-therapy reocclusion.
  • the effective dose of tPA, SK, UK or pUK may be from 0.5 to 5 mg/kg and the effective dose of the peptide may be from about 0.1 to 25 mg/kg.
  • kits for convenient administration of the inhibitor and the fibrinolytic agent at the same or different times, comprising, in a single container, such as a box, carton or other container, individual bottles, bags, vials or other containers each having an effective amount of the inhibitor for parenteral administration, as described above, and an effective amount of tPA, or other fibrinolytic agent, for parenteral administration, as described above.
  • kit can comprise, for example, both pharmaceutical agents in separate containers or the same container, optionally as lyophilized plugs, and containers of solutions for reconstitution.
  • a variation of this is to include the solution for reconstitution and the lyophilized plug in two chambers of a single container, which can be caused to admix prior to use.
  • the fibrinolytic and the compound of this invention may be packaged separately, as in two containers, or lyophilized together as a powder and provided in a single container.
  • both agents are provided in solution form, they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement.
  • the platelet aggregation inhibitor may be in an i.v. injectable form, or infusion bag linked in series, via tubing, to the fibrinolytic agent in a second infusion bag.
  • a patient can receive an initial bolus- type injection or infusion of the inhibitor followed by an infusion of the fibrinolytic agent.
  • the pharmacological activity of the compounds of this invention is assessed by their ability to inhibit the binding of H-SK&F 107260, a known RGD- fibrinogen antagonist, to the GPIIbllla receptor; their ability to inhibit platelet aggregation, in vitro, and their ability to inhibit thrombus formation in vivo.
  • GPIIb-IIIa Ten units of outdated, washed human platelets (obtained from Red Cross) were lyzed by gentle stirring in 3% octylglucoside, 20 mM Tris-HCl, pH 7.4, 140 mM NaCI, 2 mM CaC at 4°C for 2 h. The lysate was centrifuged at 100,000g for 1 h. The supernatant obtained was applied to a 5 mL lentil lectin sepharose 4B column (E.Y. Labs) preequilibrated with 20 mM Tris-HCl, pH 7.4, 100 mM NaCI, 2 mM CaCl2, 1% octylglucoside (buffer A).
  • Purified GPIIb-IIIa was diluted to a final concentration of 0.5 mg/mL and mixed with the phospholipids in a protein ⁇ hospholipid ratio of 1:3 (w:w). The mixture was resuspended and sonicated in a bath sonicator for 5 min. The mixture was then dialyzed overnight using 12,000-14,000 molecular weight cutoff dialysis tubing against a 1000-fold excess of 50 mM Tris-HCl, pH 7.4, 100 mM NaCI, 2 mM CaC12 (with 2 changes).
  • the GPIIb-IIIa-containing liposomes wee centrifuged at 12,000g for 15 min and resuspended in the dialysis buffer at a final protein concentration of approximately 1 mg/mL.
  • the liposomes were stored at -70C until needed.
  • GPIIb-IIIa The binding to the fibrinogen receptor (GPIIb-IIIa) was assayed by an indirect competitive binding method using [ 3 H] -SK&F- 107260 as an RGD-type ligand.
  • the binding assay was performed in a 96-well filtration plate assembly (Millipore Corporation, Bedford, MA) using 0.22 um hydrophilic durapore membranes.
  • the wells were precoated with 0.2 mL of 10 ⁇ g/mL polylysine (Sigma Chemical Co., St. Louis, MO.) at room temperature for 1 h to block nonspecific binding.
  • Various concentrations of unlabeled benzadiazapines were added to the wells in quadruplicate.
  • [ 3 H]-SK&F- 107260 was applied to each well at a final concentration of 4.5 nM, followed by the addition of 1 ⁇ g of the purified platelet GPIIb-IIIa-containing liposomes. The mixtures were incubated for 1 h at room temperature. The GPIIb-IIIa-bound [3H]- SK&F- 107260 was seperated from the unbound by filtration using a Millipore filtration manifold, followed by washing with ice-cold buffer (2 times, each 0.2 mL).
  • % aggregation [(90-CR) O (90-10)] x 100, where CR is the chart reading, 90 is the baseline, and 10 is the PRP blank reading.
  • IC50's were determined by plotting [% inhibition of aggregation] vs. [concentration of compound]. Compounds were assayed at 200 mM and diluted sequentially by a factor of 2 to establish a suitable dose response curve.
  • the compounds of this invention inhibit the aggregation of human platelets stimulated with ADP with IC50 of about 0.1 to about 40 ⁇ M.
  • the compounds were incubated for 3 h (rather than 3 min) in the PRP prior to addition of the agonist.
  • Example 2 N-r4-(Aminoiminomethyl benzoyll-4-oxyacetic acid-CRV ⁇ -ffR.SV hvdroxymethvDproIylbenzene
  • a solution consisting of N-carbobenzoxy-[4-(aminoiminomethyl) benzoyl]- 4-[oxy(t-butyl)acetate]-(R)- ⁇ -((R,S)-hydroxymethyl)prolylbenzene (150mg, 0.26 mmol), 10% palladium on carbon (25mg), and 1M hydrochloric acid solution in diethyl ether (0.3 mL, 0.3 mmol) and ethanol (10 mL) was hydrogenated at 50 psi for 1 hour.
  • Example 3 fRVr4-f ri-r4-rAminoiminomethyl benzoyll-2- pyrrolidinylcarbonv ⁇ - 2-('2- phenylethyDphenoxyl acetic acid.
  • 2-(2-phenylethyl)anisole A 500-mL flask was charged with ortho-anisaldehyde (lOg, 74 mmol), benzyltriphenylphosphonium chloride (28.7g, 74 mmol) and methylene chloride (100 mL). To this was added dropwise at room temperature. 50% sodium hydroxide solution (38.7 mL, 740 mmol) over a 20 minutes period (exothermic).
  • N-Carl_>obenzoxy-[4-(aminoiminomethyl)benzoyl]-4-prolyl-(R)- ⁇ -((R,S)- hydroxymethyl)-2-(2-phenylethyl)phenoxy(t-butyl)acetate was oxidized according to the procedure of Example 1(f) to give a slighlty impure product. Purification by chromatography (silica gel, 3%methanol/chloroform) and rotary evaporation of the desired fractions afforded the title compound as a white wax (62% yield),
  • N-Carbobenzoxy-(R)[4-(aminoiminomethyl)benzoyl]-4-prolyl-2-(2- phenylethyl)phenoxy(t-butyl)acetate was deprotected according to the procedure of Example 1(g) to give the desired compound as an impure wax.
  • a preparation which contains 20 mg of the compound of Example 1 as a sterile dry powder is prepared as follows: 20 mg of the compound is dissolved in 15 ml of distilled water. The solution is filtered under sterile conditions into a 25 ml multi-dose ampoule and lyophilized. The powder is reconstituted by addition of 20 ml of 5% dextrose in water (D5W) for intravenous or intramuscular injection. The dosage is thereby determined by the injection volume. Subsequent dilution may be made by addition of a metered volume of this dosage unit to another volume of D5W for injection, or a metered dose may be added to another mechanism for dispensing the drug, as in a bottle or bag for IV drip infusion or other injection- infusion system.
  • D5W dextrose in water
  • Example 8 Oral Dosage Unit Composition A capsule for oral administration is prepared by mixing and milling 50 mg of the compound of Example 1 with 75 mg of lactose and 5 mg of magnesium stearate. The resulting powder is screened and filled into a hard gelatin capsule.
  • Example 9 Oral Dosage Unit Composition A tablet for oral administration is prepared by mixing and granulating 20 mg of sucrose, 150 mg of calcium sulfate dihydrate and 50 mg of the compound of Example 1 with a 10% gelatin solution. The wet granules are screened, dried, mixed with 10 mg starch, 5 mg talc and 3 mg stearic acid; and compressed into a tablet.

Abstract

This invention relates to compounds of the nature: N-[4-(aminoiminomethyl)benzoyl]-4-oxyacetic acid-(R)-prolylbenzene; N-[4-(aminoiminomethyl)benzoyl]-4-oxyacetic acid-(R)-γ-(R,S)-hydroxymethyl-prolylbenzene; (R)-[4-{(1-[4-(aminoiminomethyl)benzoyl]-2-pyrrolidinyl)carbonyl}-2-(2-phenylethyl)phenoxy]acetic acid; (R)-[4-{(1-[4-(aminocarbonyl)benzoyl]-2-pyrrolidinyl)carbonyl}-2-(2-phenylethyl)phenoxy]acetic acid; (R)-[4-{(1-[4-(aminomethyl)benzoyl]-2-pyrrolidinyl)carbonyl}-phenoxy]acetic acid; ethyl (R)-[4-{(1-[4-(aminoiminomethyl)benzoyl]-2-pyrrolidinyl)carbonyl}-phenoxy]acetate; or a pharmaceutically acceptable salt thereof, which are effective for inhibiting platelet aggregation, pharmaceutical compositions for effecting such an activity, and a method for inhibiting platelet aggregation.

Description

CHEMICAL COMPOUNDS
Field of the Invention This invention relates to novel compounds which inhibit platelet aggregation, pharmaceutical compositions containing the compounds and methods of using the compounds.
Background of the Invention Platelet aggregation is believed to be mediated primarily through the fibrinogen receptor, or GPIIb-IIIa platelet receptor complex, which is a member of a family of adhesion receptors referred to as integrins. It has been found that frequently the natural ligands of integrin receptors are proteins which contain an Arg-Gly-Asp sequence (RGD in single letter amino acid code). Von Willebrand factor and fibrinogen, which are considered to be natural ligands for the GPIIb-IIIa receptor, possess an RGD sequence in their primary structure. Functionally, these proteins are able to bind and crosslink GPIIb-IIIa receptors on adjacent platelets and thereby effect aggregation of platelets.
Fibronectin, vitronectin and thrombospondin are RGD-containing proteins which have also been demonstrated to bind to GPIIb-IIIa. Fibronectin is found in plasma and as a structural protein in the intracellular matrix. Binding between the structural proteins and GPIIb-IIIa may function to cause platelets to adhere to damaged vessel walls.
Linear and cyclic peptides which bind to vitronectin and contain an RGD sequence are disclosed in WO 89/05150 (PCT US88/04403). EP 0 275 748 discloses linear tetra- to hexapeptides and cyclic hexa- to octapeptides which bind to the GPIIb-IIIa receptor and inhibit platelet aggregation. Other linear and cyclic peptides, the disclosure of which are incorporated herein by reference, are reported in EP-A 0 341 915. However, the peptide like structures of such inhibitors often pose problems, such as in drug delivery, metabolic stability and selectivity. Inhibitors of the fibrinogen receptor which are not constructed of natural amino acid sequences are disclosed in EP-A 0372,486, EP-A 0 381 033 and EP-A 0478 363. WO 92/07568 (PCT/US91/08166) discloses fibrinogen receptor antagonists which mimic a conformational γ-turn in the RGD sequence by forming a monocyclic seven-membered ring structure. There remains a need, however, for novel fibrinogen receptor antagonists (e.g. inhibitors of the GPIIb-IIIa protein) which have potent in vivo and in vitro effects and lack the peptide backbone structure of amino acid sequences. The present invention discloses novel compounds that inhibit binding to the GPIIb-IIIa receptor and inhibit platelet aggregation.
Summary of the Invention In one aspect this invention is a compound comprising a five- or six- membered ring as described hereinafter in formula (I).
This invention is also a pharmaceutical composition for inhibiting platelet aggregation or clot formation, which comprises a compound of formula (I) and a pharmaceutically acceptable carrier. This invention is further a method for inhibiting platelet aggregation in a mammal in need thereof, which comprises internally administering an effective amount of a compound of formula (I).
In another aspect, this invention provides a method for inhibiting reocclusion of an artery or vein in a mammal following fibrinolytic therapy, which comprises internally administering an effective amount of a fibrinolytic agent and a compound of formula (I). This invention is also a method for treating stroke, transient ischemia attacks, myocardial infarction, or atherosclerosis.
Detailed Description of the Invention This invention discloses novel compounds which inhibit platelet aggregation.
The novel compounds comprise a five- or six-membered ring. The five- or six- membered ring may contain heteroatoms, such as nitrogen, oxygen and sulfur. The five- or six-membered ring system is believed to interact favorably with the GPIIb- IIIa receptor and to orient the substituent sidechains on the five- or six-membered rings so that they may also interact favorably with the receptor.
Although not intending to be bound to any specific mechanism of action, these compounds are believed to inhibit the binding of fibrinogen to the platelet- bound fibrinogen receptor GPIIb-IIIa, and may interact with other adhesion proteins via antagonism of a putative RGD binding site. The compounds of this invention are compounds of formula (I):
Figure imgf000004_0001
wherein: A is -(CH2)r> Het, Ar, or C3_7cycloalkyl;
D is R'R"N-, R'R"NR'N-, R'R"NR'NCO-, R'NR'NC(=NR')-, RONR'C(=NR')- , R'R"NC(=NR');
R'R"NCO-,
R"R'N
Figure imgf000005_0001
'-, or ®
E is -CR'2-, -CR'2CR'2-, -CR'=CR'-, -CR'=CR'-CR'2-, -(CR'2)3-, -CR'2CH(CH3)-, -COCR'2-, -CH(OH)CR'2-, or -CR^COCR^-;
X is -CONR'-, NR'CO-, -SO2NR'-, -NR'SO2-, -CO-, -CH(OH)-, -SO2-, -OC(=O)-, or -(CR'2)m-;
Qi to Q" form any accessible substituted five- or six-membered ring, which may be saturated or unsaturated, optionally containing up to two heteroatoms chosen from the group of O, S, and N wherein S and N may be optionally oxidized;
Y is -CO-, -CR'(OH)-, -SO2-, -CR'2-, -CONR'-, -NR'CO-, SO2NR', or -NR'SO2-;
G is -CR'2-, -CR'2CR'2-, or -CR*=CR'-;
M is Het, Ar, or C3_7cycloalkyl, each of which is unsubstituted or substituted by R3;
Z is -OCR'2-, -NR'CR'2-, -CR'2CR'2-, -CH(CH3)CR'2-, -CR'2-, -CR'=CR'-, or -C(CH3)=CH-;
each R' independently is hydrogen, Cι_4alkyl, C3.7cycloalkyl-Cθ-4alkyl, or
Ar-Co-4alkyl; each R" independently is R', -C(O)R', or -C(O)OR15;
R2 is absent or present as C^alkyl, CO2 ', Z-CO2R', C^alkoxy, hydroxy, C^alkylthio, CONR'R', CN, CF3, halo, or NR'R';
R3 is Cι_4al yl, C^alkoxy, Cj^alkylthio, hydroxy, halo, CO2R', Z-CO2R', NR'R', CN CONR'R', CF3, or Ar-Co-4alkyl;
Rl4 is -, C(O)R', CN, NO2, SO2R', or C(O)OR15;
each R*5 independently is Cj.galkyl, C3.7cycloalkyl-Cθ-4alkyl, or Ar-Co-4alkyl;
J is absent or present as S or O;
T is absent or present as N=CR', CO, or O;
m is 1 or 2;
each n independently is 0 or 1; and
each t independently is 0 to 2;
or a pharmaceutically acceptable salt thereof.
Also included in this invention are complexes or prodrugs of the compounds of this invention. Prodrugs are considered to be any covalently bonded carriers which release the active parent drug according to formula (I) in vivo. In cases wherein the compounds of this invention may have one or more chiral centers, unless specified, this invention includes each unique nonracemic compound which may be synthesized and resolved by conventional techniques. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, such as
Figure imgf000007_0001
and tautomers of guanidine-type groups, such as NR" NR'2
R-R'N I NR'-X- and R.R.N NR'-X-, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or locked in one form by appropriate substitution with R'. The meaning of any substituent at any one occurrence is independent of its meaning, or any other substituent's meaning, at any other occurrence, unless specified otherwise.
With reference to formula (I), suitably, Q^ to Q" optionally substituted by R2 are any accessible combination of the following:
Q1 is CH, C, or N;
Q2 is CH, C, or N;
Q3 is CH2, CHR2, C(R2)2, CH, CR2, NH, NR2, N, O, or S;
Q4 is CH2, CHR2, C(R2)2, CH, CR2, NH, NR2, N, O, or S;
Q5 is absent or present as CH2, CHR2, C(R2)2, CH, CR2, NH, NR2, N, O, or S; and
Q6 is CH2, CHR2, C(R2)2, CH, CR2, NH, NR2, N, O, or S.
Representative compounds of this invention wherein Q* to Q" optionally substituted by R2 are given by each of formulae (II) - (XVII):
Figure imgf000008_0001
(ID (III)
Figure imgf000008_0002
,
(IV) (V)
Figure imgf000008_0003
(VI) (VII)
Figure imgf000008_0004
(VIII) (IX)
Figure imgf000008_0005
(X) (XI)
Figure imgf000009_0001
(XIV) (XV)
Figure imgf000009_0002
(XVI ) (XVII )
Preferably, Q1 to Q^ optionally substituted by R2 is
Figure imgf000009_0003
( ID (III)
Figure imgf000009_0004
(IV) (V)
Most preferably, the compounds of this invention are represented by formula n:
D is R*R"NC (=NR , R'R"NCO-, or R'R"N-; A is phenyl;
X is -CO-, -CH(OH)-, -SO2-, -CONR'- or -NR'CO-; Y is -CO-, -CH(OH)-, -SO2-, -CONR'- or -NR'CO-; M is phenyl; Z is -OCH2-, NR'CH2-, -CH2CH2, or -CH=CH-;
R2 is absent; t is O to 1; each n independently is 0; and Q1 to Q6 is
Figure imgf000010_0001
Particular compounds of the invention include, but are not limited to, the following:
N-[4-aminoiminomethyl)benzoyl]-4-oxyacetic acid-(R)-prolylbenzene; N-[4-aminoiminomethyl)benzoyl]-4-oxyacetic acid-(R)-Ψ-(R,S)- hydroxymethyl-prolylbenzene;
(R)-[4- { ( 1 -[4-(aminoiminomethyl)benzoyl]-2-pyrrolidinyl)carbonyl } -2-(2- phenylethyl)phenoxy] acetic acid;
(R)-[4- { ( 1 -[4-(aminocarbonyl)benzoyl]-2-pyrrolidinyl)carbonyl } -2-(2- phenylethyl)phenoxy] acetic acid;
(R)-[4- { ( 1 - [4-(aminomethyl)benzoyl]-2-pyrrolidinyl)carbonyl } - phenoxy] acetic acid; ethyl (R)-[4-{(l-[4-(aminoiminomethyl)benzoyl]-2-pyrrolidinyl)carbonyl}- phenoxy]acetate; or ethyl (R)-[4-{(l-[4-(aminoiminomethyl)benzoyl]-2-pyrrolidinyl)carbonyl}- phenoxy]acetate; or a pharmaceutically acceptable salt thereof.
The most prefered compound of this invention is N-[4- aminoiminomethyl)benzoyl]-4-oxyacetic acid-(R)-prolylbenzene or a pharmaceutically acceptable salt thereof.
Cι_4alkyl as applied herein means carbon chains which are branched or unbranched and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t- butyl. Ci-ό-alkyl additionally includes pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. Co-4alkyl additionally indicates that no alkyl group need be present (e.g. that a covalent bond is present).
Ar, or aryl, as applied herein, means phenyl or naphthyl, or phenyl or naphthyl substituted by one to three moieties R2. In particular, R2 may be Ci^alkyl, Ci^alkoxy, C^alkythio, CF3, OH, CI, Br, I, F, Cθ2R', or Z-CO2R', wherein Z and R' are as defined in formula (I).
Het, or heterocycle, indicates an optionally substituted five or six membered monocyclic ring, or a nine or ten-membered bicyclic ring containing one to three heteroatoms chosen from the group of nitrogen, oxygen and sulfur, which are stable and available by conventional chemical synthesis. Illustrative heterocycles are imidazole, benzimidazole, pyrrole, indole, pyridine, pyrimidine, pyrazine, quinoline, benzofuran, furan, benzopyran, benzothiophene, thiophene, thiazole, benzothiazole, indoline, moipholine, piperidine, piperazine, pyrrolidine, isoquinoline, and tetra- and perhydro- quinoline and isoquinoline. Any accessible combination of up to three substituents, such as chosen from R2, on the Het ring that is available by chemical synthesis and is stable is within the scope of this invention.
C3_.7cycloalkyl refers to an optionally substituted carbocyclic system of three to seven carbon atoms, which may contain up to two unsaturated carbon-carbon bonds. Typical of C3-7cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl and cycloheptyl. Any combination of up to three substituents, such as chosen from R2, on the cycloalkyl ring that is available by conventional chemical synthesis and is stable, is within the scope of this invention. Any accessible substituted five- or six-membered ring as referred to herein is any saturated or unsaturated five- or six-membered ring which (i) is optionally substituted by R2, wherein the substituents may be present on any atom or heteroatom that results in a stable structure, and (ii) contains up to two heteroatoms selected from the group of N, O and S, wherein S and N may optionally be oxidized, and (iii) is stable and may be synthesized by one skilled in the chemical arts. Typical of accessible five-membered rings are the common saturated and unsaturated rings of cyclopentane, cyclopentene, furan, thiophene, imidazole, pyrrole, thiazole, oxazole, pyrrolidine, pyrroline, and pyrazole. Typical of accessible six-membered rings are the common saturated and unsaturated rings of cyclohexane, cyclohexene, benzene, pyridine, pyrimidine, pyridazine, pyrazine, piperidine, piperazine, morpholine, and dihydropyridine. Θ. as used herein indicates a nitrogen heterocycle, which may be a saturated or an unsaturated stable five-, six- or seven -membered monocyclic ring, or a seven- to ten membered bicyclic ring, containing up to three nitrogen atoms or containing one nitrogen atom and a heteroatom chosen from oxygen and sulfur, and which may be substituted on any atom that results in a stable structure, and wherein the nitrogen heteroatom may be optionally quaternized. Representative of _ are pyrroline, pyrrolidine, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, piperidine, piperazine, morpholine, pyridine, tetrahydropyridine, tetrahydro- and hexahydro-azepine. In particular, _ may be pyrolidinyl, piperidinyl, tetrahydropyridinyl, or piperidinyl. t-Bu refers to the tertiary butyl radical, Boc refers to the t-butyloxycarbonyl radical, Fmoc refers to the fluorenylmethoxycarbonyl radical, Ph refers to the phenyl radical, Cbz refers to the benzyloxycarbonyl radical, BrZ refers to the o-bromobenzyloxycarbonyl radical, C1Z refers to the o-chlorobenzyloxycarbonyl radical, Bzl refers to the benzyl radical, 4-MBzl refers to the 4-methyl benzyl radical, Me refers to methyl, Et refers to ethyl, Ac refers to acetyl, Alk refers to Cι_4alkyl, Nph refers to 1- or 2-naphthyl and cHex refers to cyclohexyl.
DCC refers to dicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, DIEA refers to diisopropylethyl amine, EDC refers to N- ethyl-N'(dimethylaminopropyl)carbodiimide. HOBt refers to
1-hydroxybenzotriazole, THF refers to tetrahydrofuran, DIEA refers to diisopropylethylamine, DMF refers to dimethyl formamide, NBS refers to N- bromosuccinimide, Pd/C refers to a palladium on carbon catalyst, PPA refers to 1- propanephosphonic acid cyclic anhydride, DPPA refers to diphenylphosphoryl azide, BOP refers to benzotriazol-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate, HF refers to hydrofluoric acid, TEA refers to triethylamine, TFA refers to trifluoroacetic acid, PCC refers to pyridinium chlorochromate. .
The compounds of formula (I) .are generally prepared by reacting a compound :
Figure imgf000012_0001
(xvπi) (xrx) wherein Q Q4, Y, G, M, Z-R, R*, R2, D, A, E, and n are as defined in formula (I), with any reactive functional groups protected;
L 1 and L2 are groups which are capable of reacting to form the linkage -X-, wherein X is as defined in formula (I); or L* is hydrogen, Ql is nitrogen, and L2 is a functional group which is capable of reacting with the Q* nitrogen to from certain X linkages; and thereafter removing any protecting groups, and optionally forming a pharmaceutically acceptable salt. It will be apparent that the precise identity of L1 and L2 will be dependent upon the site of the linkage being formed. For instance, if X is CONH, L1 may be -NH2 L2 and may be COOH or COC1. When L2 is COOH, a coupling agent is used. Similarly, if X is NHCO, L1 may be -CO2H or COC1 and L2 may be -NH2.
Particularly, if X is CO, L1 may be H and Q1 is nitrogen and L2 may be COOH or COC1.
Where X is NR1SO2, L1 may be SO2CI and L2 may be -NH2. Where X is SO2NH, L1 may be -NH2 and L2 may be SO2CI. Methods to prepare such sulfonyl chlorides are disclosed, for instance, in J. Org. Chem., 23, 1257 (1958).
Coupling reagents as used herein denote reagents which may be used to form peptide bonds. Typical coupling methods employ carbodiimides, activated anhydrides and esters and acyl halides. Reagents such as EDC, DCC, DPPA, PPA, BOP reagent, HOBt, N-hydroxysuccinimide and oxalyl chloride are typical.
Coupling methods to form peptide bonds are generally well known to the art. The methods of peptide synthesis generally set forth by Bodansky et al., THE PRACTICE OF PEPTIDE SYNTHESIS, Springer- Verlag, Berlin, 1984, Ali et al. in J. Med. Chem., 29, 984 (1986) and J. Med. Chem., 30, 2291 (1987) are generally illustrative of the technique and are incorporated herein by reference.
Solution synthesis for the formation of amide or peptide bonds is accomplished using conventional methods used to form amide bonds. Typically, the amine or aniline is coupled via its free amino group to an appropriate carboxylic acids substrate using a suitable carbodiimide coupling agent, such as N,N' dicyclohexyl carbodiimide (DCC), optionally in the presence of catalysts such as 1- hydroxybenzotriazole (HOBt) and dimethylaminopyridine (DMAP). Other methods, such as the formation of activated esters, anhydrides or acid halides, of the free carboxyl of a suitably protected acid substrate, and subsequent reaction with the free amine of a suitably protected amine, optionally in the presence of a base, are also suitable. For example, a protected Boc-amino acid or Cbz-amidino benzoic acid is treated in an anhydrous solvent, such as methylene chloride or tetrahydrofuran(THF), in the presence of a base, such as N-methylmorpholine, DMAP or a trialkylamine, with isobutyl chloroformate to form the "activated anhydride", which is subsequently reacted with the free amine of a second protected amino acid or aniline.
Compounds of formula (XIX) are prepared by conventional methods known in the art from commercially available materials. D is generally a basic functional group attached to A, optionally via an alkyl chain, and is protected during the synthesis, where necessary. For example, compounds of formula (XIX) or formula (I) wherein D is a suitably substituted R'R"N-, R"R'NC(=NR'), R'2N(R13)C=N-, R"N=(R13)C-NR'-, R'2N(R'2N)C=N- or R"R'N(R'N=)C-NR', are prepared by conventional methods including those disclosed in EP-A 0 372486, EP-A 0 381 033 or EP-A 0478 363, which are incorporated herein by reference.
Compounds of formula (XIX) wherein DD iiss Θ_ ; are prepared, inter alia, by methods disclosed in EP-A 0478 363.
Compounds wherein D is R'2N(R'2N)C=N-X- or R"R'N(R'N=)C-NR'-X-, .and X is O are prepared, ter alia, by methods disclosed in J. Org. Chem., 51, 5047 (1986).
Compounds wherein D is R'2N(R'2N)C=N-X- or R"R'N(R'N=)C-NR'-X-, and X is N=CR', are prepared, inter alia, by methods disclosed in United States Patent 3,714,253 and Eur. J. Med. Chem.-Chim. Ther., 20, 25 (1985).
Compounds wherein D isR'2N(R'2N)C=N-X- or R"R'N(R'N=)C-NR'-X-, and X is C(O), are prepared, inter alia, by methods disclosed in United States Patent 3,714,253 and Can. J. Chem., 43, 3103 (1965). Compounds wherein D is R'ONR'C(=NR')- may be prepared, inter alia, by methods disclosed in J. Het. Chem., 16, 1063 (1979) or J. Het. Chem., 26, 125 (1989).
Compounds wherein D is R'2NR'NC(=NR')- are prepared by conventional methods including those disclosed in Syn., 583 (1974). Compounds wherein D is R'R"NR'N- are prepared, inter alia, by methods disclosed in J. Prakt. Chem., 36, 29 (1967).
Compounds wherein D is R'R"NR'NCO- are prepared, inter alia, by methods disclosed in Bull. Chem. Soc. Jpn., 43, 2257 (1970).
Compounds wherein D is R"R'NC(=NR')Y, and Y is S, are prepared, inter alia, by methods disclosed in Chem. Lett., 1379 (1986). Compounds of formula (XIX) or formula (I), wherein D is R"R'NC(=NR')Y and Y is O, are prepared by conventional methods including those disclosed in Japanese Patent 2022751.
The reactive functional groups of the sidechains of each synthetic fragment are suitably protected as known in the art. Suitable protective groups are disclosed in Greene, PROTECTIVE GROUPS IN ORGANIC CHEMISTRY, John Wiley and Sons, New York, 1981. For example, the Boc, Cbz, phthaloyl orFmoc group may be used for protection of an amino or amidino group. The Boc group is generally preferred for protection of an α-amino group. A t-Bu, cHex or benzyl ester may be used for the protection of the side chain carboxyl. A benzyl group or suitably substituted benzyl group (eg. 4-methoxy-benzyl or 2,4-dimethoxy-benzyl) is used to protect the mercapto group or the hydroxyl group. The tosyl group may be used for protection of the imidazolyl group and tosyl or nitro group for protection of the guanidino group. A suitably substituted carbobenzyloxy group or benzyl group may be also be used for the hydroxyl group or amino group. Suitable substitution of the carbobenzyloxy or benzyl protecting groups is ortho and/or para substitution with chloro, bromo, nitro or methyl, and is used to modify the reactivity of the protective group. Except for the Boc group, the protective groups for the amino moiety are, most conveniently, those which are not removed by mild acid treatment. These protective groups are removed by such methods as catalytic hydrogenation, sodium in liquid ammonia or HF treatment, as known in the art.
Modification of amino groups may be accomplished by alkylation, sulfonylation, cyanation or acylation as is generally known in the art.
SCHEME I
Figure imgf000016_0001
(1) (2)
Figure imgf000016_0002
(4)
Figure imgf000016_0003
Scheme I provides a method of preparing compounds wherein Q^ is absent, trogen, Q2 is CH, Q3, OA and Q^ are CH2, and A and M are each phenyl. me I, R2' and R3', D', and R* indicate R2, R3, D, and R, respectively, or a suitable precursor thereof, wherein any functional groups are protected as known in the art. In formula (6), Y' represents -CH(OH)- and -CO- groups.
According to Scheme I, a suitably protected prolylhalide of formula (I), such as N-trifluoroacetyl-D-prolylchloride, is reacted with an aromatic compound of formula (2), such as anisole, in the presence of a Lewis acid catalyst, such as aluminum chloride, aluminum bromide, titanium tetrachloride or boron trifluoride, particularly aluminum chloride, in a suitable solvent, such as methylene chloride. Subsequent deprotection of the N-protecting group of proline with concurrent reduction of the amide linkage of formula (3) compounds using, for example, sodium borohydride in a suitable solvent, such as ethanol, results in the formation of compounds of formula (4). These prolyl compounds are coupled with an appropriately substituted D'-(CH2)t-A-carboxylic acid compound, such as 4-N-Cbz- amidinobenzoic acid, in the presence of a suitable amide coupling agent, such as N- ethyl-N'(dimethylaminopropyl)carbodiimide, and optionally in the presence of a catalyst, such as 1-hydroxybenzotriazole, in a suitable solvent, such as dimethylfor amide. The formula (5) compounds may then be deprotected using conventional chemical techniques, for example, using hydrogen in the presence of a suitable catalyst, such as palladium on carbon, when a Cbz-protecting group is present, or using trifluoroacetic acid in a suitable solvent, such as methylene chloride, when a t-butyl ester is present.
Based on Scheme I, it should be appreciated that a suitably protected and substituted Q* to Q^ heterocyclic or heteroaryl compound may be used in place of the prolyl compounds of formula (1). Conventional oxidizing reagents, such as pyridinium dichromate, and reducting reagnets, such as sodium borohydride, allow for the preparation of the various X and Y spacer groups.
Acid addition salts of the compounds of formula (I) are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic or methanesulfonic. The acetate salt form is especially useful. Certain of the compounds form inner salts or zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine. Cations such as Li+, Na+, K+, Ca++, Mg++ and NH4+ are specific examples of cations present in pharmaceutically acceptable salts. This invention provides a pharmaceutical composition which comprises a compound according to formula (I) and a pharmaceutically acceptable carrier. Accordingly, the compounds of formula (I) may be used in the manufacture of a medicament. Pharmaceutical compositions of the compounds of formula (I) prepared as hereinbefore described may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation may be a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate. Alternately, these compounds may be encapsulated, tableted or prepared in a emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule. For rectal administration, the compounds of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
This invention also provides a method of inhibiting platelet aggregation and clot formation in a mammal, especially a human, which comprises the internal administration of a peptide of formula (I) and a pharmaceutically acceptable carrier. Indications for such therapy include acute myocardial infarction (AMI), deep vein thrombosis, pulmonary embolism, dissecting aneurysm, transient ischemia attack (TIA), stroke and other infarct-related disorders, and unstable angina. Chronic or acute states of hyper-aggregability, such as disseminated intravascular coagulation (DIC), septicemia, surgical or infectious shock, post-operative and post-partum trauma, cardiopulmonary bypass surgery, incompatible blood transfusion, abruptio placenta, thrombotic thrombocytopenic purpura (TTP), snake venom and immune diseases, are likely to be responsive to such treatment. In addition, the compounds of this invention may be useful in a method for the prevention of metastatic conditions, the prevention or treatment of fungal or bacterial infection, inducing immunostimulation, and the prevention or treatment of diseases in which bone resorption is a factor.
The compounds of mis invention are administered either orally or parenterally to the patient, in a manner such that the concentration of drug in the plasma is sufficient to inhibit platelet aggregation, or other such indication. The pharmaceutical compositions containing the compounds of this invention are administered at a dose between about 0.2 to about 50 mg kg in a manner consistent with the condition of the patient. For acute therapy, parenteral administration is preferred. For persistent states of hyperaggregability, an intravenous infusion of the compound in 5% dextrose in water or normal saline is most effective, although an intramuscular bolus injection may be sufficient. For chronic, but noncritical, states of platelet aggregability, oral administration of a capsule or tablet, or a bolus intramuscular injection is suitable. The compounds of this invention are administered one to four times daily at a level of about 0.4 to about 50 mg/kg to achieve a total daily dose of about 0.4 to about 200 mg/kg/day. This invention further provides a method for inhibiting the reocclusion of an artery or vein following fibrinolytic therapy, which comprises internal administration of a compound of formula (I) and a fibrinolytic agent. It has been found that administration of certain compounds in fibrinolytic therapy either prevents reocclusion completely or prolongs the time to reocclusion. When used in the context of this invention the term fibrinolytic agent is intended to mean any compound, whether a natural or synthetic product, which directly or indirectly causes the lysis of a fibrin clot. Plasminogen activators are a well known group of fibrinolytic agents. Useful plasminogen activators include, for example, anistreplase, urokinase (UK), pro-urokinase (pUK), streptokinase (SK), tissue plasminogen activator (tPA) and mutants, or variants, thereof, which retain plasminogen activator activity, such as variants which have been chemically modified or in which one or more amino acids have been added, deleted or substituted or in which one or more or functional domains have been added, deleted or altered such as by combining the active site of one plasminogen activator with the fibrin binding domain of another plasminogen activator or fibrin binding molecule. Other illustrative variants include tPA molecules in which one or more glycosylation sites have been altered. Preferred among plasminogen activators are variants of tPA in which the primary amino acid sequence has been altered in the growth factor domain so as to increase the serum half-life of the plasminogen activator. tPA Growth factor variants are disclosed, e.g., by Robinson et al, EP-A 0 297 589 and Browne et al., EP-A 0 240 334. Other variants include hybrid proteins, such as those disclosed in EP 0 028 489, EP 0 155 387 and EP 0 297 882, all of which are incorporated herein by reference. Anistreplase is a preferred hybrid protein for use in this invention. Fibrinolytic agents may be isolated from natural sources, but are commonly produced by traditional methods of genetic engineering. Useful formulations of tPA, SK, UK and pUK are disclosed, for example, in EP-A 0 211 592, EP-A 0092 182 and U.S. Patent 4,568,543, all of which are incorporated herein by reference. Typically the fibrinolytic agent may be formulated in an aqueous, buffered, isotonic solution, such as sodium or ammonium acetate or adipate buffered at pH 3.5 to 5.5. Additional excipients such as polyvinyl pyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene, glycol, mannitol and sodium chloride may also be added. Such a composition can be lyophilized.
The pharmaceutical composition may be formulated with both the compound of formula (I) and fibrinolytic in the same container, but formulation in different containers is preferred. When both agents are provided in solution form they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement.
Indications for such therapy include myocardial infarction, deep vein thrombosis, pulmonary embolism, stroke and other infarct-related disorders. The compound of this invention is administered just prior to, at the same time as, or just after parenteral administration of tPA or other fibrinolytic agent. It may prove desirable to continue treatment with the claimed compounds for a period of time well after reperfusion has been established to maximally inhibit post-therapy reocclusion. The effective dose of tPA, SK, UK or pUK may be from 0.5 to 5 mg/kg and the effective dose of the peptide may be from about 0.1 to 25 mg/kg. For convenient administration of the inhibitor and the fibrinolytic agent at the same or different times, a kit is prepared, comprising, in a single container, such as a box, carton or other container, individual bottles, bags, vials or other containers each having an effective amount of the inhibitor for parenteral administration, as described above, and an effective amount of tPA, or other fibrinolytic agent, for parenteral administration, as described above. Such kit can comprise, for example, both pharmaceutical agents in separate containers or the same container, optionally as lyophilized plugs, and containers of solutions for reconstitution. A variation of this is to include the solution for reconstitution and the lyophilized plug in two chambers of a single container, which can be caused to admix prior to use. With such an arrangement, the fibrinolytic and the compound of this invention may be packaged separately, as in two containers, or lyophilized together as a powder and provided in a single container. When both agents are provided in solution form, they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement. For example, the platelet aggregation inhibitor may be in an i.v. injectable form, or infusion bag linked in series, via tubing, to the fibrinolytic agent in a second infusion bag. Using such a system, a patient can receive an initial bolus- type injection or infusion of the inhibitor followed by an infusion of the fibrinolytic agent.
The pharmacological activity of the compounds of this invention is assessed by their ability to inhibit the binding of H-SK&F 107260, a known RGD- fibrinogen antagonist, to the GPIIbllla receptor; their ability to inhibit platelet aggregation, in vitro, and their ability to inhibit thrombus formation in vivo.
Inhibition of RGD-mediated GPIIb-IIIa binding
Purification of GPIIb-IIIa Ten units of outdated, washed human platelets (obtained from Red Cross) were lyzed by gentle stirring in 3% octylglucoside, 20 mM Tris-HCl, pH 7.4, 140 mM NaCI, 2 mM CaC at 4°C for 2 h. The lysate was centrifuged at 100,000g for 1 h. The supernatant obtained was applied to a 5 mL lentil lectin sepharose 4B column (E.Y. Labs) preequilibrated with 20 mM Tris-HCl, pH 7.4, 100 mM NaCI, 2 mM CaCl2, 1% octylglucoside (buffer A). After 2 h incubation, the column was washed with 50 mL cold buffer A. The lectin-retained GPIIb-IIIa was eluted with buffer A containing 10% dextrose. All procedures were performed at 4°C. The GPIIb-IIIa obtained was >95% pure as shown by SDS polyacrylamide gel electrophoresis. Incorporation of GPIIb-IIIa in Liposomes. A mixture of phosphatidylserine (70%) and phosphatidylcholine (30%) (Avanti Polar Lipids) were dried to the walls of a glass tube under a stream of nitrogen. Purified GPIIb-IIIa was diluted to a final concentration of 0.5 mg/mL and mixed with the phospholipids in a proteinφhospholipid ratio of 1:3 (w:w). The mixture was resuspended and sonicated in a bath sonicator for 5 min. The mixture was then dialyzed overnight using 12,000-14,000 molecular weight cutoff dialysis tubing against a 1000-fold excess of 50 mM Tris-HCl, pH 7.4, 100 mM NaCI, 2 mM CaC12 (with 2 changes). The GPIIb-IIIa-containing liposomes wee centrifuged at 12,000g for 15 min and resuspended in the dialysis buffer at a final protein concentration of approximately 1 mg/mL. The liposomes were stored at -70C until needed.
Competitive Binding to GPIIb-IIIa The binding to the fibrinogen receptor (GPIIb-IIIa) was assayed by an indirect competitive binding method using [3H] -SK&F- 107260 as an RGD-type ligand. The binding assay was performed in a 96-well filtration plate assembly (Millipore Corporation, Bedford, MA) using 0.22 um hydrophilic durapore membranes. The wells were precoated with 0.2 mL of 10 μg/mL polylysine (Sigma Chemical Co., St. Louis, MO.) at room temperature for 1 h to block nonspecific binding. Various concentrations of unlabeled benzadiazapines were added to the wells in quadruplicate. [3H]-SK&F- 107260 was applied to each well at a final concentration of 4.5 nM, followed by the addition of 1 μg of the purified platelet GPIIb-IIIa-containing liposomes. The mixtures were incubated for 1 h at room temperature. The GPIIb-IIIa-bound [3H]- SK&F- 107260 was seperated from the unbound by filtration using a Millipore filtration manifold, followed by washing with ice-cold buffer (2 times, each 0.2 mL). Bound radioactivity remaining on the filters was counted in 1.5 mL Ready Solve (Beckman Instruments, Fullerton, CA) in a Beckman Liquid Scintillation Counter (Model LS6800), with 40% efficiency. Nonspecific binding was determined in the presence of 2 μM unlabeled SK&F- 107260 and was consistently less than 0.14% of the total radioactivity added to the samples. All data points are the mean of quadruplicate determinations. The compounds of this invention inhibit [3H]-SK&F 107260 binding with Ki in the range of about 5 nM to about 10 μM. Inhibition of Platelet Aggregation Blood was collected (citrated to prevent coagulation) from, naive, adult mongrel dogs. Platelet rich plasma, PRP, was prepared by centrifugation at 150 x g for 10 min at room temperature. Washed platelets were prepared by centrifuging PRP at 800 x g for 10 min. The cell pellet thus obtained was washed twice in
Tyrode's buffer (pH 6.5) without Ca++ and resuspended in Tyrode's buffer (pH 7.4) containing 1.8 mM Ca"*"*" at 3 x 10^ cells/ml. Compounds were added 3 min prior to the agonist in all assays of platelet aggregation. Final agonist concentrations were 0.1 unit/ml thrombin and 2 mM ADP (Sigma). Aggregation was monitored in a Chrono-Log Lumi-Aggregometer. Light transmittance 5 min after addition of the agonist was used to calculate percent aggregation according to the formula % aggregation = [(90-CR) O (90-10)] x 100, where CR is the chart reading, 90 is the baseline, and 10 is the PRP blank reading. IC50's were determined by plotting [% inhibition of aggregation] vs. [concentration of compound]. Compounds were assayed at 200 mM and diluted sequentially by a factor of 2 to establish a suitable dose response curve.
The compounds of this invention inhibit the aggregation of human platelets stimulated with ADP with IC50 of about 0.1 to about 40 μM.
To assess the stability of the compounds to plasma proteases, the compounds were incubated for 3 h (rather than 3 min) in the PRP prior to addition of the agonist.
In Vivo Inhibition of Platelet Aggregation In vivo inhibition of thrombus formation is demonstrated by recording the systemic and hemodynamic effects of infusion of the peptides into anesthetized dogs according to the methods described in Aiken et al., Prostaglandins, 19, 629 (1980). The examples which follow are intended to in no way limit the scope of this invention, but are provided to illustrate how to make and use the compounds of this invention. Many other embodiments will be readily apparent and available to those skilled in the art.
EXAMPLES
Example 1 N-r4-(Aminoiminomethyl benzoyll-4-oxyacetic acid-fRVprolylbenzene a) N-trifluoroacetyl-(2-and-4-)-methoxyprolylbenzene
A cold (0°C) solution of N-trifluoroacetyl-D-prolylchloride (15.0g, 69.5 mmol), and anisole (7.56g, 70 mmol) in methylene chloride (250 mL) was made. Aluminum chloride (9.34g, 70 mmol) was sluπϊed in methylene chloride (25 mL) and added in portions under a stream of argon. The temperature. The dark mixture was then quenched with 6N hydrochloric acid (200 mL), the layers were separated, and the aqueous layer was extracted twice with 100 mL portions of methylene chloride.resulting mixture was heated to reflux for 24 hours and then allowed to cool to room The dark mixture was then quenched with 6N hydrochloric acid solution (200 mL), the layers were separated, and the aqueous layer was extracted twice with 100 mL portions of methylene chloride. The combined organic layers were washed with 5% sodium bicarbonate solution, brine, dried over magnesium sulfate, and concentrated by rotary evaporation to give a brown oil which was purified by chromatography (silica gel, 10% hexane/methylene chloride) to afford the title compound as a mixture of isomers (para/ortho, 80/20) (10.9g, 50% yield), MH+=301.
b) N-trifluroacetyl-4-hydroxyprolylbenzene
To a cold (-78°C) rapidly stirred solution of N-trifluroacetyl-(2-and-4-)- methoxyprolylbenzene (7.4g, 24 mmol) in methylene chloride (175 mL) was added a solution of 1M boron tribromide (30mL, 30 mmol, in methylene chloride) under a stream of argon. The solution was allowed to come to room temperature and stirring was continued under a calcium chloride drying tube for 3 hours. The resulting suspension was diluted with ice (50 mL), and the layers were separated. The aqueous layer was extracted twice with diethyl ether, and the combined organic extracts were washed with brine, dried over magnesium sulfate, and concentrated by rotary evaporation to give a white solid. The desired isomer (para) was then isolated by chromatography (silica gel, 2% methanol/chloroform) to afford the title compound as white flakes (3.68g, 53% yield), MH+= 287.
c) N-trifluroacetyl-4-[oxy(t-butyl)acetic acid]prolylbenzene
A mixture of N-trifluoroacetyl-4-hydroxyprolylbenzene (720mg, 2.5 mmol), t-butylbromoacetate (448mg, 2.5 mmol), and potassium carbonate (773mg, 5.6 mmol) in dimethylformamide (25 mL) was stirred under argon for 3 hours. The reaction mixture was then diluted with equal volumes of diethyl ether and water (100 mL) and separated. The aqueous phase was extracted twice with diethyl ether and the combined organic extracts were washed with water and brine, dried over magnesium sulfate, and concentrated by rotary evaporation to give an oil. Further purification by multiple triturations with hexane afforded the title compound as a clear oil (950mg, 95% yield), MH+= 401. d) 4-[oxy(t-butyl)acetate]-(R)-Ψ- ((R,S)-hydroxymethyl)prolylbenzene
A solution of N-trifluroacetyl-4-[oxy(t-butyl)acetic acid] prolylbenzene (3.90g, 9.7 mmol) in ethanol (45 mL) was made under argon. Sodium borohydride (1.47g, 38.89 mmol) was added portionwise. After 1 hour the reaction mixture was cooled to 0°C and acetone (45 mL) was added cautiously. After the addition was complete, the solution was allowed to come to room temperature for 15 minutes. After this, the reaction was diluted with diethyl ether washed with brine and saturated sodium bicarbonate, dried over magnesium sulfate, and concentrated by rotary evaporation. Final purification by trituration with ethyl acetate afforded the title compound as a white solid (2.9g, 97% yield), MH+= 307.
e) N-carbobenzoxy-[4-(aminoiminomethyl)benzoyl]-4-[oxy(t-butyl)acetate]- (R)-Ψ-((R,S)-hydroxymethyl)prolylbenzene To a cold (0°C) solution consisting of 4-[oxy(t-butyl)acetate]-(R)-Ψ-((R,S)- hydroxymethyl) prolylbenzene (2.0g, 6.5 mmol) in dimethylformamide (50 mL) was added consecutively 1-hydroxybenzotriazole (0.97g, 7.2 mmol), 4-N-Cbz- amidinobenzoic acid (2.12g, 7.2 mmol), and water-soluble carbodiimide (N-ethyl- N'(dimethylaminopropyl)carbodimide, 1.38g, 7.2 mmol) under argon. The resulting solution was stirred at room temperature for 4 hours, after which, the solution was concentrated by rotary evaporation, and the resulting residue was treated with 10% potassium carbonate solution, filtered, and washed with water until neutral. The wax was disolved in ethyl acetate and washed successively with 5% citric acid, 5% sodium bicarbonate solution, and brine. Concentration by rotary evaporation yielded the title compound as a white solid (2.17g, 57% yield), MH+=587.
f) N-carbobenzoxy-[4-(aminoiminomethyl)benzoyl]-4-oxy(t-buty)acetate-(R)- prolylbenzene
To a cold (0°C) stirred solution of N-carbobenzoxy-[4-(aminoiminomethyl)- benzoyl]-4-[oxy(t-butyl)acetate]-(R)-Ψ-((R,S)-hydroxymethyl)prolylbenzene
(1.91g, 3.4 mmol) in methylene chloride (60 mL) was added pyridinium dichromate (3.76g, 10 mmol). The resulting dark suspension was stirred at room temperature for 4 hours. The solution was concentrated, and the resulting black residue was taken up into ethyl acetate and filtered through Celite®. The filtrate was then washed with brine, dried over magnesium sulfate, and concentrated by rotary evaporation. Evaporation from chloroform, and trituration with diethyl ether afforded the title compound (1.40 g, 75%), MH+= 585. g) N-[4-(arninoiminomethyl)benzoyl]-4-oxyacetic acid-(R)-prolylbenzene
A solution consisting of N-carbobenzoxy-[4-(aminoiminomethyl) benzoyl]- 4-oxy(t-butyl)acetate-(R)-prolylbenzene (1.10g, 1.9 mmol), 10% palladium on carbon (100 mg), 1M hydrochloric acid in diethyl ether (1.9 mL, 1.9 mmol) and ethanol (35 mL) was hydrogenated at 50 psi for 1 hour. The mixture was filtered through Celite® and concentrated to give the unprotected amidino ester. The ester was then hydrolyzed by treatment with 20% trifluroacetic acid in methylene chloride (40 mL) for 1.5 hours. Concentration by rotary evaporation followed by trituration with diethyl ether gave an impure product which was purified by high pressure liquid chromatography to afford the title compound as the (reverse phase silica, 16% acetonitrile/water/0.1% trifluoroacetic acid) salt (620 mg, 83% yield), MH+= 395, Anal. Calcd. for C21H21N3O5 • C2HO2F3: C.49.16 ;H,4.01; N.7.07. found: C.49.14; H,4.01; N.7.07.
Example 2 N-r4-(Aminoiminomethyl benzoyll-4-oxyacetic acid-CRVΨ-ffR.SV hvdroxymethvDproIylbenzene A solution consisting of N-carbobenzoxy-[4-(aminoiminomethyl) benzoyl]- 4-[oxy(t-butyl)acetate]-(R)-Ψ-((R,S)-hydroxymethyl)prolylbenzene (150mg, 0.26 mmol), 10% palladium on carbon (25mg), and 1M hydrochloric acid solution in diethyl ether (0.3 mL, 0.3 mmol) and ethanol (10 mL) was hydrogenated at 50 psi for 1 hour. The mixture was filtered through Celite® and concentrated to give the unprotected amidino ester. The ester was then hydrolyzed by treatment with 20% trifluroacetic acid in methylene chloride (15 mL) for 1.5 hours. Concentration by rotary evaporation followed by trituration with diethyl ether yielded a white wax which was purified by high pressure liquid chromatography column to afford the title compound as the high pressure liquid chromatography (reverse phase silica, 16% acetonitrile/water/0.1% trifluoro acid) TFA salt (65 mg, 50% yield), MH+= 397, Anal. Calcd .for C21H23N3O5 • 1.5(C2HO2F ): C,49.92;H,4.45;N,7.28. Found C,50.16;H,4.77;N,7.45.
Example 3 fRVr4-f ri-r4-rAminoiminomethyl benzoyll-2- pyrrolidinylcarbonvπ- 2-('2- phenylethyDphenoxyl acetic acid. a) 2-(2-phenylethyl)anisole A 500-mL flask was charged with ortho-anisaldehyde (lOg, 74 mmol), benzyltriphenylphosphonium chloride (28.7g, 74 mmol) and methylene chloride (100 mL). To this was added dropwise at room temperature. 50% sodium hydroxide solution (38.7 mL, 740 mmol) over a 20 minutes period (exothermic). After the addition was complete, stirring was continued for an additional 30 minutes. The layers were diluted with water/methylene chloride (1/1) and separated. The aqueous layer was extracted twice with methylene chloride and the combined organic layers were dried over magnesium sulfate and concentrated by rotary evaporation to give a white wax. Purification by trituration with hexane, removal of the solid impurities by filtration, and concentration of the fitrate, affords the vinyl intermediate as a clear oil (15.0g, 96%).
The oil was then taken into ethanol (40 mL), treated with 10% palladium on carbon (lg), and hydrogenated at 50 psi for 2 hours. The catalyst was filtered off through Celite®, and the filtrate was concentrated by rotary evaporation to yield a crude oil. Purification by flash chromatography (silica gel, hexane) afforded the title compound (13.24g, 84% yield), MH+=212.
b) N-trifluoroacetyl-(4-and-6)-prolyl-2-(2-phenylethyl)anisole
A cold (0°C) solution of N-trifluoroacetyl-D-prolylchloride (14.43g, 63 mmol), 2-(2-phenylethyl)anisole (13.24g, 63 mmol) in methylene chloride (500 mL) was made. Aluminum chloride (8.4g, 63 mmol) was slurried in methylene chloride (25 mL) and added in portions under a stream of argon. The resulting mixture was heated to reflux for 24 hours and then allowed to cool to room temperature. The dark mixture was then quenched with 6N hydrochloric acid solution (200 mL), the layers were separated, and the aqueous layer was extracted twice with 100 mL portions of methylene chloride. The combined organic layers were washed with 5% sodium bicarbonate, brine, dried over magnesium sulfate, and concentrated by rotary evaporation to give a brown oil, which was purified by chromatography (silica gel, 10% hexane/methylene chloride) to afford the title compound as a mixture of isomers (12.55g, 53% yield), MH+=405.
c) N-trifluoroacetyl-4-prolyl-2-(2-phenylethyl)phenol N-Trifluoroacetyl-(4-and-6)-prolyl-2-(2-phenylethyl)anisole was deprotected according to the procedure of Example 1(b) to give the title compound as a white solid (77% yield), MH+=391.
d) N-trifluoroacetyl-4-prolyl-2-(2-phenylethyl)phenoxy (t-buty)acetate N-Trifluoroacetyl-4-prolyl-2-(2-phenylethyl)phenol was O-alkylated according to the procedure of Example 1(c) to give the title compound (96% yield), MH+=505.
e) 4-prolyl-(R)-Ψ-((R,S)-hydroxymethyl)-2-(2-phenylethyl)phenoxy(t- butyl)acetate
N-Trifluoroacetyl-4-prolyl-2-(2-phenylethyl)phenoxy(t-buty)acetate was deprotected according to the procedure of Example 1(d) to afford a crude product. Purification by chromatography (silica gel, diethyl ether) and concentration of the desired fractions by rotary evaporation afforded the title compound (93% yield), MH+=411.
f) N-carbobenzoxy-[4-(aminoiminomethyl)benzoyl]-4-prolyl-(R)-Ψ-((R,S)- hydroxymethyl)-2-(2-phenylethyl)phenoxy(t-butyl)acetate 4-Prolyl-(R)-Ψ-((R,S)-hydroxymethyl)-2-(2-phenylethyl)phenoxy(t- butyl)acetate was coupled to 4-Cbz-amidinobenzoic acid according to the procedure of Example 1(e) to give the title compound as a light yellow solid (86% yield), MH+=691.
g) N-carbobenzoxy-[4-(aminoiminomethyl)benzoyl]-4-prolyl-2-(2- phenylethyl)phenoxy(t-butyl)acetate
N-Carl_>obenzoxy-[4-(aminoiminomethyl)benzoyl]-4-prolyl-(R)-Ψ-((R,S)- hydroxymethyl)-2-(2-phenylethyl)phenoxy(t-butyl)acetate was oxidized according to the procedure of Example 1(f) to give a slighlty impure product. Purification by chromatography (silica gel, 3%methanol/chloroform) and rotary evaporation of the desired fractions afforded the title compound as a white wax (62% yield),
MH+=689.
h) (R)-[4-{[l-[4-(aminoiminomethyl)benzoyl]-2-pyrrolidinylcarbonyl}-2-(2- phenylethyl)phenoxy]acetic acid
N-Carbobenzoxy-(R)[4-(aminoiminomethyl)benzoyl]-4-prolyl-2-(2- phenylethyl)phenoxy(t-butyl)acetate was deprotected according to the procedure of Example 1(g) to give the desired compound as an impure wax. A portion of the product was further purified by high pressure liquid chromatography (reverse phase silica, 28% acetonitrile/water/0.1% trifuluoroacetic acid) column to afford the title compound as the major component, MH+=499, Anal. Calcd. for C29H29N3O5 • 1.5(C2HO2F3): C.57.31; H,4.58; N,6.27; found: Q57.10; H.4.60; N,6.20. Example 4 CR)- \4- rπ - r4-(Aminocarbonyl benzoyl1 -2- pyrrolidinyDcarbonyl ) -2-C2- phenylethyPphenoxylacetic acid A minor component from Example 3 (y) was also isolated by high pressure liquid chromatography to afford the title compound, MH+=500 Anal. Calcd. for C29H28N2O6 • C2HO2F3 • water : C,58.86; H.4.94; N.4.43. Found: C.58.76; H,5.04; N;4.43.
Example 5 R -r4-f ri-r4-(Aminomethyl)benzoyll-2- pyrrolidinyDcarbonyl Iphenoxyl acetic acid a) N-trifluoroacetyl-(2-and-4-)-methoxyprolylbenzene
Anisole was acylated according to the procedure of Example 1(a) to give the title compound as before, MH +=301.
b) N-trifluroacetyl-4-hydroxyprolylbenzene
N-Trifluoroacetyl-(2-and-4-)-methoxyprolylbenzene was deprotected according to the procedure of Example 1(b) to give the title compound as a white solid (77% yield), MH+=287
c) N-t-butylcarbamate-4-hydroxyprolylbenzene
To a solution of N-trifluroacetyl-4-hydroxyprolylbenzene (6.2g, 21.3 mmol) in methanol (20 mL) was added IN sodium hydroxide solution ( 25.6 mL, 25.6 mmole). The resulting mixture was allowed to stir at room temperature for 2 hours. di-tert-butyldicarbonate (1.09g, 50 mmol) was added in portions, and the resulting solution was allowed to stir overnight. The solution was concentrated by rotary evaporation, taken into ethyl acetate, and washed with 5% citric acid, water, brine, dried over magnesium sulfate, and concentrated to afford a yellow oil. Trituration with hexane followed by drying in vacua gave the title compound as a white wax (5.72g, 93% yield), MH+291.
d) 4-hydroxyprolylbenzene N-t-Butylcarbamate-4-hydroxyprolylbenzene (2.73g, 7.3 mmol) was treated with 20% trifluoroacetica acid in methylene chloride (25 mL) for 1.5 hours. The solvents were removed and the residue was evaporated twice from methylene chloride . Trituration with diethyl ether, then drying in vacuo gave the title compound as a trifluoroacetic acid salt (1.7g, 81% yield), MH+= 191.
e) (R)-[4-{[l-[4-[N-t-butylcarbamate(aminomethyl)]benzoyl]-2- pyrrolidinyl } carbonyl]phenol To a cold (0°C) solution of 4-hydroxyprolylbenzene £4}(800mg, 2.9 mmol) in dimethylformamide (25 mL) was aded diisopropylethylamine until the pH was equaled at 7.5. To the resulting system was added consecutively 1-hydroxy- benzotriazole (430mg, 3.19 mmol), 4-[N-t-butylcarbamate(aminomethyl)]benzoic acid (800mg, 3.19 mmol) and water-soluble carbodiimide (N-ethyl-N'(dimethyl- aminopropyl)carbodimide, 61 lmg, 3.19 mmol). The resulting solution was stirred at room temperature for 18 hours. The solution was concentrated by rotary evaporation, and the residue was taken into ethyl acetate. The solution was then washed with 5% sodium bicarbonate solution and brine, dried over magnesium sulfate. Concentration, followed by precipitation of the material from ethyl acetate/hexane gave the title compound as a white solid (1.15g, 94% yield), MH+=424.
f) ethyl (R)-[4- { [l-[4-[N-t-butylcarbamate(aminomethyl)]benzoyl]-2- pyrrolidinyl } carbonyl)phenoxy] acetate (R)-[4-{[l-[4-[N-t-butylcarbamate(aminomethyl)]benzoyl]-2-pyrrolidinyl} carbonyl] phenol was O-alkylated with ethyl bromoacetate according to the procedure of Example 1(c) to give the title compound as a white solid (1.05g, 81% yield), MH+= 510.
g) ethyl(R)-[4-{[l-[4-(aminomethyl)benzoyl]-2- pyrrolidinyl)carbonyl }phenoxy] acetate
Ethyl(R)-[4- { [ l-[4-[N-t-butylcarbamate(aminomethyl)]benzoyl]-2- pyrrolidinyl)carbonyl}phenoxy] acetate was N-deprotected according to the procedure of Example 5(d). The resulting white solid was further purified by chromatography (silica gel, 8% methanol/chloroform) to give the title compound as the trifluoroacetic acid salt (789mg, 73% yield), MH+= 410, Anal. Calcd. for C23H26N2θ5-C2HO2F3 : C,60.8; H,7.17; N.3.90. Found: C.61.01; H.7.09; N.3.88. h) (R)-[4- { [ 1 -[4-(aminomethyl)benzoyl]-2-pyrrolidinyl)carbonyl } - phenoxyjacetic acid
To a solution of ethyl (R)-[4-{[l-[4-(aminomethyl)benzoyl]-2-pyrrolidinyl] carbonyljphenoxy] acetate ( 380mg, 0.72 mmol) in methanol (10 mL) was added 2N sodium hydroxide solution (7.2 mL, 1.45 mmol). After 2 hours the reaction was neutralized with IN hydrochloric acid solution and the solution was concentrated by rotary evaporation. Purification by chromatography (silica gel, 10% meth.anol/chloroform) afforded material which was then desalted by non-ionic XAD-2 absorbant (equilibrated with water, eluted with 30% acetonitrile) to give the title compound (60mg, 22% yield), MH+= 382, Anal. Calcd. for C21H22N2O5 •1.5(water): C.58.19; H.5.81; N.6.80. Found: Q58.30; H.5.62; N,6.80.
Example 6 fR -r4-f π-r4-(Aminoiminomethyl benzoyl1-2-pyrrolidinyl carbonyl)- phenoxvl acetate a) 4-hydroxyprolylbenzene
Preparation of the title compound was carried out according to the procedures of Example 5(a) through 5(d).
b) (R)-[4-{ l-[4-(aminoiminomethyl)benzoyl]-2-pyrrolidinyl}carbonyl]phenol To a cold (0°C) solution of 4-hydroxyprolylbenzene (470mg, 1.6 mmol) in dimethylformamide (25 mL) was aded diisopropylethylamine until the pH was equaled at 7.5. To the resulting system was added consecutively 1-hydroxy- benzotriazole (243mg, 1.8 mmol), 4-N-Cbz-amidinobenzoic acid (536mg, 1.8 mmol) and water-soluble carbodiimide (N-ethyl-N'(dimethylaminopropyl)- carbodimide, 343mg, 1.8 mmol). The resulting solution was stirred at room temperature for 18 hours. The solution was concentrated by rotary evaporation, and the residue was taken into ethyl acetate. The solution was then washed with 5% citric acid, 5% sodium bicarbonate, Brine, and dried over magnesium sulfate.
Concentration, followed by precipitation of the material from ethyl acetate/hexane gave the title compound as a white solid (500mg, 59% yield), MH+=473.
fR)-r4-f ri-rN-carbobenzoxy-4-(aminoiminomethyl benzoyIl-2-pyrrolidinyl)- carbonyl ) phenoxyl acetate (R)-[4-{ l-[4-(Aminoiminomethyl)benzoyl]-2-pyrrolidinyl}carbonyl}phenol was O-alkylated according to the procedure of Example 5(f) to give the title compound as an oil (88% yield), MH+=555.
(RVr4-f ri-r4-(aminoiminomethyl benzovn-2-pyrrolidinyl carbonyπ- phenoxvlacetate
Ethyl (R)-[4-{ [l-[N-carbobenzoxy-4-(aminoiminomethyl)benzoyl]-2- pyιτolidinyl)carbonyl}phenoxy] acetate was deprotected according to the procedure of Example 5(g) to give a white impure solid. Purification by chromatography (silica gel, 10%methanol/chloroform) gave the titie compound as an white powder (24% yield), MH+=423, Anal. Calcd. for C23H25N3O5 • 2(hydrochloric acid) : C48.77; H,5.52; N/7.42. Found: Q48.48; H,5.52; N,7.20.
Example 7 Parenteral Dosage Unit Composition
A preparation which contains 20 mg of the compound of Example 1 as a sterile dry powder is prepared as follows: 20 mg of the compound is dissolved in 15 ml of distilled water. The solution is filtered under sterile conditions into a 25 ml multi-dose ampoule and lyophilized. The powder is reconstituted by addition of 20 ml of 5% dextrose in water (D5W) for intravenous or intramuscular injection. The dosage is thereby determined by the injection volume. Subsequent dilution may be made by addition of a metered volume of this dosage unit to another volume of D5W for injection, or a metered dose may be added to another mechanism for dispensing the drug, as in a bottle or bag for IV drip infusion or other injection- infusion system.
Example 8 Oral Dosage Unit Composition A capsule for oral administration is prepared by mixing and milling 50 mg of the compound of Example 1 with 75 mg of lactose and 5 mg of magnesium stearate. The resulting powder is screened and filled into a hard gelatin capsule.
Example 9 Oral Dosage Unit Composition A tablet for oral administration is prepared by mixing and granulating 20 mg of sucrose, 150 mg of calcium sulfate dihydrate and 50 mg of the compound of Example 1 with a 10% gelatin solution. The wet granules are screened, dried, mixed with 10 mg starch, 5 mg talc and 3 mg stearic acid; and compressed into a tablet.
The foregoing is illustrative of the making and using of this invention. This invention, however, is not limited to the precise embodiments described herein, but encompasses all modifications within the scope of the claims which follow.

Claims

What is claimed is:
1. A compound of the formula:
Figure imgf000034_0001
wherein:
A is -(CH2 , Het, Ar, or C3_7cycloalkyl;
D is R*R"N-, R'R"NR'N-, R'R"NR'NCO-, R1NR'NC(=NR')-, R'ONR'C(=NR')-, R'R"NC(=NR'); R'R"NCO-,
R"R'N
Figure imgf000034_0002
, or ®
E is -CR'2-, -CR'2CR'2-, -CR=CR-, -CR'=CR'-CR'2-, -(CR'2)3-,
-CR'2CH(CH3)-, -COCR'2-, -CH(OH)CR'2-, or -CR'2COCR'2-;
X is -CONR'-, NR'CO-, -SO2NR'-, -NR'SO2-, -CO-, -CH(OH)-, -SO2-, -OC(=O)-, or -(CR'2)m-;
Qi to Q^ form any accessible substituted five- or six-membered ring, which may be saturated or unsaturated, optionally containing up to two heteroatoms chosen from the group of O, S, and N wherein S and N may be optionally oxidized;
Y is -CO-, -CR'(OH)-, -SO2-, -CR'2-, -CONR'-, -NR'CO-, SO2NR', or
-NR'SO2-; G is -CR'2-, -CR'2CR'2-, or -CR'=CR'-;
M is Het, Ar, or C3_.7cycloalkyl, each of which is unsubstituted or substituted by R3;
Z is -OCR'2-, -NR'CR'2-, -CR'2CR'2-, -CH(CH3)CR'2-, -CR' -, -CR'=CR'-, or -C(CH3)=CH-;
each R' independently is hydrogen, Cι_4alkyl, C3_7cycloalkyl-Co_.4alkyl, or Ar-Co-4alkyl;
each R" independently is R', -C(O)R', or -C(O)OR15;
R2 is absent or present as Cι_4alkyl, CO2R', Z-CO2R', Cj^alkoxy, hydroxy, Cι. alkylthio, CONR'R', CN, CF3, halo, or NR'R';
R3 is Cι_4alkyl, C^^alkoxy, Cι_4alkylthio, hydroxy, halo, CO2R', Z-CO2R', NR'R', CN CONR'R', CF3, or Ar-C0__4alkyl;
Rl4 is R', C(O)R\ CN, NO2, SO2R*, or C(O)OR15;
each Rl5 independently is Ci.galkyl, C3_7cycloalkyl-C()-4alkyl, or Ar-Co-4alkyl;
J is absent or present as S or O;
T is absent or present as N=CR', CO, or O;
m is 1 or 2;
each n independently is 0 or 1; and
each t independently is 0 to 2;
or a pharmaceutically acceptable salt thereof.
2. The compound according to claim 1, wherein Ql to Q^ optionally substituted by R2 are any accessible combination of the following:
Q1 is CH, C, or N; Q2 is CH, C, or N;
Q3 is CH2, CHR2, C(R2)2, CH, CR2, NH, NR2, N, 0, or S; Q4 is CH2, CHR2, C(R2)2, CH, CR2, NH, NR2, N, O, or S; Q5 is absent or present as CH2, CHR2, C(R2)2, CH, CR2, NH, NR2, N, O, or S; and
Q6 is CH2, CHR2, C(R2)2, CH, CR2, NH, NR2, N, O, or S.
3. The compound according to claim 2, wherein Q and Q2 optionally substituted by R2 is:
Figure imgf000036_0001
Figure imgf000036_0002
Figure imgf000036_0003
Figure imgf000037_0001
Figure imgf000037_0002
4. The compound according to claim 3, wherein Q to Q^ optionally substituted by R2 is:
Figure imgf000038_0001
(X) (X)
Figure imgf000038_0002
5. The compound according to claim 4, wherein: D is R'R"NC (=NR , R'R"NCO-, or R*R"N-; A is phenyl;
X is -CO-, -CH(OH)-, -SO2-, -CONR'- or -NR'CO-;
Y is -CO-, -CH(OH)-, -SO2-, -CONR'- or -NR'CO-;
M is phenyl;
Z is -OCH2-, NR'CH2-, -CH2CH2, or -CH=CH-;
R2 is absent; t is 0 to 1; each n independendy is 0; and
Q1 to Q6 is
Figure imgf000038_0003
6. The compound according to claim 5 which is N-[4- aminoiminomethyl)benzoyl]-4-oxyacetic acid-(R)-prolylbenzene or a pharmaceutically acceptable salt thereof.
7. The compound according to claim 5 which is:
N-[4-aminoiminomethyl)benzoyl]-4-oxyacetic acid-(R)-Ψ-(R,S)- hydroxymethyl-prolylbenzene;
(R)- [4- { ( 1 - [4-(aminoiminomethy l)benzoyl] -2-pyrrolidinyl)carbonyl } -2- (2- phenylethyl)phenoxy]acetic acid; (R)-[4- { ( 1 -[4-(aminocarbonyl)benzoyl]-2-pyrrolidinyl)carbonyl } -2-(2- phenylethyl)phenoxy]acetic acid; (R)-[4- { (1 -[4-(aminomethyl)benzoyl]-2-pyrrolidinyl)carbonyl } - phenoxy] acetic acid; ethyl (R)-[4-{(l-[4-(aminoiminomethyl)benzoyl]-2-pyrrolidinyl)carbonyl}- phenoxy] acetate; or ethyl (R)-[4-{(l-[4-(aminoiminomethyl)benzoyl]-2-pyrrolidinyl)carbonyl}- phenoxy]acetate; or a pharmaceutically acceptable salt thereof.
8. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier.
9. A pharmaceutical composition comprising a compound according to claim 6 and a pharmaceutically acceptable carrier.
10. A pharmaceutical composition comprising a compound according to claim 7 and a pharmaceutically acceptable carrier.
11. A compound according to claim 1 for use in the manufacture of a medicament.
12. A method for effecting inhibition of platelet aggregation which comprises administering to a subject in need thereof an effective amount of a compound according to claim 1.
13. A method of treating stroke which comprises administering to a subject in need thereof an effective amount of a compound according to claim 1.
14. A method of treating transient ischemia attacks which comprises administering to a subject in need thereof an effective amount of a compound according to claim 1.
15. A method of treating myocardial infarction which comprises administering to a subject in need thereof an effective amount of a compound according to claim 1.
16. A method of treating atherosclerosis which comprises administering to a subject in need thereof an effective amount of a compound according to claim 1.
PCT/US1993/012531 1992-12-30 1993-12-22 Chemical compounds WO1994015913A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0656348A2 (en) * 1993-12-03 1995-06-07 F. Hoffmann-La Roche Ag Aceric acid derivatives as medicaments
US7235690B2 (en) 2001-04-28 2007-06-26 Sanofi-Aventis Deutschland Gmbh Anthranilamides and methods of their use

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0505868A2 (en) * 1991-03-26 1992-09-30 F. Hoffmann-La Roche Ag N-Acyl-alpha-aminoacids derivatives

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0505868A2 (en) * 1991-03-26 1992-09-30 F. Hoffmann-La Roche Ag N-Acyl-alpha-aminoacids derivatives

Non-Patent Citations (2)

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Title
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See also references of EP0677041A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0656348A2 (en) * 1993-12-03 1995-06-07 F. Hoffmann-La Roche Ag Aceric acid derivatives as medicaments
EP0656348A3 (en) * 1993-12-03 1995-09-06 Hoffmann La Roche Aceric acid derivatives as medicaments.
US5726185A (en) * 1993-12-03 1998-03-10 Hoffmann-La Roche Inc. Acetic acid derivatives
US5973188A (en) * 1993-12-03 1999-10-26 Hoffmann-La Roche Inc. Acetic acid derivatives
US7235690B2 (en) 2001-04-28 2007-06-26 Sanofi-Aventis Deutschland Gmbh Anthranilamides and methods of their use

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