Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
  • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
  • C07D513/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
• • 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

• the invention relates to novel heterocycles containing aminobenzothiazole and aminobenzoxazole derivatives which are effective platelet ADP receptor inhibitors. These derivatives may be used in various pharmaceutical compositions. In particular, the derivatives may be used in pharmaceutical compositions effective for the prevention and/or treatment of cardiovascular diseases, particularly those diseases related to thrombosis.
• thrombotic cytopenic purpura examples include acute myocardial infarction, unstable angina, chronic stable angina, transient ischemic attacks, strokes, peripheral vascular disease, preeclampsia/eclampsia, deep venous thrombosis, embolism, disseminated intravascular coagulation and thrombotic cytopenic purpura.
• Thrombotic and restenotic complications also occur following invasive procedures, e.g., angioplasty, carotid endarterectomy, post CABG (coronary artery bypass graft) surgery, vascular graft surgery, stent placements and insertion of endovascular devices and protheses. It is generally thought that platelet aggregates play a critical role in these events.
• Blood platelets which normally circulate freely in the vasculature, become activated and aggregate to form a thrombus with disturbed blood flow caused by ruptured atherosclerotic lesions or by invasive treatments such as angioplasty, resulting in vascular occlusion. Platelet activation can be initiated by a variety of agents, e.g., exposed subendothelial matrix molecules such as collagen, or by thrombin which is formed in the coagulation cascade.
• agents e.g., exposed subendothelial matrix molecules such as collagen, or by thrombin which is formed in the coagulation cascade.
• ADP adenosine 5'- diphosphate
• P 2T receptors Hourani et al., Trends Pharmacol. Sci. 15, 103 (1994); Savi et al., Med. Res. Rev. 16, 159 (1996); Mills, Thromb, Hemost. 76, 835 (1996); Gachet et al., Thromb. Hemost. 78, 271 (1997)).
• Platelet ADP receptors mediating aggregation are activated by ADP and some of its derivatives and antagonized by ATP (adenosine 5'-triphosphate) and some of its derivatives. Therefore, platelet ADP receptors are members of the family of P2 receptors activated by purine and/or pyrimidine nucleotides (Harden et al., Annu. Rev. Pharmacol. Toxicol. 35, 541 (1995); North et al, Curr. Opin. Neurobiol. 7, 346 (1997)).
• the orally active antithrombotic thienopyridines ticlopidine and clopidogrel inhibit ADP-induced platelet aggregation, binding of radiolabeled ADP receptor agonist 2-methylthioadenosine 5 '-diphosphate to platelets, and other ADP-dependent events indirectly, probably via formation of an unknown metabolite, in humans or animals (Savi et al., Med. Res. Rev. 16, 159 (1996)).
• Some derivatives of the endogenous antagonist ATP, e.g., ARL (formerly FPL) 67085 are selective platelet ADP receptor antagonists which inhibit ADP-dependent platelet aggregation and are effective in animal thrombosis models (Mills, Thromb. Hemost.
• platelet ADP receptor inhibitors having antithrombotic activity that are useful in the prevention and/or treatment of cardiovascular diseases, particularly those related to thrombosis.
• the invention provides pharmaceutical compositions for preventing or treating thrombosis in a mammal containing a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
• the invention further provides a method for preventing or treating thrombosis in a mammal by administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
• C,-C 6 alkyl refers to a straight or branched hydrocarbon containing one to six carbon atoms.
• C 3 -C 8 cycloalkyl refers to a cyclic aliphatic hydrocarbon containing three to eight carbon atoms.
• phenyl refers to a six carbon containing aromatic ring which can be variously mono- or poly-substituted with H, C,-C 6 alkyl, hydroxyl, C,-C 6 alkoxy, amino, mono-C,-C 6 alkylamino, di-C,-C 6 alkylamino, nitro, fluoro, chloro, bromo, iodo, hydroxycarbonyl, or C,-C 6 alkoxy carbonyl.
• C,-C 6 alkoxy refers to an ether moiety whereby the oxygen is connected to a straight or branched chain of carbon atoms of the number indicated.
• phenoxy refers to an ether moiety whereby the oxygen is connected to a phenyl substituent, the latter being defined as above.
• mono-C,-C 6 alkylamino refers to an amino moiety whereby the nitrogen is substituted with one H and one C,-C 6 alkyl substituent, the latter being defined as above.
• di-C,-C 6 alkylamino refers to an amino moiety whereby the nitrogen is substituted with two C,-C 6 alkyl substituents as defined above.
• monoarylamino refers to an amino moiety whereby the nitrogen is substituted with one H and one aryl substituent, such as a phenyl, the latter being defined as above.
• diarylamino refers to an amino moiety whereby the nitrogen is substituted with two aryl substituents, such as phenyl, the latter being defined as above.
• C,-C 6 alkylsulfonyl refers to a dioxosulfur moiety with the sulfur atom also connected to one C,-C 6 alkyl substituent, the latter being defined as above.
• C,-C 6 alkoxy carbonyl refers to a hydroxycarbonyl moiety whereby the hydrogen is replaced by a C r C 6 alkyl substituent, the latter being defined as above.
• heterocyclic group refers to any saturated or unsaturated mono- or bicyclic ring system, containing from one to five heteroatoms. Each heteroatom may independently be nitrogen, oxygen or sulfur.
• suitable heterocyclic groups include, but are not limited to, piperidyl, pyrrolidinyl, pyridyl, piperazinyl, piperidonyl, thiazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, pyridoxazolyl, pyridothiazolyl, pyridazinoxazolyl, pyridazinothiazolyl, pyrimidothiazolyl, pyrimidoxazolyl, pyrazinothiazolyl, pyrazinoxazolyl, triazinothiazolyl, and triazinoxazolyl.
• a “pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable.
• the salts may be formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or organic acids such as, but not limited to, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
• “pharmaceutically acceptable base addition salts” include but are not limited to those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum bases, and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts.
• Salts derived from pharmaceutically acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
• Particularly preferred organic nontoxic bases are isopropyl amine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
• Bio property for the purposes herein means an in vitro or in vivo biological effect or an antigenic function or activity that is directly or indirectly performed by a compound of the invention. Effect or functions include receptor or ligand binding, any enzyme activity or enzyme modulatory activity, any carrier binding activity, any hormonal activity, any activity in promoting or inhibiting adhesion of cells to an extracellular matrix or cell surface molecules, including the aggregation of platelets or any structural role. Antigenic functions include possession of an epitope or antigenic site that is capable of reacting with antibodies raised against it.
• W is carbon or nitrogen, wherein at least one W is a carbon;
• Y is nitrogen, oxygen, or sulfur;
• R is, independently, H, C,-C 6 alkyl, C 3 -C 8 cycloalkyl, phenyl, pyridyl, pyrimidinyl, hydroxyl, C,-C 6 alkoxy, phenoxy, amino, mono-C,-C 6 alkylamino, di-C,-C 6 alkylamino, monoarylamino, diarylamino, nitro, fluoro, chloro, bromo, iodo, C,-C 6 alkylsulfonyl, hydroxycarbonyl, C,-C 6 alkoxycarbonyl, absent if W is a nitrogen, or adjacent Rj groups together may form a five- or six-membered alicyclic ring, a six-membered aromatic ring, or a six-membered heteroaromatic ring containing one or two nitrogens, with the proviso that when a sequence of three W-R, groups form a N(R,)-C(R,
• R 2 and R 3 are, independently, H, C,-C 6 alkyl, C 3 -C 8 cycloalkyl, or R 2 and R 3 together form an alicyclic ring containing 3 to 8 carbon atoms;
• R 4 is a substituted or unsubstituted heterocyclic group containing at least one heteroatom of nitrogen, oxygen, or sulfur. Suitable substituents of R 4 include those groups encompassed by R,.
• W is carbon or nitrogen, wherein at least one W is a carbon
• Y is oxygen or sulfur
• R is, independently, H, C,-C 6 alkyl, phenyl, pyridyl, pyrimidinyl, C,-C 6 alkoxy, phenoxy, amino, mono-C,-C 6 alkylamino, di-C,-C 6 alkylamino, C C 6 alkylsulfonyl, absent if W is a nitrogen, or adjacent R, groups together form a six-membered aromatic ring, or a six- membered heteroaromatic ring containing one or two nitrogens, with the proviso that when a sequence of three W-R, groups form a N(R
• R 2 and R 3 are, independently, H or C j - alkyl
• R 4 is a substituted or unsubstituted heterocyclic group containing at least one heteroatom of nitrogen, oxygen, or sulfur. Suitable substituents of R 4 include those groups encompassed by R, as described herein.
• W is carbon
• Y is sulfur;
• R is, independently, H, pyridyl, pyrimidinyl, amino, mono-C,-C 6 alkylamino, or di-
• R at the 8-position is C,-C 6 alkyl, pyridyl, pyrimidinyl, hydroxyl, C,-C 6 alkoxy, amino, mono-C,-C 6 alkylamino, di-C,-C 6 alkylamino, or C,-C 6 alkylsulfonyl;
• R 2 and R 3 are each a hydrogen; and R 4 is a substituted or unsubstituted heterocyclic group containing at least one heteroatom of nitrogen, oxygen, or sulfur. Suitable substituents of R 4 include those groups encompassed by R, as described herein.
• R 4 groups of a compound of formula (I) include, but are not limited to, piperidyl, pyrrolidinyl, pyridyl, piperazinyl, piperidonyl, thiazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, pyridoxazolyl, pyridothiazolyl, pyridazinoxazolyl, pyridazinothiazolyl, pyrimidothiazolyl, pyrimidoxazolyl, pyrazinothiazolyl, pyrazinoxazolyl, triazinothiazolyl, and triazinoxazolyl.
• Preferred R 4 groups include, but are not limited to, benzothiazolyl, benzoxazolyl, pyrido[2,3- d][l,3]oxazolyl, pyrido[2,3-d][l,3]thiazolyl, pyrido[3,4-d][l,3]oxazolyl, pyrido[3,4- d][l,3]thiazolyl, pyrido[4,3-d][l,3]oxazolyl, ⁇ yrido[4,3-d][l,3]thiazolyl, pyrido[3,2- d][l,3]oxazolyl, pyrido[3,2-d][l,3]thiazolyl, pyridazino[3,4-d][l,3]oxazolyl, pyridazino [3 ,4- d][l,3]thiazolyl, pyrid
• Another preferred embodiment of the compound of formula (I) is a compound of formula (II):
• W, Y, R réelle R 2 , and R 3 are each as defined above.
• a compounds of formula (I) may be prepared by reacting an aminoazole and chlorosulfonylacetyl chloride in an organic solvent in the presence of a molar excess of a tertiary amine base.
• the molar ratio of aminoazole to chlorosulfonylacetyl ranges from about a 1 : 1 , as shown by Scheme A, to about a 2: 1 , as shown by Scheme B.
• the aminoazole may be any commercially available aminoazole, including for example, substituted 2-aminobenzothiazole or 2-aminobenzoxazole derivatives.
• the aminoazole may also be prepared synthetically using techniques known in the art.
• substituted 2-aminobenzoxazoles may be prepared according to the method outlined in Scheme I, where a substituted o-aminophenol is reacted with cyanogen bromide (Sam et al., Journal of Pharmaceutical Sciences 53, 538 (1964)):
• substituted 2-aminobenzothiazoles may be prepared according to the method outlined in Scheme II, where a substituted aniline is reacted with ammonium thiocyanate in the presence of bromine or iodine (Mangold et al., Journal of Medicinal Chemistry 25, 630 (1982); Allen et al., Organic Synthesis Collective 3, 76 (1955)):
• pyrido-fused and pyrimido-fused aminoazoles by, for example, starting with commercially available materials such as 2-amino-3-hydroxypyridine or 4-aminopyrimidine.
• pure aminoazoles may be isolated using typical isolation and purification techniques known in the art, such as solvent-solvent extraction and normal phase chromatography on silica gel.
• the pure aminoazole compounds may then be reacted in the usual manner as described above with chlorosulfonylacetyl chloride in the presence of a tertiary amine base to produce a compound of formula (I).
• any tertiary amine base capable of acting as a neutralizing agent for the HCl generated upon reaction of the aminoazole with chlorosulfonylacetyl chloride may be used.
• the tertiary amine base is triethylamine or diisopropylethylamine.
• the organic solvent may be any solvent common to the practice of organic chemistry such as, for example, tetrahydrofuran, dichloromethane, chloroform, acetonitrile, and N,N-dimethylformamide.
• the organic solvent is tetrahydrofuran.
• carbon atoms to which four non- identical substituents are bonded are asymmetric.
• a compound of formula (I) may exist as enantiomers, diastereomers or a mixture thereof.
• the enantiomers and diastereomers may be separated by chromatographic or crystallization methods, or by other methods known in the art.
• the asymmetric carbon atom when present in a compound of formula (I) of the invention may be in one of two configurations (R or S) and both are within the scope of the invention.
• compounds of formula (I) may be further treated to form pharmaceutically acceptable salts.
• Treatment of a compound of the invention with an acid or base may form, respectively, a pharmaceutically acceptable acid addition salt and a pharmaceutically acceptable base addition salt, each as defined above.
• Various inorganic and organic acids and bases known in the art including those defined herein may be used to effect the conversion to the salt.
• the invention also relates to pharmaceutically acceptable isomers, hydrates, and solvates of compounds of formula (I).
• Compounds of formula (I) may also exist in various isomeric and tautomeric forms including pharmaceutically acceptable salts, hydrates and solvates of such isomers and tautomers.
• This invention also encompasses prodrug derivatives of the compounds of formula (I).
• prodrug refers to a pharmacologically inactive derivative of a parent drug molecule that requires biotransformation, either spontaneous or enzymatic, within the organism to release the active drug.
• Prodrugs are variations or derivatives of the compounds of formula (I) of this invention which have groups cleavable under metabolic conditions. Prodrugs become the compounds of the invention which are pharmaceutically active in vivo when they undergo solvolysis under physiological conditions or undergo enzymatic degradation.
• Prodrug compounds of this invention may be called single, double, triple, etc., depending on the number of biotransformation steps required to release the active drug within the organism, and indicating the number of functionalities present in a precursor-type form.
• Prodrug forms often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam (1985); Silverman, 77 ⁇ e Organic Chemistry of Drug Design and Drug Action, pp. 352-401, Academic Press, San Diego, CA (1992)).
• Prodrugs commonly known in the art include acid derivatives well known to practitioners of the art, such as, for example, esters prepared by reaction of the parent acids with a suitable alcohol, or amides prepared by reaction of the parent acid compound with an amine, or basic groups reacted to form an acylated base derivative.
• the prodrug derivatives of this invention may be combined with other features herein taught to enhance bioavailability.
• a compound of formula (I) or formula (II) according to the invention may be formulated into pharmaceutical compositions. Accordingly, the invention also relates to a pharmaceutical composition for preventing or treating thrombosis in a mammal, particularly those pathological conditions involving platelet aggregation, containing a therapeutically effective amount of a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof, each as described above, and a pharmaceutically acceptable carrier or agent.
• a pharmaceutical composition of the invention contains a compound of formula (I) or formula (II) or a salt thereof in an amount effective to inhibit platelet aggregation, more preferably, ADP-dependent aggregation, in a mammal, in particular, a human.
• Pharmaceutically acceptable carriers or agents include those known in the art and are described below.
• compositions of the invention may be prepared by mixing the compound of formula (I) or formula (II) with a physiologically acceptable carrier or agent.
• Pharmaceutical compositions of the invention may further include excipients, stabilizers, diluents and the like and may be provided in sustained release or timed release formulations.
• Acceptable carriers, agents, excipients, stablilizers, diluents and the like for therapeutic use are well known in the pharmaceutical field, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., ed. A.R. Gennaro (1985).
• Such materials are nontoxic to the recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate and other organic acid salts, antioxidants such as ascorbic acid, low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidinone, amino acids such as glycine, glutamic acid, aspartic acid, or arginine, monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, counterions such as sodium and/or nonionic surfactants such as TWEEN, or polyethyleneglycol.
• buffers such as phosphate, citrate, acetate and other organic acid salts
• antioxidants such as ascorbic acid,
• Methods for preventing or treating thrombosis in a mammal embraced by the invention administer a therapeutically effective amount of a compound of formula (I) or formula (II) alone or as part of a pharmaceutical composition of the invention as described above to a mammal, in particular, a human.
• Compounds of formula (I) or formula (II) and pharmaceutical compositions of the invention containing a compound of formula (I) or formula (II) of the invention are suitable for use alone or as part of a multi-component treatment regimen for the prevention or treatment of cardiovascular diseases, particularly those related to thrombosis.
• a compound or pharmaceutical composition of the invention may be used as a drug or therapeutic agent for any thrombosis, particularly a platelet-dependent thrombotic indication, including, but not limited to, acute myocardial infarction, unstable angina, chronic stable angina, transient ischemic attacks, strokes, peripheral vascular disease, preeclampsia/eclampsia, deep venous thrombosis, embolism, disseminated intravascular coagulation and thrombotic cytopenic purpura, thrombotic and restenotic complications following invasive procedures, e.g., angioplasty, carotid endarterectomy, post CABG (coronary artery bypass graft) surgery, vascular graft surgery, stent placements and insertion of endovascular devices and protheses.
• invasive procedures e.g., angioplasty, carotid endarterectomy, post CABG (coronary artery bypass graft) surgery, vascular graft surgery
• Compounds and pharmaceutical compositions of the invention may also be used as part of a multi-component treatment regimen in combination with other therapeutic or diagnostic agents in the prevention or treatment of thrombosis in a mammal.
• compounds or pharmaceutical compositions of the invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice such as anticoagulant agents, thrombolytic agents, or other antithrombotics, including platelet aggregation inhibitors, tissue plasminogen activators, urokinase, prourokinase, streptokinase, heparin, aspirin, or warfarin.
• Coadministration may also allow for application of reduced doses of the thrombolytic agents and therefore minimize potential hemorrhagic side-effects.
• Compounds and pharmaceutical compositions of the invention may also act in a synergistic fashion to prevent reocclusion following a successful thrombolytic therapy and or reduce the time to reperfusion.
• the compounds and pharmaceutical compositions of the invention may be utilized in vivo, ordinarily in mammals such as primates, (e.g., humans), sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.
• mammals such as primates, (e.g., humans), sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.
• the biological properties, as defined above, of a compound or a pharmaceutical composition of the invention can be readily characterized by methods that are well known in the art such as, for example, by in vivo studies to evaluate antithrombotic efficacy, and effects on hemostasis and hematological parameters.
• Compounds and pharmaceutical compositions of the invention may be in the form of solutions or suspensions.
• the compounds or pharmaceutical compositions of the invention may also be in such forms as, for example, tablets, capsules or elixirs for oral administration, suppositories, sterile solutions or suspensions or injectable administration, and the like, or incorporated into shaped articles.
• Subjects (typically mammalian) in need of treatment using the compounds or pharmaceutical compositions of the invention may be administered dosages that will provide optimal efficacy.
• Dosage formulations of compounds of formula (I) or formula (II) or pharmaceutical compositions of the invention to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile membranes such as 0.2 micron membranes, or by other conventional methods. Formulations typically will be stored in a solid form, preferably in a lyophilized form.
• the dosage formulations of compounds of formula (I) or formula (II) or pharmaceutical compositions of the invention may also be administered by injection, intravenously (bolus and/or infusion), subcutaneously, intramuscularly, colonically, rectally, nasally, transdermally or intraperitoneally.
• a variety of dosage forms may be employed as well including, but not limited to, suppositories, implanted pellets or small cylinders, aerosols, oral dosage formulations and topical formulations such as ointments, drops and dermal patches.
• the compounds of formula (I) or formula (II) and pharmaceutical compositions of the invention may also be incorporated into shapes and articles such as implants which may employ inert materials such biodegradable polymers or synthetic silicones as, for example, SILASTIC, silicone rubber or other polymers commercially available.
• the compounds and pharmaceutical compositions of the invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
• Liposomes can be formed from a variety of lipids, such as cholesterol, stearylamine or phosphatidylcholines.
• Therapeutically effective dosages may be determined by either in vitro or in vivo methods.
• the range of therapeutically effective dosages will be influenced by the route of administration, the therapeutic objectives and the condition of the patient. For injection by hypodermic needle, it may be assumed the dosage is delivered into the bodily fluids. For other routes of administration, the absorption efficiency must be individually determined for each compound by methods well known in pharmacology. Accordingly, it may be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.
• the determination of effective dosage levels that is, the dosage levels necessary to achieve the desired result, i.e., platelet ADP receptor inhibition, will be readily determined by one skilled in the art.
• a pharmaceutically acceptable carrier used in a pharmaceutical composition of the invention, typically, about 5 to 500 mg of a compound of formula (I) or formula (II) is compounded with a pharmaceutically acceptable carrier as called for by accepted pharmaceutical practice including, but not limited to, a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, dye, flavor, etc.
• the amount of active ingredient in these compositions is such that a suitable dosage in the range indicated is obtained.
• Typical adjuvants which may be incorporated into tablets, capsules and the like include, but are not limited to, binders such as acacia, corn starch or gelatin, and excipients such as microcrystalline cellulose, disintegrating agents like corn starch or alginic acid, lubricants such as magnesium stearate, sweetening agents such as sucrose or lactose, or flavoring agents.
• binders such as acacia, corn starch or gelatin
• excipients such as microcrystalline cellulose
• disintegrating agents like corn starch or alginic acid
• lubricants such as magnesium stearate
• sweetening agents such as sucrose or lactose
• flavoring agents such as sucrose or lactose
• a dosage form is a capsule, in addition to the above materials it may also contain liquid carriers such as water, saline, or a fatty oil.
• Other materials of various types may be used as coatings or as modifiers of the physical form of the dosage unit.
• dissolution or suspension of the active compound in a vehicle such as an oil or a synthetic fatty vehicle like ethyl oleate, or into a liposome may be desired.
• a vehicle such as an oil or a synthetic fatty vehicle like ethyl oleate
• Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.
• the instrument used for data collection was a Waters (Bedford, MA) Model 600 controller connected to a Waters Model 996 photodiode array detector interfaced with a Waters Model 717 autosampler. Data collection and analysis were computer-controlled using the Millenium software package proprietary to the Waters system. Preparative HPLC data were obtained using a 5.0 cm diameter C 18 column under the solvent elution conditions indicated in the specific examples.
• the instrument used for sample preparation was a Waters Model 600 controller connected to a Waters Model 490 four- wavelength detector interfaced with an X- Y stripchart recorder to monitor peak elu-ion as a function of time.
• the reaction was quenched by addition of water.
• the biphasic solution was acidified with 10%) HCl and extracted twice with ethyl acetate.
• the combined organic extracts were washed twice with saturated brine, dried with magnesium sulfate, and concentrated in vacuo. There was obtained a yellow-brown solid.
• the reaction was quenched by addition of water.
• the biphasic solution was acidified with 10%) citric acid and extracted twice with ethyl acetate.
• the combined organic extracts were washed twice with saturated brine, dried with magnesium sulfate, and concentrated in vacuo. There was obtained a yellow-orange solid.
• the reaction was quenched by addition of water.
• the biphasic solution was acidified with 10%) citric acid and extracted twice with ethyl acetate.
• the combined organic extracts were washed twice with saturated brine, dried with magnesium sulfate, and concentrated in vacuo. There was obtained a yellow-orange solid.
• the reaction was quenched by addition of water.
• the biphasic solution was acidified with 10%) citric acid and extracted with ethyl acetate.
• a heavy, yellow-orange precipitate formed which was collected by vacuum filtration.
• the filtrate contained only trace amounts of material after phase separation and evaporation of organic solvent.
• the reaction was quenched by addition of water.
• the biphasic solution was acidified with 10%) citric acid and extracted twice with ethyl acetate.
• the combined organic extracts were washed twice with saturated brine, dried with magnesium sulfate, and concentrated in vacuo. A yellow solid was obtained.
• the reaction was quenched by addition of water.
• the biphasic solution was acidified with 10%) citric acid and extracted twice with ethyl acetate.
• the combined organic extracts were washed twice with saturated brine, dried with magnesium sulfate, and concentrated in vacuo. An orange-brown film was obtained.
• the reaction was quenched by addition of water.
• the biphasic solution was acidified with 10%) citric acid and extracted twice with ethyl acetate.
• the combined organic extracts were washed twice with saturated brine, dried with magnesium sulfate, and concentrated in vacuo. An orange foam was obtained.
• test Compounds 1-8 of, respectively, Examples 1-8 on ADP-induced human platelet aggregation was assessed in 96-well microtiter assay.
• Human venous blood was collected from healthy, drug-free volunteers into ACD (85 mM sodium citrate, 111 mM glucose, 71.4 mM citric acid) containing PGI 2 (1.25 ml ACD containing 1.6 ⁇ M PGI 2 /10 ml blood; PGI 2 was from Sigma, St. Louis, MO).
• ACD 85 mM sodium citrate, 111 mM glucose, 71.4 mM citric acid
• PGI 2 (1.25 ml ACD containing 1.6 ⁇ M PGI 2 /10 ml blood; PGI 2 was from Sigma, St. Louis, MO).
• Platelet-rich plasma (PRP) was prepared by centrifugation at 160 x g for 20 minutes at room temperature.
• Washed platelets were prepared by centrifuging PRP for 10 minutes at 730 x g and resuspending the platelet pellet in CGS (13 mM sodium citrate, 30 mM glucose, 120 mM NaCl; 2 ml CGS/10 ml original blood volume) containing lU/ml apyrase (grade V, Sigma, St. Louis, MO).
• CGS 13 mM sodium citrate, 30 mM glucose, 120 mM NaCl; 2 ml CGS/10 ml original blood volume
• lU/ml apyrase grade V, Sigma, St. Louis, MO
• the platelets were collected by centrifugation at 730 x g for 10 minutes and resuspended at a concentration of 3xl0 8 platelets/ml in Hepes-Tyrode's buffer (10 mM Hepes, 138 mM NaCl, 5.5 mM glucose, 2.9 mM KC1, 12 mM NaHCO 3 , pH 7.4) containing 0.1% bovine serum albumin, 1 mM CaCl 2 and 1 mM MgCl 2 . This platelet suspension was kept >45 minutes at 37°C before use in aggregation assays.
• ADP was added to a final concentration of 2 ⁇ M which induces submaximal aggregation. Buffer was added instead of ADP to one set of control wells (ADP " control).
• the OD of the samples was then determined at 490 nm using a microtiter plate reader (Softmax, Molecular Devices, Menlo Park, CA) resulting in the 0 minute reading.
• Table 2 show the mean of 3-12 independent IC 50 experiments each performed in duplicate.
• Compounds 1-8 inhibited ADP-dependent aggregation of human platelets with IC 50 s from 180 nM to > 120 ⁇ M.
• Compound 1 was also tested in the presence of 8-sulphophenyltheophylline, an adenosine receptor antagonist. The potency of compound 1 was not reduced indicating that the anti-platelet activity was not mediated by platelet adenosine receptors.
• Outdated platelet suspensions were diluted with 1 volume of CGS and platelets pelleted by centrifugation at 1900 x g for 45 minutes. Platelet pellets were resuspended at 3- 6xl0 9 platelets /ml in CGS containing 1 U/ml apyrase (grade V, Sigma, St. Louis, MO) and incubated for 15 minutes at 37°C. After centrifugation at 730 x g for 20 minutes, pellets were resuspended in Hepes-Tyrode's buffer containing 0.1%) BSA (Sigma, St. Louis, MO) at a concentration of 6.66x10 8 platelets/ml. Binding experiments were performed after > 45 minutes resting of the platelets.
• binding experiments were performed with fresh human platelets prepared as described in I. (Inhibition of ADP-Mediated Platelet Aggregation in vitro), except that platelets were resuspended in Hepes-Tyrode's buffer containing 0.1 %> BSA (Sigma , St. Louis, MO) at a concentration of 6.66x10 8 platelets/ml. Very similar results were obtained with fresh and outdated platelets (see below).
• a platelet ADP receptor binding assay using the tritiated potent agonist ligand [ 3 H]2- MeS-ADP, fresh platelets from rats and rapid filtration has been described (Savi et al., J. Pharmacol. Exp. Ther. 269, 772 (1994)).
• a binding assay in a 96-well microtiter format using outdated or fresh human platelets and the radioligand [ 3 H]2-MeS-ADP [ 3 H]2- methylthioadenosine-5'-diphosphate, ammonium salt; specific activity 49 Ci/mmole, obtained by custom synthesis from Amersham Life Science, Inc., Arlington Heights, IL
• All steps were performed at room temperature unless indicated otherwise.
• Binding Wash Buffer (10 mM Hepes pH 7.4, 138 mM NaCl) using a 96-well cell harvester (Minidisc 96, Skatron Instruments, Sterling, VA) and 8x12 GF/C glassfiber filtermats (Printed Filtermat A, for 1450 Microbeta, Wallac Inc., Gaithersburg, MD). The platelet-bound radioactivity on the filtermats was determined in a scintillation counter (Microbeta 1450, Wallac Inc., Gaithersburg, MD). Specific binding was determined by subtraction of non-specific binding from total binding, and specific binding in the presence of test compounds was expressed as %> of specific binding in the absence of test compounds dilutions.
• the data in Table 2 provide the mean of 2-8 independent IC 50 experiments each performed in duplicate with outdated platelets.
• Compounds 1-8 inhibited binding of 1 nM [ 3 H]2-MeS-ADP to human platelets with IC 50 s from 170 nM to 37 ⁇ M.
• There was a good correlation between the IC 50 s of these compounds for ADP-dependent platelet aggregation and [ 3 H]2- MeS-ADP binding suggesting that the anti-platelet activity was specifically mediated by ADP receptors.
• hP2Y. Receptor Activity Assay Platelet ADP receptors are considered members of the P2 family of cell surface receptor subtypes that are activated by purine and/or pyrimidine nucleotides (North et al., Curr. Opin. Neurobiol. 7, 346 (1997); Harden et al, Annu. Rev. Pharmacol. Toxicol. 35, 541 (1995)). Recent studies with cells expressing a cloned member of this family, the human P2Y, receptor (hP2Y,), suggest that its pharmacological profile might be very similar to platelet ADP receptors mediating aggregation (Gachet et al., Thromb. Hemost. 78, 271 (1997)).
• hP2Y, receptor activity of test compounds was assessed by measuring agonist-induced intracellular calcium mobilization in a mammalian cell line expressing the cloned receptor gene.
• a genomic fragment encompassing the entire open reading frame of the human P2Y, receptor plus 220 bp of 3' untranslated region and 10 bp 5' to the ATG initiation codon was isolated from human genomic DNA using standard molecular biology techniques. The deduced amino acid sequence was as described (Schachter et al., Br. J. Pharmacol. 118, 167 (1996)).
• This fragment was cloned into the mammalian expression vector pcINeo (Promega, Madison, WI) and transfected into Jurkat cells (American Type Culture Collection, Rockville, MD) using standard procedures resulting in the clonal cell line hP2Yl-JA7 stably expressing the hP2Y, receptor.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Animal Behavior & Ethology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Veterinary Medicine (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Medicinal Chemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Public Health (AREA)
• Pharmacology & Pharmacy (AREA)
• General Health & Medical Sciences (AREA)
• Heart & Thoracic Surgery (AREA)
• Cardiology (AREA)
• Urology & Nephrology (AREA)
• Vascular Medicine (AREA)
• Diabetes (AREA)
• Hematology (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)

Applications Claiming Priority (5)

Publications (1)

Family

ID=26677116

Family Applications (1)

Country Status (5)

Families Citing this family (14)

Family Cites Families (1)

• 1999
  • 1999-01-08 WO PCT/US1999/000518 patent/WO1999036425A1/en not_active Application Discontinuation
  • 1999-01-08 JP JP2000540141A patent/JP2002509152A/ja active Pending
  • 1999-01-08 AU AU22195/99A patent/AU2219599A/en not_active Abandoned
  • 1999-01-08 EP EP99902148A patent/EP1047699A1/en not_active Withdrawn
  • 1999-01-08 CA CA002318199A patent/CA2318199A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events