EP1943261A2 - Stents pour élution de médicament avec composé antagoniste récepteur p2y12 non nucléotide - Google Patents

Stents pour élution de médicament avec composé antagoniste récepteur p2y12 non nucléotide

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Publication number
EP1943261A2
EP1943261A2 EP06827504A EP06827504A EP1943261A2 EP 1943261 A2 EP1943261 A2 EP 1943261A2 EP 06827504 A EP06827504 A EP 06827504A EP 06827504 A EP06827504 A EP 06827504A EP 1943261 A2 EP1943261 A2 EP 1943261A2
Authority
EP
European Patent Office
Prior art keywords
compound
formula
alkyl
tetrahydro
purin
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP06827504A
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German (de)
English (en)
Inventor
José L. BOYER
James G. Douglass, Iii
Sammy R. Shaver
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Inspire Pharmaceuticals Inc
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Inspire Pharmaceuticals Inc
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Filing date
Publication date
Priority claimed from US11/267,941 external-priority patent/US7749981B2/en
Priority claimed from PCT/US2006/017781 external-priority patent/WO2006119507A2/fr
Application filed by Inspire Pharmaceuticals Inc filed Critical Inspire Pharmaceuticals Inc
Publication of EP1943261A2 publication Critical patent/EP1943261A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/82—Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2250/00—Special features of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2250/0058—Additional features; Implant or prostheses properties not otherwise provided for
    • A61F2250/0067—Means for introducing or releasing pharmaceutical products into the body

Definitions

  • This invention relates to non-nucleotide compounds and methods of making and using such compounds in the prevention or treatment of diseases or conditions associated with platelet aggregation, including thrombosis, stroke and myocardial infarction in humans and other mammals, and for inhibition of platelet aggregation in blood and blood-related products.
  • This invention also relates to drug-eluting stents, wherein a therapeutically effective amount of a non-nucleotide P2Yi2 receptor antagonist compound is eluted continuously from the stent to the local environment of the stent, when the stent is placed in a narrowed or damaged arterial vessel.
  • BACKGROUND OF THE INVENTION Hemostasis is the spontaneous process of arresting bleeding from damaged blood vessels. Upon injury, precapillary vessels contract within seconds, and thrombocytes, or blood platelets, bind to the exposed subendothelial matrix of an injured vessel by a process called platelet adhesion. Platelets also stick to each other in a phenomenon known as platelet aggregation to form stable platelet aggregates that quickly help stop or slow blood outflow from injured vessels.
  • An intravascular thrombus can result from pathological disturbances of hemostasis. or by the rupture of atherosclerotic plaques. Platelet adhesion and aggregation are critical events in intravascular thrombosis. Activated under conditions of high shear blood flow in diseased vessels or by the release of mediators from other circulating cells and damaged endothelial cells lining the vessel, platelets and other cells accumulate at a site of vessel injury to form a thrombus, and recruit more platelets to the developing thrombus. The thrombus can grow to sufficient size to block off arterial blood vessels. Thrombi can also form in areas of stasis or slow blood flow in veins.
  • Venous thrombi can easily detach portions of themselves, creating emboli that travel through the circulatory system. This process can result in blockade of other vessels, such as pulmonary arteries. Blockages of this sort can result in pathological outcomes such as pulmonary embolism. Thus, arterial thrombi cause serious disease by local blockade, whereas the morbidity and mortality associated with venous thrombi arise primarily after distant blockade, or embolization.
  • Conditions associated with pathological thrombus formation include venous thromboembolism, thrombophlebitis, deep vein thrombosis, arterial embolism, coronary and cerebral arterial thrombosis, unstable angina, myocardial infarction, stroke, transient ischemic attack, cerebral embolism, renal embolism and pulmonary embolism.
  • GP Ilb/IIIa glycoprotein Ilb/IIIa
  • integrin ⁇ ubf ⁇ glycoprotein Ilb/IIIa
  • GP Ilb/IIIa antagonists include function-blocking antibodies like Abciximab (ReoPro ® ), cyclic peptides and peptidomimetic compounds (The EPIC investigators; Califf, R.
  • Thrombin can produce platelet aggregation independently of other pathways but substantial quantities of thrombin are unlikely to be present without prior activation of platelets by other mechanisms.
  • Thrombin inhibitors such as hirudin, are highly effective antithrombotic agents. However, functioning as both antiplatelet and anti-coagulant agents, thrombin inhibitors again can produce excessive bleeding (The TIMI 9a Investigators, Circulation, 90: 1624-1630 (1994); The GUSTO Ha Investigators, Circulation, 90: 1631- 1637 (1994); Neuhaus, et al., Circulation, 90: 1638-1642 (1994)).
  • Various antiplatelet agents have been studied as inhibitors of thrombus formation.
  • agents such as aspirin and dipyridamole have come into use as prophylactic antithrombotic agents, and others have been the subjects of clinical investigations.
  • therapeutic agents such as the disintegrins, and the thienopyridines ticlopidine (TICLID ® ) and clopidogrel (PLAVIX ® ) have been shown to have utility as platelet aggregation inhibitors, although they can produce a substantial number of side effects and have limited effectiveness in some patients.
  • ADP adenosine 5'-diphosphate
  • ADP induces inhibition of adenylyl cyclase and modulation of intracellular signaling pathways such as activation of phosphoinositide-3 kinase (PI3K), influx and mobilization of intracellular Ca +2 , secretion, shape change, and platelet aggregation (Dangelmaier, et al. Thromb Haemost. 85: 341-348 (2001 )).
  • PI3K phosphoinositide-3 kinase
  • the P2Xj receptor is a ligand-gated cation channel that is activated by ATP, resulting in a transient influx of extracellular calcium. This receptor has been implicated in the regulation of platelet shape change, and recent evidence suggests its participation in thrombus formation in small arteries under high shear forces. (Jagroop, et al, Platelets 14: 15-20 (2003); Hechler, et al, J. Exp. Med. 198: 661-667 (2003)).
  • the P2Yj receptor is a G protein-coupled receptor that is activated by ADP, and is responsible for calcium mobilization from intracellular stores, platelet shape change and initiation of aggregation.
  • the P2Yi 2 receptor also referred to as the P2Y ac and P2 ⁇ receptor, is a G protein-coupled receptor that is activated by ADP and is responsible for inhibition of adenylyl cyclase and activation of PI3K. Activation of P2Yi 2 is required for platelet secretion and stabilization of platelet aggregates (Gachet, Thromb. Haemost. 86: 222-232 (2001); Andre, et al, J. Clin. Invest, 112: 398-406 (2003)).
  • ADP-induced platelet aggregation requires the simultaneous activation of both P2Yi and P2Yj2 receptors, and therefore, aggregation can be inhibited by blockade of either receptor.
  • ATP adenosine triphosphate
  • Zamecnik USPN 5,049,550 has disclosed a method for inhibiting platelet aggregation by administration of a diadenosine tetraphosphate-like compound, App(CH 2 ) ⁇ pA.
  • Nucleotide P2Y i2 antagonists have been developed, however, there is still a need for compounds that have improved oral bioavailability and blood stability.
  • Thienopyridines, ticlopidine and clopidogrel react covalently with the P2Yi 2 receptor and produce irreversible platelet inhibition in vivo (Quinn and Fitzgerald, Circulation 100: 1667-1672 (1999); Geiger, et al, Arterioscler. Thromb. Vase. Biol. 19: 2007-2011 (1999); Savi, et al, Thromb Haemost. 84: 891-896 (2000)).
  • Patients treated with thienopyridines usually require 2-3 days of therapy to observe significant inhibition of platelet aggregation, however, and maximal inhibition usually is observed between 4 to 7 days after initiation of treatment.
  • thienopyridines persists up to 7-10 days after the therapy is discontinued, and both ticlopidine and clopidogrel produce a significant prolongation of the bleeding time (from 1.5 to 2-fold over control). Because of the prolonged effect of thienopyridines, these drugs need to be discontinued for 7 to 10 days prior to elective surgery, leaving the patient unprotected from a possible thrombotic event during that period. Recently, the association of thienopyridine treatment with events of thrombotic thrombocytopenic purpura has been reported (Bennett, et al, N. Engl. J. Med. 342: 1773- 1777 (2000); Bennett, et al, Ann. Intern. Med. 128: 541-544 (1998)).
  • Guile, et al disclose the use of triazolo[4,5-d]pyrimidine compounds in therapy.
  • Brown, et al (USPN 6,369,064) disclose the use of Triazolo(4,5-d)pyrimidine compounds in the treatment of myocardial infarction and unstable angina.
  • Dixon, et al. (WO 02/096428) disclose the use of 8-azapurine derivatives in combination with other antithrombotic agents for antithrombotic therapy.
  • AZD6140 as a potent, selective, orally active P2Y ⁇ receptor antagonist which is now in Phase I clinical trials (Abstracts of Papers, 225 th ACS National Meeting, New La, LA; March, 2003; MEDI-Ol 6).
  • WO 02/016381 discloses a method of preventing or treating diseases or conditions associated with platelet aggregation using mononucleoside polyphosphates and dinucleoside polyphosphates.
  • Stents are typically slotted metal tubes, which can be expanded by a balloon in an angioplastied artery, providing a rigid structural support for the arterial wall.
  • the use of coronary stents for the treatment of patients with acute coronary syndrome has increased significantly during the past years. With coronary stents implanted in more than 2 million people worldwide, some doctors and researchers are now concerned about a long-term problem of blood clots inside the stents that is observed in some patients who have received stents. In-stent restenosis is caused primarily due to hyperplasia of smooth muscle cells in the intimal layer of the vessel wall (so-called neointimal hyperplasia) and, to a much lesser extent, mural thrombus.
  • both macrophages and polymorphonuclear neutrophils migrate to the site of damage, where they release chemokines.
  • These chemokines serve to increase the amount of matrix metalloproteinase, which leads to ⁇ emodeling of the extracellular matrix and stimulate smooth muscle cell migration.
  • the wound healing reaction stimulates platelets, growth factor and smooth muscle cell activation, followed by smooth muscle cell and fibroblast migration and proliferation into the injured area. Smooth muscle cells are also stimulated to increase the expression of genes involved in cell division.
  • neointimal hyperplasia and in-stent restenosis which are characterized by a marked proliferative response produced by the stent as has been demonstrated by histological examinations. Stenting also raises the systemic levels of inflammatory markers such as C-reactive protein and interleukin-6.
  • stents are coated with agents that reduce or prevent exaggerated neointimal proliferation, and thereby, restenosis.
  • paclitaxel-eluting stents inhibit the proliferation of smooth muscle cells
  • sirolimus-eluting stents inhibits the inflammation response of the arterial wall.
  • the drugs also inhibit the regeneration of the endothelium destroyed during the expansion of the narrowed artery, creating a potential risk of thrombosis.
  • the placement of these stents often requires the treatment by systemic administration of antithrombotic drugs.
  • This invention is directed to methods of preventing or treating diseases or conditions associated with platelet aggregation or where the aggregation of platelets inhibits treatment options.
  • This invention is directed to methods of preventing or treating thrombosis and related disorders.
  • This invention is further directed to methods of inhibiting platelet aggregation in blood and blood products comprising platelets, such as stored blood.
  • the method comprises administering to a mammalian subject or to a sample comprising blood or platelet-comprising material, a composition comprising one or more non-nucleotide P2Y 12 receptor antagonist compound that effectively binds to P2Yi 2 receptors on platelets, preferably in a reversible manner, and thereby causes an inhibition of the ADP- induced platelet aggregation response in blood or in a platelet-comprising material.
  • the compounds useful for the methods are compounds of general Formula I, III-XII, and/or tautomers thereof, and/or pharmaceutically-aeceptable hydrates, solvates, and/or salts thereof.
  • the invention also provides novel compounds and pharmaceutical compositions.
  • the compounds of Formulae I, and III-XII are useful in that they possess antagonist activity at platelet P2Yj2 receptors.
  • the compounds of this invention can be used in combination with other compounds useful for the treatment of platelet aggregation disorders or diseases.
  • the present invention also provides a drug-eluting stent, wherein the stent is coated with one or more non-nucleotide P2Y ⁇ 2 receptor antagonist compounds of general Formula I, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.
  • P2Yj 2 receptor antagonist compound When the stent is placed in a vessel, a therapeutically effective amount of the P2Yj 2 receptor antagonist compound is eluted to the local environment of the stent.
  • the P2Yi2 receptor antagonist compound-eluting stents are useful in preventing thrombosis and restenosis, and are effective in inhibiting thrombus formation, inhibiting the contraction of vascular smooth muscle cells, inhibiting cell proliferation, and reducing inflammation.
  • Halo substituents are taken from fluorine, chlorine, bromine, and iodine.
  • Alkyl groups are from 1 to 12 carbon atoms inclusively, either, straight chained or branched, are more preferably from 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbon atoms inclusively.
  • Alkylene chains are from 2 to 20 carbon atoms inclusively, have two points of attachment to the to the molecule to which they belong, are either straight chained or branched, can contain one or more double and/or triple bonds, are more preferably from 4 to 18 atoms inclusively, and are most preferably from 6 to 14 atoms inclusively.
  • Alkenyl groups are from 1 to 12 carbon atoms inclusively, either straight or branched containing at least one double bond but can contain more than one double bond.
  • Alkynyl groups are from 1 to 12 carbon atoms inclusively, either straight or branched containing at least one triple bond but can contain more than one triple bond, and additionally can contain one or more double bonded moieties.
  • Alkoxy refers to the group alkyl-O- wherein the alkyl group is as defined above including optionally substituted alkyl groups as also defined above.
  • Aryl refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms inclusively having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the like.
  • Arylalkyl refers to aryl -alkyl- groups preferably having from 1 to 6 carbon atoms inclusively in the alkyl moiety and from 6 to 10 carbon atoms inclusively in the aryl moiety. Such arylalkyl groups are exemplified by benzyl, phenethyl and the like.
  • Arylalkenyl refers to aryl -alkenyl- groups preferably having from 1 to 6 carbon atoms in the alkenyl moiety and from 6 to 10 carbon atoms inclusively in the aryl moiety.
  • Arylalkynyl refers to aryl -alkynyl- groups preferably having from 1 to 6 carbon atoms inclusively in the alkynyl moiety and from 6 to 10 carbon atoms inclusively in the aryl moiety.
  • Aryloxy refers to the group aryl-O- wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.
  • Cycloalkyl refers to cyclic alkyl groups of from 3 to 12 carbon atoms inclusively having a single cyclic ring or multiple condensed rings which can be optionally substituted with from 1 to 3 alkyl groups.
  • Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1- methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and the like, or multiple ring structures such as adamantyl, and the like.
  • Cycloalkenyl refers to cyclic alkenyl groups of from 4 to 12 carbon atoms inclusively having a single cyclic ring or multiple condensed rings and at least one point of internal unsaturation, which can be optionally substituted with from 1 to 3 alkyl groups.
  • suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3- enyl, cyclooct-3-enyl and the like.
  • Cycloalkylalkyl refers to cycloalkyl -alkyl- groups preferably having from 1 to 6 carbon atoms inclusively in the alkyl moiety and from 6 to 10 carbon atoms inclusively in the cycloalkyl moiety. Such cycloalkylalkyl groups are exemplified by cyclopropyhnethyl, cyclohexyl ethyl and the like.
  • Heteroaryl refers to a monovalent aromatic carbocyclic group of from 1 to 10 carbon atoms inclusively and 1 to 4 heteroatoms inclusively selected from oxygen, nitrogen and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or fkryl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).
  • Heteroarylalkyl refers to heteroaryl -alkyl- groups preferably having from 1 to 6 carbon atoms inclusively in the alkyl moiety and from 6 to 10 carbon atoms inclusively in the heteroaryl moiety. Such arylalkyl groups are exemplified by pyridylmethyl and the like.
  • Heteroarylalkenyl refers to heteroaryl -alkenyl- groups preferably having from 1 to 6 carbon atoms inclusively in the alkenyl moiety and from 6 to 10 carbon atoms inclusively in the heteroaryl moiety.
  • Heteroarylalkynyl refers to heteroaryl -alkynyl- groups preferably having from 1 to 6 carbon atoms inclusively in the alkynyl moiety and from 6 to 10 carbon atoms inclusively in the heteroaryl moiety.
  • Heterocycle refers to a saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 8 carbon atoms inclusively and from 1 to 4 hetero atoms inclusively selected from nitrogen, sulfur or oxygen within the ring.
  • Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl).
  • Preferred heterocycles include piperidinyl, pyrrolidinyl and tetrahydro&ryl.
  • heterocycles and heteroaryls include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline and the like.
  • Positions occupied by hydrogen in the foregoing groups can be further substituted with substituents exemplified by, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, alkyl, substituted alkyl, thio, thioalkyl, acyl, carboxyl.
  • Pharmaceutically acceptable salt forms include various polymorphs as well as the amorphous form of the different salts derived from acid or base additions.
  • the acid addition salts can be formed with inorganic or organic acids.
  • Illustrative but not restrictive examples of such acids include hydrochloric, hydrobromic, sulfuric, phosphoric, citric, acetic, propionic, benzoic, napthoic, oxalic, succinic, maleic, malic, adipic, lactic, tartaric, salicylic, methanesulfonic, 2- hydroxyethanesulfonic, toluenesulfonic, benzenesulfonic, camphorsulfonic, and ethanesulfonic acids.
  • the pharmaceutically acceptable base addition salts can be formed with metal or organic counterions and include, but are not limited to, alkali metal salts such as sodium or potassium; alkaline earth metal salts such as magnesium or calcium; and ammonium or tetraalkyl ammonium salts, i.e., NX 4 + (wherein X is CM).
  • Tautomers are compounds that can exist in one or more forms, called tautomeric forms, which can interconvert by way of a migration of one or more hydrogen atoms in the compound accompanied by a rearrangement in the position of adjacent double bonds. These tautomeric forms are in equilibrium with each other, and the position of this equilibrium will depend on the exact nature of the physical state of the compound. It is understood that where tautomeric forms are possible, the current invention relates to all possible tautomeric forms. Solvates are addition complexes in which a compound is combined with a pharmaceutically acceptable cosolvent in some fixed proportion.
  • Cosolvents include, but are not limited to, water, methanol, ethanol, 1-propanol, isopropanol, 1-butanol, isobutanol, tert- butanol, acetone, methyl ethyl ketone, acetonitrile, ethyl acetate, benzene, toulene, xylene(s), ethylene glycol, dichloromethane, 1 ,2-dichloroethane, N-methylformamide, N, N- dimethylformamide, N-methylacetamide, pyridine, dioxane, and diethyl ether . Hydrates are solvates in which the cosolvent is water. It is to be understood that the definition of the compound of the present invention encompasses all possible hydrates and solvates, in any proportion, which possess the stated activity.
  • P2Yi 2 receptor antagonist compounds useful for preventing or treating diseases or conditions associated with platelet aggregation and/or platelet activation include compound of general Formula I, and/or tautomers thereof, or a pharmaceutically acceptable salt, solvate, or hydrate thereof:
  • R a and R b are each independently selected from the group consisting of: hydrogen, Ci. 8 alkyl, Ci-s alkenyl, Ci- 8 alkynyl, C 3 _ 7 cycloalkyl, C.j. 7 cycloalkenyl, aralkyl, aralkenyl, aralkynyl, aryl, and saturated or unsaturated C 3 .. 6 heterocycle; where all rings or chains optionally can bear one or more desired substituents; or
  • R a and R b are taken together to form a ring of 3 to 7 members, with or without substitution, and with or without heteroatoms in place of ring carbon atoms;
  • R 0 H, C 1-S alkyl, C 3 J 7 cycloalkyl, aralkyl, aryl, or heterocycle, or R(CO)-; where R is selected from the group consisting of: Ci-S alkyl, C 1-S alkenyl, Ci -8 alkynyl, C3J7 cycloalkyl, C4 -7 cycloalkenyl, aryl, aralkenyl, aralkynyl, heteroaryl, and saturated or unsaturated C 3-6 heterocycle; where all rings or chains optionally bear one or more desired substituents;
  • G O, S, or NR d , where R d is defined as below;
  • R f H, halogen, Ci- 8 alkyl, Ci-s alkenyl, Ci.g alkynyl, C 3 -7 cycloalkyl, C4-7 cycloalkenyl, C 4 .11 alkylcycloalkyl, C S - ⁇ alkylcycloalkenyl, with 1 to 4 carbons in the alkyl portion, aryl, aralalkyl, aralkenyl, aralkynyl, heteroaryl, saturated or unsaturated C3-6 heterocycle, -OH, Ci -6 alkoxy, aryloxy, -SH, Ci -6 thioalkyl, thioaryl, -[(CO)OR], -[(CO)NRR], amino, -N- substituted amino, or N,N-disubstituted amino; wherein each said substituent on said N- substituted-amino group, or N,N-disubstituted-amino-group of Rf is
  • R f is -NRR, -[NH(CO)NRR], -[N(Ci -8 alkyl)(CO)NRR], -[N(aryl)(CO)NRR], or [N(aralkyl)(CO)NRR]
  • a and B are each independently selected from the group consisting of: C, N, substituted N, O, S, S(O), SO2, -Ct-3 alkylene-, -Cioheteroalkylene, wherein each said -C 1 -3 alkylene- unit of A and B independently can be saturated or unsaturated, and each carbon of a -Ci-3 alkylene- unit of A or B independently can be substituted with O to 2 fluorine groups, O to 2 alkyl groups, O to 2 -[(CO)OR] groups, O to 2 -(OR) groups, O to 1 (OH) groups, or O to 1 cycloalkyl groups, whether bridging both A and B or affixed to either A or B; or wherein each said -Ci-3heteroalkylene unit of A and B independently comprises NH, substituted N, O, S, S(O), SO 2 , or; A and B are independently CH 2 , CF 2 ,-, -(CO)-;
  • X H, -OR, -COOH 5 -COOR 3 -SR, -S(O)RL, -S(O 2 )RL 5 -SO 3 H 5 -S(O 2 )NRR, - S(O 2 )NR(CO)RL, -NRR, -NR(CO)RL, -N[(CO)RL] 2 , -NR(SO 2 )RL, -NR(CO)NR(SO 2 )RL, - NR(SO 2 )NRR, or -NR(SO 2 )NR(CO)RL; wherein L is: H, -CF 3 , -CF 2 CF 3 , C 1-8 alkyl, Ci -8 alkenyl, C 1-8 alkynyl, C3-7 cycloalkyl, C 4 .7 cycloalkenyl, C 4-H alkylcycloalkyl, C 5 _i 1 alkylcycloalkenyl, with 1 to 4 carbons in the alky
  • Xe is the attachment point to the moiety defined by A-B; the ring defined by Xi-Xg is taken to mean a ring with or without unsaturation;
  • X 1 -Xe are independently C, N, O, or S; and when any of X 1 -X 5 is C, the carbon atom when doubly bonded in an unsaturated ring (as, for example, an aromatic ring), bears a H, or a substituent M, or a moiety comprising CO2R11 as defined below; or when any of Xi-Xs is C, the carbon atom when singly bonded in a saturated ring (as, for example, a cycloalkyl ring), bears two H's, or one H and a moiety comprising COaR h as defined below, or one H plus one substituent M, or two substituents M without H, with the proviso that any such moiety with one or two M substituents is of sufficient chemical stability; when any of Xi-Xs is N in an saturated ring, the nitrogen atom bears an H or substituents such as alkyl or acyl; any of X1-X5 can be absent, with
  • Rh is H, a physiologically-relevant cation forming a carboxylate salt, alkyl, aryl, or aralkyl, with the resultant moiety C(O)OR h preferably having an adjacent relationship to the attachment point of A; preferably Rh is H or alkyl (such as ethyl); M is selected from the group consisting of: halogen (such as F, Cl, Br) 5 -CF 3 , Ci-s alkyl, Ci-s alkenyl, CL 8 alkynyl, C 3 -7 cycloalkyl, C4-7 cycloalkenyl, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, saturated or unsaturated C 3 -6 heterocycle, -OH, cyano, nitro, saturated or unsaturated Ci ⁇ alkoxy, aralkoxy, aryloxy, -SH 5 C 1- O thioalkyl, thioaryl, -[(CO
  • the furanosyl moiety in Formula I has the T- and 3 '-oxygen- groups in a cis-orientation relative to one another on the furanose ring.
  • a furanosyl moiety which supports a 2 ⁇ 3'-acetal or -ketal group is, preferably, derived from ribose; other furanose derivatives can be used, however.
  • a preferred stereochemical embodiment of this invention includes, but is not limited to (D)-ribose-(2', 3'-acetal or -ketal) compounds of Formula I, such as found in acetals derived from (-)-adenosine.
  • the compound of Formula I is selected from the group consisting of: 4- ⁇ 2,2-Dimethyl-6-[6-(3-phenyl-ureido)-purin-9-yl]-tetrahydro-furo[3,4- d][l,3]dioxol-4-ylmethoxy ⁇ -isophthalic acid (1), 5-Amino-2- ⁇ 2-benzyl-6-[6-(3-phenyl- ureido)-purin-9-yl]-tetrahydro-furo[3,4-d][l,3]dioxol-4-ylmethoxy ⁇ -benzoic acid (2), 3- ⁇ 2,2- Dimethyl-6-[6-(3-phenyl-ureido)-purin-9-yl]-tetrahydro-furo[3,4-d][l,3]dioxol-4- ylrnethoxy ⁇ -isoxazole-5-carboxylic acid (3)
  • the compounds depicted in the following structures falling under the definitions of Formulae III- XII represent either one of the two possible diastereomers (which arise from the resultant chiral carbon of the acetal) in pure form, or a mixture of the two diastereomers in any proportion.
  • the compounds as depicted represent the pure forms of the diastereomers. Diastereomers are distinct compounds, each with potentially different chemical and biological properties; thus pure forms are preferred as pharmaceutical agents.
  • a mixture of two diastereomers having different acetal stabilities can be subjected to aqueous acidic conditions, which leads to decomposition of the less-stable diastereomer, while leaving the more stable diastereomer intact.
  • the single diastereomer that survives the decomposition is preferred, since chemical stability is an important attribute for a pharmaceutical product.
  • an important aspect of this acetal modification is that the resultant bicyclic structure (i.e. the bicyclic ring system arising from the fusion of the ribose residue and acetal ring at the 2' and 3 ' carbons of the ribose) is much more stable compared with the native nucleoside structure where the 2' and 3' hydroxyl groups are free.
  • the chemical and biological degradation processes that nucleosides with free 2' and 3' hydroxyls typically undergo are greatly impaired, thus the stability of the compound is improved.
  • High overall chemical and biological stability are important attributes for pharmaceutical products, especially for those being designed for chronic treatment delivered via oral administration, where the pharmaceutical product will be subject to a broad range of chemical and biological environments.
  • removal of the acetal functionality from the compounds of the present invention substantially diminishes or completely abolishes the activity at P2Y t2 .
  • compounds of the present invention contain an acetal bearing an aromatic ring
  • the aromatic ring is optionally substituted with moieties, the nature of which as previously defined.
  • These substituents can be chosen to enhance the pharmaceutical properties of the molecules of interest. For example, replacement of a hydrogen atom on the phenyl ring of an aromatic acetal with fluorine can be done to prevent hepatic metabolism at that position following in vivo administration of the compound. Alternately, replacement of a hydrogen atom on the phenyl ring with a basic or acidic moiety can be done to improve the solubility of the molecules of interest, or to enhance the ability of the compound to be absorbed from the digestive tract when given orally.
  • a hydrogen atom on the phenyl ring can be replaced with a moiety that will be metabolized in a predictable fashion, leading to an active form of the compound in vivo, or leading to a species that has a preferred mode of excretion.
  • X is a ring falling under the definition of Formula IL
  • the compound of Formula I is a compound of Formula III: Formula III
  • R 3 , Rb, Rc, G, Rd, Rd', Re, Rf, J 9 Rg, and Rh are as defined in Formulae I and II;
  • Ai is CH 2 , O, S 3 S(O), SO 2 , NH, or substituted N;
  • D is O or CH 2 ;
  • Xi is selected from the group consisting of: N (nitrogen) and C-M;
  • M is independently selected from the group consisting of: halogen, -CF 3 , Ci-s alkyl, cyano, Ci-g alkenyl, Ci ⁇ alkynyl, C3 -7 cycloalkyl, Cj -7 cycloalkenyl, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, saturated or unsaturated C 2 - 6 heterocycle, -OH, saturated or unsaturated C 1-6 alkoxy, aralkoxy, aryloxy, -SH, Ci -6 thioalkyl, thioaryl, -[(CO)OR'], -[(CO)NR 5 R"], amino, -N-substituted amino, and N,N-disubstituted amino;
  • D is O. In another embodiment, D is CH 2 .
  • D O or CH2, with O being preferred; alkyl, or C3.6 cycloalkyl; R e is absent;
  • Rb trans-phenyl, cis-phenyl, cis-benzyl, or trans-styryl; where cis- or trans- refers to the configuration of hydrogen atom on the acetal carbon of the dioxolane ring relative to the hydrogens on the 2' and 3' carbons of the ribose ring; wherein the hydrogen on each phenyl ring is optionally substituted (such as by fluorine); and
  • M halogen, C1-4 alkyl, C 1 ⁇ alkoxy, CF 3 , cyano, carboxy, or amino.
  • the compound of Formula I is a compound of Formula IV: Formula IV
  • R a , Rh, R 0 , G, D, Rd, Rd',Re, Rf 5 J 5 M, y, R g and Rh are as defined in Formulae I and
  • M and -CO2R11 groups are independently and optionally attached to any carbon of the pyrrolidine ring; and when M is attached to a carbon that is bonded to the pyrrolidine nitrogen atom (alpha position), then M is not a halogen, hydroxyl, sulfhydryl, or amino group.
  • D is CH 2
  • D is O
  • Preferred compounds of Formula IV are wherein: ;
  • R tT Ci -4 alkyl, or C3 4 cycloalkyl
  • the compound of Formula I is a compound of Formula V:
  • R a , Rb, R 0 , D, G, Rd, Rd ' ,Re, R f5 J, M, y, X 1 ⁇ R g and R h are as defined in Formulae I and II;
  • Ri and Rj are independently H, F, alkyl from 1 -3 carbons, or are CH 2 joined by a bond (cyclopropyl);
  • a 2 is C, O, S, S(O), SO 2 , or N 5 where C bears H, F, doubly bonded O, OH, or alkyl, and N bears H 5 alkyl, or acyl; or A 2 is absent.
  • D is CH 2 , and in another embodiment, D is O.
  • D CH 2 , or O, with O being preferred; H; H, CH3, F 5 or cyclopropyl;
  • Rh H or ethyl
  • Rd' Ci.4 alkyl, or C3-6 cycloalkyl;
  • a 2 is CH 2 , CH(OH) , CF 2 , C(O), O, NH, N-methyl, N-acetyl, or absent;
  • J C;
  • M halogen, CF 3 , cyano, or amino.
  • the compound is a compound of Formula VI:
  • R a , Rb, Rc, D, G, Rd, Rd' s Re, Rf, J, M, y, Xi, R g and Rh are as defined in Formulae I and II;
  • D is O
  • a 3 is C, where C can be substituted with H or alkyl; or
  • a 3 is absent
  • Ri is H or alkyl.
  • D is CHb, and in another embodiment, D is O.
  • Rd ' Ci- 4 alkyl, or C3-6 cycloalkyl
  • the compound of Formula I is a compound of Formula VII:
  • R a , Rb, R 0 , D, G, Rd, Rd ' jRe, Rf, J, Rg and Rj, are as defined in Formulae I and II; qt and q 2 are independently 0, 1, or 2;
  • Az is as previously defined for Formula V, with the proviso that when q ⁇ and/or q 2 are 0 , A 2 is C; or
  • A2 is absent.
  • D is CH 2 , and in another embodiment, D is O.
  • Preferred compounds of Formula VII are wherein:
  • R e is absent;
  • R h H or ethyl; alkyl, or C3-6 cycloalkyl;
  • the compound of Formula I is a compound of Formula VIII:
  • R a , Rb, Rc, D, G, K ⁇ , Rd', Re 5 Rf, J, Rg and Rj, are as defined in Formulae I and II; q 3 is 1, 2, or 3; and Ri is H or alkyl.
  • D is CH 2 , and in another embodiment, D is O.
  • D CH2, or O, with O being preferred;
  • R e is absent
  • R h H or ethyl
  • R b trans-phenyl, cis-phenyl, cis-benzyl, or trans-styryl; where cis- or trans- refers to the configuration of the hydrogen atom on the acetal carbon of the dioxolane ring relative to the hydrogens on the 2' and 3' carbons of the ribose ring; wherein the hydrogen on each phenyl ring is optionally substituted (such as by fluorine).
  • the compound of Formula I is a compound of Formula IX:
  • R 3 , Rb, Rc 3 G, D, Rd, Rd'jRe, Rf 5 J, Rg and Rj, are as defined in Formulae I and II;
  • a 4 and Ae are independently C, N, O, or S, with the proviso that A 4 can be absent;
  • G' is O 5 or S; such that the moiety described by A4/ C(G')/ A & is an amide, thioamide, carbamate, thiocarbamate, urea, thiourea, ketone, or thioketone; and q ! is 0 ,1, or 2.
  • D is CH 2 , and in another embodiment, D is O.
  • Preferred compounds of Formula IX are wherein:
  • the compound of Formula I is a compound of Formula X:
  • R a , Rb, Rc, G, D, Rd, Rds Re» Rf j J > M, y, X 1 , R g and Rh are as defined in Formulae I and II; G' is O or S; Ae is C, N, O, S, or absent; and Ri is H or alkyl; such that the moiety described by A O / C(G')/ NR J is an amide, thioamide, carbamate, thiocarbamate, urea, or thiourea.
  • D is CH 2 , and in another embodiment, D is O.
  • the compound of Formula I is a compound of Formula XI:
  • R 3 , Rb Rc, G, D, Rd, Rd' ,Re, Rf 5 J, M, y, R g and R 1 , are as defined in Formulae I and II;
  • G' is O or S, such that the moiety C(C)-NR; is an amide or thioamide;
  • Xi is C or N.
  • D is CH 2 , and in another embodiment, D is O.
  • Preferred compounds of Formula XI are wherein:
  • D CH2, or O, with O being preferred;
  • Re is absent; H or methyl
  • Rd' CM alkyl, or C3-6 cycloalkyl
  • the compound of Formula I is a compound of Formula XII:
  • R 35 Rb, Rc, G, D, Rd, Rd' ⁇ R 6 , Rf, J, M, y, R g and R h are as defined in Formulae I and II;
  • X 1 , X2, X4, Xs, and X 6 are independently selected from the group consisting of: N, C, S, O, or absent; and are taken to form a ring from three to five atoms, with or without unsaturation; qi and q2 are independently 0, l,or 2;
  • a 2 is C 5 O 5 S 5 S(O), SO 2 , or N 5 where C bears H, F, doubly bonded O, OH 5 or alkyl, and N bears H 5 alkyl, or acyl; or
  • D is CH2, and in another embodiment, D is O.
  • Preferred compounds of Formula XII are wherein: (saturated cyclopentyl ring);
  • a 2 CH 2 , O, NH, or absent;
  • Rd > Ci-4 alkyl, or C3-6 cycloalkyl;
  • the present invention additionally provides novel pharmaceutical formulations comprising a pharmaceutically acceptable carrier and compounds of Formula I 5 HI-XI, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.
  • Pharmaceutically acceptable carriers can be selected by those skilled in the art using conventional criteria.
  • Pharmaceutically acceptable carriers include, but are not limited to, saline solution, aqueous electrolyte solutions, isotonicy modifiers, water polyethers such as polyethylene glycol, polyvinyls such as polyvinyl alcohol and povidone, cellulose derivatives such as methylcellulose and hydroxypropyl methylcellulose, polymers of acrylic acid such as carboxypolymethylene gel, polysaccharides such as dextrans, and glycosaminoglycans such as sodium hyaluronate and salts such as sodium chloride and potassium chloride.
  • the pharmaceutical formulation of the present invention provides an aqueous solution comprising water, suitable ionic or non-ionic tonicity modifiers, suitable buffering agents, and a compound of Formula I, or III-XII.
  • the compound is at 0.005 to 3% w/v
  • the aqueous solution has a tonicity of 200-400 mOsm/kG and a pH of 4-9.
  • the pharmaceutical formulation can be sterilized by filtering the formulation through a sterilizing grade filter, preferably of a 0.22-micron nominal pore size.
  • the pharmaceutical formulation can also be sterilized by terminal sterilization using one or more sterilization techniques including but not limited to a thermal process, such as an autoclaving process, or a radiation sterilization process, or using pulsed light to produce a sterile formulation.
  • the pharmaceutical formulation is a concentrated solution of the active ingredient; the formulation can be serially diluted using appropriate acceptable sterile diluents prior to intravenous administration.
  • the tonicity modifier is ionic such as NaCl, for example, in the amount of 0.5-0.9 % w/v, preferably 0.6-0.9 % w/v.
  • the tonicity modifier is non-ionic, such as mannitol, dextrose, in the amount of at least 2%, or at least 2.5%, or at least 3%, and no more than 7.5%; for example, in the range of 3-5 %, preferably 3.5-5%, and more preferably 4.2-5% w/v.
  • non-ionic such as mannitol, dextrose
  • adenosine ⁇ -adenosine, 2', 3'- isopropylidineadenosine, 5'-acetyl-2 ⁇ 3'-isopropylidineadenosine, N 6 -(2- isopentenyl)adenosine, 2-chloroadenosine, 2-amino-6-chloropurine riboside, 6-chloropurine riboside, inosine, 8-bromoguanosine, 8-bromoadenosine, 8-azidoadenosine, 8-azaguanine, 8- azaadenine, protected ribonic acid lactone derivatives and protected furanose derivatives.
  • Other appropriate intermediates can be purchased from commercial sources and used as starting materials for compounds of the present invention, or can be synthesized as described in the chemical literature.
  • Scheme 1 discloses a useful method for the synthesis of 5'-aryl- or 5'- heteroaryl- ether derivatives by substitution of an appropriately fimctionalized adenosine analogue or 8-azapurine derivative for a halogen on an appropriately-substituted halogenated aromatic compound or a related heteroaromatic derivative.
  • Groups not defined in Scheme 1 are defined as in Formula I.
  • Preferred substituents at M of the aromatic-/heteroa ⁇ ornatic- group in Scheme 1 are hydrogen, or halogen, or groups containing carboxylic acid derivatives such as: -CO 2 R.
  • halogen or esters or amides of alkylcarboxylic acids, arylcarboxylic acids, -O-(alkylcarboxylic acids), -NR-(alkyl carboxylic acids), and the like.
  • M is halogen in Scheme 1, preferred halogens are chloro and fluoro.
  • 5 '-Substituted aryl derivatives can also be prepared via Mitsunobu coupling of phenols (Mitsunobu, Synthesis 1-28 (1981); Brown, et al., J. Med. Chem. 37 (5), 674-88 (1994); Santosh and Balasubramanian, Synthetic Communications, 24(8), 1049-62 (1994)) to derivatives of adenosine, 8-azaadenosine, guanosine, 8-azaguanosine, etc., as provided in Scheme 2.
  • Groups in Scheme 2 are defined as in Formula I.
  • Some preferred substituents at M of the aromatic/heteroaromatic-group in Scheme 2 independently can be hydrogen, halogen, alkyl, alkoxy, aryl or groups containing carboxylic acid derivatives such as: -CO2R3; but esters or amides of alkylcarboxylic acids, arylcarboxylic acids, -O-(alkylcarboxylic acids), -NR-(alkylcarboxylic acids), and the like are also included.
  • M is halogen in Scheme 2
  • preferred halogens are chloro and fluoro.
  • the Mitsunobu coupling can be carried out using hydroxyisoxazoles as provided in Scheme 3.
  • Groups in Scheme 3 are defined as in Formula I.
  • preferred substituents at M of the isoxazole derivatives of Scheme 3 independently include hydrogen, alkoxy, or halogen, or groups containing carboxylic acid derivatives such as: -CO2R3; but esters or amides of alkylcarboxylic acids, arylcarboxylic acids, -O-(alkylcarboxylic acids), -NR-(alkylcarboxylic acids), and the like are also included.
  • M is halogen in Scheme 3 preferred halogens are chloro and fluoro.
  • esters When the products of any of Schemes 1, 2 or 3 comprise esters, said esters can be useful in the invention.
  • Said ester derivatives can be purified by methodologies well-known in the art, such as by normal phase, or reverse phase chromatography, or in suitable circumstances, using crystallization techniques.
  • said ester derivatives can be used in the synthesis of other derivatives such as amides, hydroxamic acids, and different alkyl esters by well-known methods in the art.
  • esters can be hydrolysed under basic conditions, or cleaved using other methods known in the art, which cleave esters selectively in the presence of ketals or acetals to provide acid salts. These salts are also useful in the invention.
  • said acid salts can be converted into acids upon mild acid treatment. Workup by common techniques, and purification by methods well known in the art, including purification by crystallization or chromatography can be used to give the purified acids. Said acids can also be converted into other useful derivatives such as amides, hydroxamic acids, aryl esters, etc., by methods known to those skilled in the art of chemical synthesis. These acid derivatives are also useful in the invention, and are also purified using well-known methods such as crystallization or chromatography.
  • R a . and R ⁇ denote different R a and/ or R b
  • Diversity using common intermediates can be introduced into the 2', 3'-acetal or - lcetal position of compounds encompassed in Formula I, as well as at the N 6 -position of compounds of Formula I using solid-phase synthetic methods.
  • Schemes 4 and 5 exemplify transketalization procedures using polymer-bound relatives of compounds of Formula I. These methods can be used to transform one class of 2% 3 '-ketal or -acetal into other useful 2 ⁇ 3 '-acetal or -ketal derivatives of adenosine, guanosine, 8-azaadenosine, etc.
  • Scheme 5 shows another variation of compound preparation employing resin-bound materials of Formula I. It outlines a solid-phase procedure that is useful for the introduction of functionality at the 6-position, as well as for transketalization at the 2% 3'-position, if desired. It should be noted that the chemistry procedures used in these solid phase approaches are similar to methods known and used for solution-phase chemical transformations involving the synthesis of adenosine, guanosine, 8-azaadenosine, etc., derivatives.
  • the primary differences are the necessity of attachment of a starting material to a resin, the simplicity of resin-based purification techniques (filtration and washing by compatible solvents) compared to solution-phase techniques (chromatography, crystallization, etc.), and the requirement for cleavage of a compound of the invention, or an intermediate useful for the synthesis of a compound of the invention from a resin prior to final purification and/or use of a compound so cleaved.
  • an early intermediate such as a 6-chloroadenosine-2% 3'-ketal or -acetal derivative, or a 6-chloro-8-aza-adenosine-2% 3'-ketal or -acetal derivative is attached to a resin such as polystyrene resin via a ⁇ -thioethanol linker (e.g., hydroxy ethylsulfanylmethyl polystyrene; HESM polystyrene resin; Garcia-Echeverria, Tetrahedron Lett., 38, 8933-7 (1997)).
  • a resin such as polystyrene resin via a ⁇ -thioethanol linker
  • the resin bound material is treated with a primary amine, ammonia or a hydroxylamine derivative to introduce an amino group via displacement of the 6-chloride.
  • a useful solvent like dimethylformamide (DMF)
  • subsequent introduction of an ureido-, thioureido-, or guanidino-group at N 6 can be made in one step using an excess of an appropriate isocyanate, isothiocyanate, carbodiimide, carbamoyl chloride, or 2-alkyl-2-thiopseudourea; or using a chemical equivalent of such materials.
  • a two-step approach can be used to introduce a group at N 6 , which comprises treatment of an appropriate 6-amino derivative, synthesized as in Scheme 5, with a solution of a small excess of phosgene or thiophosgene and a tertiary amine such as diisopropylethylamine in a suitable solvent such as dichloromethane or toluene at a temperature which allows reaction, followed by treatment with an excess of a primary or secondary amine to give a resin-bound urea or thiourea after workup.
  • transketalization can be performed on a bound substrate of Scheme 5 in a manner similar to that shown in Scheme 4.
  • Preferred oxidizing agents include peracids like m-chloroperbenzoic acid (MCPBA) and peracetic acid, but other oxidizing agents like hydrogen peroxide, permanganate salts, or persulfate salts can be used to oxidize a thioether to a sulfone.
  • Preferred elimination conditions include DBU in dichloromethane, and 10% ammonium hydroxide in trifluoroethanol.
  • Preferred aldehydes, aldehyde acetals and ketone ketals useful in the transketalization methods shown in Schemes 4 and 5 comprise the below said carbonyl compounds and/or derivatives of: benzaldehyde, bi ⁇ henyl-3-carboxaldehyde, biphenyl-4-carboxaldehyde, biphenyl-4-yl-acetaldehyde, 2-bromobenzaldehyde, benzo[b]thiophene-3-carbaldehyde, cyclohexanecarbaldehyde, cyclopentanecarbaldehyde, 2,5-dimethylbenzaldehyde, 2,6- difluorobenzaldehyde, 2-fluorobenzaldehyde, naphthalene-2-carbaldehyde, phenyl acetaldehyde, phenyl propynal, 3 -phenyl propenal, 3 -phenyl prop
  • Scheme 6 exemplifies the preparation of 5'- isoxazole ethers, introducing ammonia, various amines or hydroxylamine derivatives at the 6- position of the purine/8-azapurine ring by displacment of a chloride leaving group (a 6- chloride is shown in Schemes 5 and 6, but the leaving group at C 6 could also be another type, useful for such a transformation, e.g., a 6-bromide or 6-mesylate moiety) by such materials.
  • a 6- chloride is shown in Schemes 5 and 6, but the leaving group at C 6 could also be another type, useful for such a transformation, e.g., a 6-bromide or 6-mesylate moiety
  • Amines and amine-like compounds useful for displacement of a 6-halogen intermediate as contemplated for these schemes comprise ammonia, methylamine and other N-alkyl amines; N-aralkylamines; N-cyclopropylamine and other N-cycloalkylamines; anilines; ethers and other O-derivatives of hydroxylamine; aminopyridines and other heteroaromatic amines; heterocyclic compounds having a pendant -NHR c -group; and N-alkyl amines which have one or more heteroatom units like O, NR, and/or S substituted for carbon units in the alkyl chain.
  • N 6 -products can be further transformed into ureas, thioureas, or guanidines by literature methods or by methods disclosed for such transformations in Schemes 5 and 6.
  • the materials can be purified by methods typically used in the literature, such as by chromatography or, in certain cases, by crystallization Preferred substituents at N 6 are ureas.
  • M of an isoxazole derivative as provided in Scheme 6 contains an ester group [e.g., - C(CO)O-(alkyl), -C(CO)O-(aryl), -(CH 2 ) m C(CO)O-(alkyl), -O(CH 2 ) m C(CO)O-(aryl), etc., where "m" defines a carbon chain length of a compound of Formula I], said ester can be used in the present invention, or it can be converted into an acid using a method which is compatible with an acetal or ketal moiety and a desired group at N 6 .
  • an ester of Scheme 6 can be hydrolysed at room temperature (RT) in several hours using an excess of aqueous 2M lithium hydroxide solution dissolved in dioxane and/or methanol to give a carboxylate salt. Purification of said salt or a corresponding acid of said salt can be accomplished as previously disclosed. These acids and acid derivatives are also useful in this invention.
  • Scheme 6 is exemplified using ammonia and primary amines (including hydroxylamine derivatives) as choices for nucleophiles in the displacement of a leaving group (like chloride) at C 6 . Further modification of the N 6 -group of a compound of Scheme 6 with a-[(CG)NRdRd']-group yields a compound useful in the invention.
  • Condition c L 3 SO 2 NHL 4 , DCC, DMAP,
  • the acid moiety which is at, or linked to, the 5 '-position of the furanose derivative can be coupled to a solid phase resin such as a 4-sulfamylbutyryl resin, hydroxymethyl resin, or a pegylated-hydroxy resin, etc., using techniques well-known in the peptide literature and/or solid phase organic synthesis literature to give a resin-bound material.
  • a solid phase resin such as a 4-sulfamylbutyryl resin, hydroxymethyl resin, or a pegylated-hydroxy resin, etc.
  • a protecting group is used on the N 6 -position of such an acid, it can be removed subsequent to a transketalization procedure, and a N 6 - amine so formed can be converted into a urea, thiourea or guanidine by methods disclosed herein, or by methods in the literature. Cleavage from the solid support using known methods then yields a compound of the invention which can be purified, if needed, by employing commonly-used techniques.
  • Condition f L 3 SO 2 NHL 4 , DCC, DMAP,
  • Scheme 8 shows an adenine unit, it will be understood by chemistry practioners that the methods of Scheme 8 are generally applicable to substituted members of the adenine family and also to the 8-azaadenines. Preferred amines and amine derivatives for the 5 '-amide-forming reactions shown in
  • Schemes 7 and 8 are: trifluoromethanesulfonamide, methanesulfonamide, serine, glycine, proline, anthranilic acid and its regioisomers, and methyl anthanilate and its regioisomers.
  • Scheme 9 Amino acid amides of 5'-adenosinecarboxylic acid derivatives and 8- azaadenosine-5'-carboxylic acid derivatives using solid phase techniques.
  • R a > and R b denote different R a and/ or R b
  • Amide derivatives of 5'-carboxylic acids e.g., those shown in Scheme 7 and Scheme 8
  • amide derivatives of acid moieties linked at the 5 '-position of Formula I can also include amides derived from amino acids, peptides, aminoalcohols and the like.
  • a convenient way of attaching naturally-occurring, as well as synthetically-derived amino acids and peptides, or derivatives, to a 5'-carboxylic acid or related homologue is exemplified in Scheme 9 using the amino acid, proline.
  • the said coupled product if it bears an amine at the 6- ⁇ osition, can then be treated with one of the various reagents described previously, or with a reagent known in the literature, to yield a urea, thiourea or guanidine at the adenosine 6-position.
  • a 5'-carboxylic acid derivative used in a coupling to a solid phase has a urea, etc., already installed at the 6-position, then the latter said modification at N 6 need not be performed.
  • a coupled 5'-amide/N 6 -derivatized product can be converted into a variety of different acetals or ketals using solid phase methods such as described for Schemes 4 and 5.
  • cleavage of a compound of the invention from a solid phase can be performed by a variety of methods known in the art; such cleavage conditions depending upon the type of linker used.
  • Cleavage methodologies useful for cleaving peptide- or amino acid derivatives of 5'-linked-adenosine compounds comprise the linker oxidation/elimination procedures given in Schemes 5 and 9; treatment with a hydroxide source, such as lithium hydroxide, using conditions as for the ester hydrolysis described in Scheme 6; and hydrolysis using potassium trimethylsilanolate, as described in Scheme 4; as well as others known in the art (including aminolysis to form amides).
  • a compound useful in this invention can be obtained in purified form by cleaving it from a resin and purifying it as described previously.
  • an amino group can be installed at the 5 '-position of an adenosine or 8-azaadenosine analogue, or on the chain of a 5'-homologue of such a material.
  • This amine can be utilized to form amide-, sulfonamide-, and other derivatives.
  • Scheme 10 illustrates how a sulfonylurea can be synthesized at the 5'-position using a 5'-amine 5 or at related positions on homologous amine derivatives.
  • an amine introduced at the 5'-position or on the 5'-chain of a homologue is also useful for the synthesis of amides, ureas, sulfonamides and other amine derivatives using methods known in the art for such processes.
  • Scheme 11 Homologation of adenosine derivatives, including steps involving oxidation, Wittig or Horner-Emmons Reaction, reduction and coupling with amines.
  • the 5 '-position of the nucleoside derivative or 8-azanucleoside derivative is homologated with one or more carbon atoms, affording compounds with different distances between the atoms of the tetrahydrofuran ring and the homologated group.
  • Scheme 11 illustrates the preparation of a a class of homologated adenosine analogs which are useful for the invention. Or, if desired, such a homologue can subsequently be coupled with an amino acid to give other compounds useful for this invention.
  • proline is used to exemplify the amide coupling, but other amines or amino acid derivatives can be employed.
  • the reduction step of Scheme 11 is omitted, which generates unsaturated homologues useful in this invention.
  • Scheme 12. Preparation of 5'-substituted ethers.
  • Scheme 12 discloses a useful method for the synthesis of aryl- or heteroaryl-nucleoside ethers from purine or 8-azapurine carbocyclic nucleoside acetals by substitution of an appropriately ftmctionalized adenosine analogue or 8-azapurine derivative with a halogenated on an appropriately-substituted halogenated aryl, alkyl, or alkylaryl compound or a related heteroaromatic derivative.
  • Preferred substituents at M of the aromatic/heteroaromatic-group in Scheme 12 are independently hydrogen, or halogen, or groups containing carboxylic acid derivatives such as: -CO 2 R3; but they can also be halogen, or esters or amides of alkylcarboxylic acids, arylcarboxylic acids, -O-(alkylcarboxylic acids), -NR-(alkylcarboxylic acids); and the like.
  • M is halogen in Scheme 1
  • preferred halogens are chloro and fluoro.
  • the method disclosed in Scheme 12 can also be extended to a wide range of alkyl or aralkyl halides, making it a useful method for the general preparation of 5' substituted ethers.
  • Scheme 13 Preparation of 2, 3-acetaIs and -ketals.
  • Preferred aldehydes, aldehyde acetals and ketone ketals useful in the transketalization methods shown in Schemes 2 comprise the below said carbonyl compounds and/or derivatives of: benzaldehyde, biphenyl-3-carboxaldehyde, biphenyl-4-carboxaldehyde, biphenyl-4-yl-acetaldehyde, 2-bromobenzaldehyde, benzo[b]thiophene-3-carbaldehyde, cyclohexanecarbaldehyde, cyclopentanecarbaldehyde, 2,5-dimethylbenzaldehyde, 2,6- difluorobenzaldehyde, 2-fluorobenzaldehyde, naphthalene-2-carbaldehyde, phenyl acetaldehyde, phenyl propynal, 3-phenyl propenal, 3 -phenyl propionaldehyde
  • This invention provides a method of preventing or treating diseases or conditions associated with platelet aggregation and/or platelet activation. This invention also provides a method for solving treatment problems or limited treatment options caused by the aggregation of platelets or by the irreversible inhibition of platelet aggregation.
  • This invention provides methods of preventing or treating thrombosis and related disorders, such as venous thrombosis, established peripheral arterial disease, thrombophlebitis, arterial embolism, coronary and cerebral arterial thrombosis, unstable angina, myocardial infarction, stroke, cerebral embolism, renal embolism, pulmonary embolism and other embolism- or thrombosis-related afflictions produced by but not limited to procedural or surgical interventions.
  • thrombosis and related disorders such as venous thrombosis, established peripheral arterial disease, thrombophlebitis, arterial embolism, coronary and cerebral arterial thrombosis, unstable angina, myocardial infarction, stroke, cerebral embolism, renal embolism, pulmonary embolism and other embolism- or thrombosis-related afflictions produced by but not limited to procedural or surgical interventions.
  • This invention further provides methods for the prevention of embolism or thrombosis during percutaneous coronary interventions, placement of coronary stents, coronary angioplasty, coronary endarectomy, carotid endarectomy, or due to platelet-aggregation complications related to atherosclerosis, inflammation, exposure of blood to artificial devices, drug effects.
  • This invention further provides methods of inhibiting platelet aggregation in blood and blood products comprising platelets, such as stored blood.
  • the method comprises administering to a subject or blood and blood products a composition comprising an effective amount of P2Y! 2 receptor antagonist compound, wherein said amount is effective to bind the P2Yi 2 receptors on platelets and inhibit platelet aggregation, preferably in a reversible manner.
  • the invention further provides useful methods of treating patients to inhibit platelet aggregation in a reversible manner, especially in patients that are subject to a procedure such as percutaneous coronary interventions, stent placement, balloon angioplasty, coronary atherectomy, coronary endarterectomy, carotid endarterectomy, thrombolytic theraphy, coronary or other vascular graft surgery, dialysis, etc.
  • a procedure such as percutaneous coronary interventions, stent placement, balloon angioplasty, coronary atherectomy, coronary endarterectomy, carotid endarterectomy, thrombolytic theraphy, coronary or other vascular graft surgery, dialysis, etc.
  • platelet aggregation inhibition can be rapidly reversed (within hours for oral administration and within minutes for intravenous administration) if necessary.
  • the method comprises the steps of: (a) providing a patient in need of rapid reversal of platelet aggregation inhibition; (b) administering a therapeutically effective amount of a compound of Formula I, III-XII to the patient; (c) submitting the patient to a procedure selected from the group consisting of: percutaneous coronary interventions, stent placement, balloon angioplasty, coronary atherectomy, coronary endarterectomy, carotid endarterectomy, thrombolytic theraphy, coronary or other vascular graft surgery, and dialysis, (d) discontinuing the administering of said compound to the patient; and (e) allowing the amount of said compound in the patient's blood to reduce to below an therapeutically effective amount.
  • the administration of the compound can be either continuous or intermittent as long as it provides a therapeutically effective amount of the compound in the patient's blood. The amount of the compound in the patient's blood is monitored.
  • the compounds of general Formulae I, III-XII are antagonists of the effect of ADP on its platelet membrane receptor, the P2Yi 2 receptor.
  • the compounds of general Formula I are useful in therapy, in particular in the prevention or treatment of platelet aggregation.
  • the compounds provide efficacy as antithrombotic agents by their ability to block ADP from acting at its platelet receptor site and thus prevent platelet aggregation.
  • the compounds provide a more efficacious antithrombotic effect than aspirin, but with less profound effects on bleeding than antagonists of the fibrinogen receptor.
  • the P2Yj 2 receptor antagonists of this invention in contrast with currently available marketed products clopidogrel (PLAVIX®) and ticlopidine (TICLID®), bind to the P2Yj 2 receptor in a reversible fashion and therefore, the effects of the treatment with compounds described in this invention are reversed by the simple discontinuation of the treatment, restoring the hemostatic functionality of the platelet as necessary. Since platelets are non- nucleated cell particles that lack the ability to synthesize new proteins, treatment of subjects with irreversible P2Yi 2 antagonists results in the impairment of platelet function that lasts for the lifespan of the platelet (approximately 8 to 10 days).
  • the ADP-induced platelet aggregation is mediated by the simultaneous activation of both P2Yi2 and P2Yi receptors, thus the combined administration of the Formula I compounds with antagonists of platelet P2Yi receptors can provide a more efficacious antithrombotic effect at concentrations of each antagonist that are below the effective concentrations to block each receptor subtype in other systems, resulting in a decrease of the potential manifestation of adverse effects.
  • these compounds can be used in conjunction with lower doses of other platelet aggregation inhibitors, which work by different mechanisms, to reduce the possible side effects of said agents.
  • the compounds of the present invention are useful as anti-thrombotic agents, and are thus useful in the treatment or prevention of unstable angina, coronary angioplasty (PTCA) and myocardial infarction.
  • PTCA coronary angioplasty
  • the compounds of the present invention are useful in the treatment or prevention of primary arterial thrombotic complications of atherosclerosis such as thrombotic stroke, peripheral vascular disease, and myocardial infarction without thrombolysis.
  • the compounds of the invention are useful for the treatment or prevention of arterial thrombotic complications due to interventions in atherosclerotic disease such as angioplasty, endarterectomy, stent placement, coronary and other vascular graft surgery.
  • the compounds of the invention are useful for the treatment or prevention of thrombotic complications of surgical or mechanical damage such as tissue salvage following surgical or accidental trauma, reconstructive surgery including skin flaps, and "reductive" surgery such as breast reduction.
  • the compounds of the present invention are useful for the prevention of mechanically-induced platelet activation in vivo, for example, caused by cardiopulmonary bypass, which results in temporary platelet dysfunction (prevention of microthromboembolism).
  • the compounds of the present invention are useful for prevention of mechanically-induced platelet activation in vitro.
  • the compounds are useful in the preservation of blood products, e.g. platelet concentrates, prevention of shunt occlusion such as renal dialysis and plasmapheresis, and thrombosis secondary to vascular damage/inflammation such as vasculitis, arteritis, glomerulonephritis and organ graft rejection.
  • the compounds of the present invention are useful in disorders with a diffuse thrombotic/platelet consumption component such as disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, heparin-induced thrombocytopenia and pre-eclampsia/eclampsia.
  • a diffuse thrombotic/platelet consumption component such as disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, heparin-induced thrombocytopenia and pre-eclampsia/eclampsia.
  • the compounds of the invention are useful for the treatment or prevention of venous thrombosis such as deep vein thrombosis, veno-occlusive disease, hematological conditions such as thrombocythemia and polycythemia, and migraine.
  • the compounds of the present invention are useful in treating a mammal to alleviate the pathological effects of atherosclerosis and arteriosclerosis, acute MI 3 chronic stable angina, unstable angina, transient ischemic attacks and strokes, peripheral vascular disease, arterial thrombosis, preeclampsia, embolism, restenosis or abrupt closure following angioplasty, carotid endarterectomy, and anastomosis of vascular grafts.
  • the compounds of the present invention are useful in treating 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.
  • DIC disseminated intravascular coagulation
  • septicemia surgical or infectious shock
  • post-operative and post-partum trauma CAD
  • cardiopulmonary bypass surgery incompatible blood transfusion
  • abruptio placenta thrombotic thrombocytopenic purpura
  • snake venom and immune diseases
  • the compounds of the present invention are useful in treating diseases or conditions associated with platelet activation and/or aggregation produced by the contact of blood with an artificial device.
  • the artificial device is a paracorporeal artificial lung and an extracorporeal membrane oxigenation device.
  • the artificial device is an internal implantable artificial heart.
  • the artificial device is an apheresis instrument used to remove or isolate a specific component of the blood, and returning the remaining blood components to the donor.
  • the artificial device is a hemodialysis instrument.
  • the compounds of the present invention are useful in vitro to inhibit the aggregation of platelets in blood and blood products, e.g. for storage, or for ex vivo manipulations such as in diagnostic or research use. In such applications, the compounds are administered to the blood or blood product.
  • the compounds of the present invention have sufficient binding affinity and bear a fluorescent moiety, they are useful as biochemical probes for the P2Yi 2 receptor.
  • the compounds are used in the treatment of unstable angina, coronary angioplasty and myocardial infarction.
  • the compounds are useful as adjunctive therapy in the prevention or treatment of thrombotic disorders, such as coronary arterial thrombosis during the management of unstable angina, coronary angioplasty and acute myocardial infarction, for example, as adjuvants of thrombolytic therapy.
  • thrombotic disorders such as coronary arterial thrombosis during the management of unstable angina, coronary angioplasty and acute myocardial infarction, for example, as adjuvants of thrombolytic therapy.
  • the compounds are also administered in combination with other antiplatelet and/or anticoagulant drugs such as heparin, aspirin, GP Ilb/IIIa antagonists, or thrombin inhibitors.
  • This invention provides a method of inhibiting platelet aggregation and clot formation in a mammal, especially a human, which comprises administering to the subject a compound of Formula (I) and a pharmaceutically acceptable carrier.
  • This invention further provides a method for inhibiting the reocclusion of an artery or vein and the formation of new blood clots following fibrinolytic therapy, which comprises administering to a subject a compound of Formula (I) and a fibrinolytic agent.
  • 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 functional domains have been added, deleted or altered such as by combining the active site of one plasminogen activator or fibrin binding domain of another plasminogen activator or fibrin binding molecule.
  • the increased clinical efficacy of the combination of the compounds described in this invention with fibrinolytic agents allows the use of lower concentrations of the fibrinolytic agent, and therefore decreases the risk of hemorrhagic events. This in turn, allows the administration of fibrinolytic therapy over an extended period of time after a heart attack or stroke.
  • Extracorporeal circulation is routinely used for cardiovascular surgery in order to oxygenate blood. Platelets adhere to surfaces of the extracorporeal circuit. Platelets released from artificial surfaces show impaired hemostatic function. Compounds of the invention can be administered to prevent adhesion.
  • the active compounds can be administered systemically to target sites in a subject in need such that the extracellular concentration of a P2Yj 2 agonist is elevated to block the binding of ADP to P2Yi2 receptor, thus inhibit the platelet aggregation.
  • systemic includes subcutaneous injection, intravenous, intramuscular, intrastemal injection, intravitreal injection, infusion, inhalation, transdermal administration, oral administration, rectal administration and intra-operative instillation.
  • the pharmaceutical formulation is prepared in a sterile medium.
  • the active ingredient depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • Adjuvants such as local anesthetics, preservatives and buffering agents can also be dissolved in the vehicle.
  • the sterile indictable preparation can be a sterile indictable solution or suspension in a nontoxic acceptable diligent or solvent.
  • acceptable vehicles and solvents that can be employed are sterile water, saline solution, or Ringer's solution.
  • compositions containing active compounds are in the form of tablets, lozenges, aqueous or oily suspensions, viscous gels, chewable gums, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • an aqueous suspension is prepared by addition of water to dispersible powders and granules with a dispersing or wetting agent, suspending agent one or more preservatives, and other excipients.
  • Suspending agents include, for example, sodium carboxymethylcellulose, methylcellulose and sodium alginate.
  • Dispersing or wetting agents include naturally-occurring phosphatides, condensation products of an allylene oxide with fatty acids, condensation products of ethylene oxide with long chain aliphatic alcohols, condensation products of ethylene oxide with partial esters from fatty acids and a hexitol, and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anydrides.
  • Preservatives include, for example, etihyl, and n-propyl p- hydroxybenzoate.
  • Other excipients include sweetening agents (e.g., sucrose, saccharin), flavoring agents and coloring agents.
  • sweetening agents e.g., sucrose, saccharin
  • excipients can be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets can be uncoated or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
  • Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.
  • Formulation for oral use can also be presented as chewable gums by embedding the active ingredient in gums so that the active ingredient is slowly released upon chewing.
  • Additional means of systemic administration of the active compound to the target platelets of the subject would involve a suppository form of the active compound, such that a therapeutically effective amount of the compound reaches the target sites via systemic absorption and circulation.
  • compositions in the form of suppositories can be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the compound.
  • suitable non-irritating excipients include cocoa butter and polyethylene glycols.
  • the active compounds can also be systemically administered to the platelet aggregation sites through absorption by the skin using transdermal patches or pads.
  • the active compounds are absorbed into the bloodstream through the skin.
  • Plasma concentration of the active compounds can be controlled by using patches containing different concentrations of active compounds.
  • respirable particles comprising the active compound, which the subject inhales.
  • the active compound would be absorbed into the bloodstream via the lungs, and subsequently contact the target platelets in a pharmaceutically effective amount.
  • the respirable particles can be liquid or solid, with a particle size sufficiently small to pass through the mouth and larynx upon inhalation; in general, particles ranging from about 1 to 10 microns, but more preferably 1-5 microns, in size are considered respirable.
  • Another method of systemically administering the active compounds to the platelet aggregation sites of the subject involves administering a liquid/liquid suspension in the form of eye drops or eye wash or nasal drops of a liquid formulation, or a nasal spray of respirable particles that the subject inhales.
  • Liquid pharmaceutical compositions of the active compound for producing a nasal spray or nasal or eye drops can be prepared by combining the active compound with a suitable vehicle, such as sterile pyrogen free water or sterile saline by techniques known to those skilled in the art.
  • Intravitreal delivery can include single or multiple intravitreal injections, or via an implantable intravitreal device that releases P2Yi 2 antagonists in a sustained capacity. Intravitreal delivery can also include delivery during surgical manipulations as either an adjunct to the intraocular irrigation solution or applied directly to the vitreous during the surgical procedure.
  • plasma concentrations of active compounds delivered can vary according to compounds; but are generally lxlO ' ⁇ -lxlO "4 moles/liter, and preferably Ixl0 "8 -lxl0 "s moles/liter.
  • P2Yi 2 antagonist compounds of this invention are indicated by their inhibition of ADP-induced platelet aggregation.
  • This widely used assay as described in S.M.O. Hourani et al. Br. J. Pharmacol 105, 453-457 (1992) relies on the measurement of the aggregation of a platelet suspension upon the addition of an aggregating agent such as ADP.
  • Coating stents with pharmaceutical agents has an inherent advantage over systemic administration, due to the ability to precisely deliver a much lower dose of the drug to the target area thus achieving high tissue concentration while minimizing the risk of systemic toxicity.
  • the present invention is also directed to a P2Yi 2 receptor antagonist compound- eluting stent, which is a stent coated with one or more P2Y t2 receptor antagonist compounds of Formula I, or III-XII, such as Compound 1-326, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.
  • a P2Yi 2 receptor antagonist compound- eluting stent which is a stent coated with one or more P2Y t2 receptor antagonist compounds of Formula I, or III-XII, such as Compound 1-326, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.
  • a therapeutically effective amount of the P2Yi 2 receptor antagonist compound is an amount that is effective in preventing thrombosis and maintaining blood flow rate of the stented vessel, by decreasing in shear forces, relaxing vascular smooth muscle, and reducing narrowing of the vascular lumen restenosis.
  • the stent is coated with the P2Yi 2 receptor antagonist compound itself (without a carrier), or the stent is coated with the compound in a carrier, i.e., the compound is in the form of a component of a mixture or matrix.
  • the stent is coated with a carrier that comprises at least one P2Yn receptor antagonist compound.
  • the carrier is usually a biocompatible and non-toxic polymer.
  • the polymer is preferably a biodegradable polymer or a biostable polymer.
  • Biodegradable polymers suitable for this invention can be chosen from, but are not limited to, polycaprolactone, polylactic acid (D/L or L), poly(lactide-co- glycolide), poly(hydroxybutyrate), poly(hydroxybutyrate-covalerate), polydioxanone.
  • Biostable polymers can be chosen from, but are not limited to, polyurethanes, polyesters, polyamides, polyolefins, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, poly(ethylene-vinyl alcohol), polyethers, silicones, acrylate polymers and copolymers, polyvinylmethyl ether, polyimide, and polyacrylonitrile.
  • the concentration of the P2Yi2 receptor antagonist compound in the stent is in general in the range of 0.001 -20, preferably 0.01 - 10, and more preferably 0.1 -5 ⁇ g/mm 2 .
  • the concentration of the P2Yi 2 receptor antagonist compound in the stent is 1- 500, preferably 10-100 ⁇ g/mm.
  • Muni, et al. (American Heart Journal, 149:415-433, (2005)) have reported stent drug carriers, drug concentrations, stent sizes, and types of lesions; the article is incorporated herein by reference in its entirety.
  • the elution of the P2Yi 2 receptor antagonist compound is slow release and long-acting, i.e., the compound is eluted constantly and provides a local therapeutically effective amount at least until the epithelium damaged by the stent placement is healed.
  • the local elution of the P2Y 12 receptor antagonist compound into the tissue surrounding the stent is preferably over a period of 3 to 6 months, and preferably 6 months.
  • the elution of the compound from the stent directly relates to the rate of degradation of the polymer.
  • P2Yj 2 receptor antagonists useful in this invention are compounds that do not require hepatic, renal or any other metabolic transformation to become pharmacologically active.
  • the compound can be a prodrug if the conversion of the prodrug into the active species is carried out locally in the release area.
  • an ester prodrug can be converted into an active drug by tissue esterases such as endothelial esterases.
  • AU ester forms of P2Y 12 receptor antagonists are included in the application.
  • P2Yi2 receptors in vascular smooth muscle (Arterioscler. Tkromb. Vase. Biol. 2004; 24: 1810-1815).
  • the activation of these P2Yi2 receptors by endogenously released ADP results in vasoconstriction.
  • This effect contributes to tonic contraction of smooth muscle cells by circulating ADP, or by released ADP from adhering platelets, endoethelial cells and leukocytes attracted to the damaged area as part of the healing process, or by ADP produced locally from hydrolysis of released ATP from endothelial cells, dmooth muscle cells or blood cells.
  • P2Yi 2 receptor activation is associated with an increase in cell proliferation and the onset of inflammation; both of these effects contribute to in-stent restenosis observed in approximately 10% of the patients treated with currently marketed stents.
  • P2Yi 2 receptor antagonists- eluting stents The elution of P2Y ⁇ 2 receptor antagonists to local stented tissues can prevent the stenosis of stented arteries by relaxing the arterial smooth muscle, which results in an increase in blood flow rate of the stented artery and a decrease in shear forces that could promote thrombosis. Additionally, the inhibition of vascular smooth muscle contraction in stented arteries can decrease the risk of ischemia and thrombosis.
  • P2Yi 2 receptor antagonists-eluting stents improves the therapeutic benefit of current stents by decreasing the incidence of thrombosis and restenosis and improving of the flow rate of perfusion of the stented artery due to the relaxing activity of smooth muscle cells.
  • P2Yi2 receptor antagonist compound-eluting stents can be used as in situ antithrombotics to decrease the risk of stent thrombosis by a constant delivery of the P2Yi2 antagonist for several months. This treatment will decrease the risk of thrombosis by inhibiting the aggregation of platelets in the stented artery promoted by damaged endothelium during deployment of the stent and opening the narrowed vessel.
  • P2Yi 2 receptor antagonist compounds are useful to coat all types of stents, including coronary stents, cerebral arterial stents (basilar or vertebral arteries), other arterial stents (aortic, carotid, renal, peripheral, etc), and vein stents (portal, renal, including vein graft conduits).
  • Peripheral artery is defined as an artery that carries blood to upper and lower extremities.
  • P2Y 12 receptor antagonist eluting stents are useful for saphenous vein grafts previously grafted in coronary arteries, which have reduced patency due to restenosis or thrombosis.
  • Preferred stents for this invention are coronary stents.
  • P2Y 12 antagonists such as PLAVIX ® and TICLID ® are not suitable for coating stents because PLAVIX ® and TICLID ® need to be metabolized in the liver in order to generate the active metabolites.
  • the P2Yi 2 receptor antagonists of the present invention do not require metabolism for activation, and therefore they are capable of exerting their antithrombotic and smooth muscle relaxing activity in situ.
  • P2Y i2 receptor antagonist compound-eluti ⁇ g stents provide the advantages of inhibiting thrombus formation, inhibiting the contraction of vascular smooth muscle cells, inhibiting cell proliferation, and reducing inflammation.
  • P2Yi 2 receptor antagonist compound-eluting stents are useful in preventing the thrombosis and restenosis observed on patients after placement of bare metal and other drug-eluting stents.
  • the present invention provides a method for treating blocked or narrowed blood vessels such as arteries and veins.
  • the method comprises the step of placing a P2Yi 2 receptor antagonist compound-eluting stent according to the present invention in a narrowed or blocked blood vessel of a patient, whereby a therapeutically effective amount of the compound is eluted to the stented area, whereby the blood flow is resumed by the stent and the restenosis and thrombosis are prevented by the P2Y 12 receptor antagonist compound.
  • the artery can be, for example, coronary artery, cerebral artery, or peripheral artery, which has been narrowed or blocked by a plaque or a plaque rupture, respectively.
  • the inserted stent delivers P2Yi2 receptor antagonist compound locally to the stented area, and decreases the incidence of thrombosis and restenosis.
  • the method optionally comprises the step of monitoring the patient to ensure patency of the stented artery.
  • the patient can be monitored by clinical symptoms of the cardie function, e.g., electrocardiogram (EKG), to determine if the blood flow in the heart muscle is restored.
  • EKG electrocardiogram
  • the patient can be monitored by ultrasound to determine if the narrowed artery is restored, and by evaluation of clinical symptoms such as headache, facial droop, loss of coordination, vertigo and depressed mental status.
  • the stent is inserted into the cerebral arteries, the patient can be monitored by neurological examinations including clinical symptoms such as headache, facial droop, loss of coordination, vertigo and depressed mental status.
  • Stents are frequently made from stainless steel.
  • Stents can be made of any biocompatible metal, including, but not limited to, steel, cobalt, titanium, tantalum, chromium, zirconium, niobium, tungsten, platinum, palladium, vanadium, silver, gold, molybdenum, nickel, or magnesium, and alloys thereof in any combination.
  • stents can be constructed of non-metallic biocompatible materials, such as bioabsorbable or biostable polymers. The preparation of drug-eluting stents has been described in Kavanagh, et al.
  • P2Yi 2 receptor antagonist compounds of the present invention are preferably not attached directly (covalently of non-covalently) to the surface of an unmodified stent.
  • the stent is preferably coated with an organic or inorganic polymer (or polymers) or some other substance (such as an inorganic coating) that is able to retain the compound to be delivered and release it at a desired rate.
  • the nature of this retention can be covalent or non- covalent, with the latter being preferred.
  • the stent is first modified by coating it with an inorganic substance or an organic or inorganic polymer which is capable of binding the compound to the stent surface.
  • an inorganic substance or an organic or inorganic polymer which is capable of binding the compound to the stent surface.
  • the stent is first coated with a substance or a polymer that bears a basic moiety, and the compound is bound to the modified stent by an ionic interaction.
  • the P2Yi 2 receptor antagonist compound bears a basic moiety
  • the stent is first coated with a substance or a polymer that bears an acidic moiety, and the compound is bound to the modified stent by an ionic interaction.
  • the P2Yi 2 receptor antagonist compound is first incorporated into a compatible polymer matrix, which is then used to coat a stent.
  • a compatible polymer matrix which is then used to coat a stent.
  • the advantage of this approach is that the elution of the P2Y 12 receptor antagonist compound from the stent depends on the property of the polymer, thus one can select a suitable polymer, which provides controlled and sustained release of the P2Yi 2 receptor antagonist compound to the site of action.
  • the polymer can be hydrophilic, hydrophobic, biodegradable, or biostable, thus one can further select a polymer to optimize the desired therapeutic effect.
  • the present invention provides a composition comprising at least one biodegradable polymer and at least one P2Yi2 receptor antagonist compound of general Formula I, or III- XII, wherein said biodegradable polymer is selected from the group consisting of polycaprolactone, polylactic acid, polyQactide-co-glycolide), poly(hydroxybutyrate), poly(hydroxybutyrate-covalerate), polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid), poly(glycolic acid-cotrimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acids), poly(trimethylene carbonate), poly(iminocarbonate), cyanoacrylates, polyalkylene oxalates, polyphosphazenes, aliphatic polycarbonates, cellulose, starch, dextran, hyaluronic acid, and collagen.
  • biodegradable polymer is selected from the group consisting of polycaprolactone
  • the present invention further provides a composition comprising at least one biostable polymer and at least one P2Yi 2 receptor antagonist compound of general Formula I, wherein said biostable polymer is selected from the group consisting of polyurethanes, polyesters, polyamides, polyolefins, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, poly(ethylene- vinyl alcohol), polyethers, silicones, acrylate polymers and copolymers, polyvinylmethyl ether, polyimide, and polyacrylonitrile.
  • biostable polymer is selected from the group consisting of polyurethanes, polyesters, polyamides, polyolefins, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, poly(ethylene- vinyl alcohol), polyethers, silicones, acrylate polymers and copolymers, polyvinylmethyl ether, polyimide, and polyacrylonitrile.
  • the P2Yi 2 receptor antagonist compound When biodegradable polymers are used, the P2Yi 2 receptor antagonist compound is incorporated into the polymer matrix and released in a controlled manner by a gradual degradation of the polymer matrix. This degradation can occur by various processes, including hydrolysis, metabolism, bulk erosion, or polymer surface erosion. When biostable polymers are used, the P2Yi 2 receptor antagonist compound is uniformly distributed in the polymer or encapsulated within the polymer, from which the compound is eluted via diffusion processes or through pores of the polymer structure.
  • the P2Yi 2 receptor antagonist compound can be incorporated into the polymer via processes known to those skilled in the art. These include, but are not limited to, encapsulation of the compound within a polymer matrix during polymer synthesis prior to application of the polymer to the stent, dissolving both polymer and the compound in an appropriate solvent and applying the solution to a stent, after which the solvent is allowed to evaporate and the stent is allowed to dry, or pre-coating a stent with a polymer, after which the therapeutic agent is applied as a solution in an appropriate solvent.
  • Application methods can include, but are not limited to, spraying, dipping, or spin coating processes.
  • the mixture was sonicated for 15 minutes then stirred at room temperature for Ih under argon.
  • the reaction mixture was cooled to 0 0 C and under argon flow diethylazodicarboxylate (0.33 g, 1.1 eq), dissolved in 1 ml of dichloromethane, was added dropwise via syringe.
  • the mixture was protected from light and stirred at 0 0 C for 30 min. then at room temperature for 20 h.
  • the resin was washed liberally with dichloromethane and methanol.
  • the organic solution obtained from the washes was concentrated to give, after flash chromatography purification, 0.740 g of the product as a white solid (95% yield).
  • Example 4 Solution phase synthesis of 5'-carboxamide adenosine analogs 2% 3'-O-Isopropylideneadenosine-5'-carboxylic acid.
  • reaction mixture was then diluted with ethyl acetate (100 ml), washed with saturated sodium bicarbonate, brine, and dried over magnesium sulfate.
  • the crude product was purified by a chromatography with 0-2 % methanol in dichloromethane to give 57 mg (50 %) of pure product and recovered starting material (10 mg).
  • Phenyl isocyanate was coupled with the amide product using a method similar to those described above, affording the intermediate phenylurea compound. MW calculated for C 30 H 29 N 7 O 7 (MH + ) 600, found 600 by LCMS.
  • dimethylaminopyridine 5 mg, 0.045 mmol
  • methanesulfonamide 0.009 mg, 0.091 mmol
  • dichloromethane 0.5 ml
  • dicyclohexylcarbodiimide 10 mg, 0.05 mmol
  • the mixture was stirred for 2 days at RT. Additional dicyclohexylcarbodiimide (10 mg, 0.05 mmol) and dimethylaminopyridine (5 mg, 0.045 mmol) were added and the reaction was continued at RT overnight.
  • HESM hydroxymethylsulfanylmethyl
  • polystyrene resin 1.4 mmol/g, 200 mesh, NovaBiochem; 2.82 g, 3.95 mmol
  • Boc-Pro-OH 3.40 g, 15.8 mmol
  • HATU 5.7 g, 15.0 mmol
  • dimethylaminopyridine 0.24 g, 1.98 mmol
  • diisopropylethylamine 3.5 mL, 19.8 mmol
  • N 5 N- dimethyl formamide was drained from the HESM resin and the solution of activated proline derivative was added to the resin.
  • the resin was agitated at RT for 17 h.
  • the solvent was then drained and the resin washed with N,N-dimethyl formamide (3 x 30 mL), dichloromethane (3 x 30 mL), methanol (3 x 30 mL), dichloromethane (2 x 30 mL), methanol (3 x 30 mL) and dried in vacuo overnight.
  • Mass of resin 3.42 g, 93% loading.
  • Resin obtained in the previous step was agitated with a 40% trifluoroacetic acid/ dichloromethane solution (75 mL) for 15 minutes.
  • the solvent was drained, and a fresh solution of 40% trifluoroacetic acid in dichloromethane was added, and resin was agitated for another 15 minutes.
  • the resin was washed with dichloromethane (5 x 40 mL), 20% diisopropylethylamine/dichloromethane (2 x 30 mL), dichloromethane (3 x 30 mL), and methanol (5 x 40 mL).
  • the resin was dried under vacuum.
  • a chloranil test indicated the presence of a free amino group, and this proline-bound resin was carried over to the next step.
  • the proline resin product from the previous step was swelled in 50 mL N,N-dimethyl formamide for 30 minutes, after that the N,N-dimethyl formamide was drained.
  • 2', 3'-0-Isopropylideneadenosine-5'-carboxylic acid (1.40 g, 4.35 mmol)
  • dichloroethane (0.91 g, 4.74 mmol)
  • HOBfH 2 O (0.73 g, 4.74 mmol
  • diisopropylethylamine 3.5 mL, 19.8 mmol
  • a small amount of resin is suspended in a solution of 5 - 6 equivalents of m- chloroperbenzoic acid in dichloromethane and agitated for 7 - 8 hours at RT. The solution is then drained, and the resin is washed 5 - 6 times with fresh dichloromethane. Then resin is suspended in a solution of 4 - 5 equivalents of DBU in dichloromethane, and agitated at RT for 4 - 5 hours. The resin is then filtered and the solution is analyzed by LC/MS, HPLC or another method. The compounds are recovered from solution by rotary evaporation.
  • the adenosine-proline amide-derivatized resin from the previous resin-synthesis step (0.5 g, 0.275 mmol) was suspended in anhydrous N,N-dimethyl formamide (10 mL). Ethyl isocyanate was added (0.43 mL, 5.5 mmol), and the reaction mixture was heated in a capped vial at 55 0 C for 16 h.
  • the resin described above (0.05 g, 0.026 mmol) was suspended in trifluoroacetic acid, then benzaldehyde (0.095 g, 0.9 mmol) was added all at once.
  • the resin was agitated in a tightly closed vial for 24 h.
  • the resin was drained, washed with dichloromethane (5 x 3 mL), 20% diisopropylethylamine/ dichloromethane (2 x 3 mL), dichloromethane (3 x 3 mL), and methanol (5 x 3 mL), then dried in vacuo for 3 h.
  • R d cyclopropyWran.s-2-phenyl
  • R 2 2-naphthyl
  • Rd n-hexyl
  • R 3 CCPh Calculated MW for C31H35N7O7: 618.65 (MH+), found: 618.0.
  • Rd cyclopro ⁇ yl-*r ⁇ ms--2-phenyl
  • R 3 CCPh Calculated MW for C34H31 N7O7: 650.65
  • Rd benzyl
  • R 3 CCPh Calculated MW for C32H29N7O7: 624.62 (MH+), found: 624.1.
  • Rd ethyl
  • R 3 CCPh Calculated MW for C27H27N7O7: 562.55 (MH+), found: 562.0.
  • R d cyclopro ⁇ yl-*r ⁇ n,s-2-phenyl
  • R 3 4-bi ⁇ henyl Calculated MW for C38H35N7O7: 702.73
  • Rd cyclopropyl-*r ⁇ «.s-2-phenyl
  • R 3 phenyl Calculated MW for C32H31N7O7: 626.63
  • This tosylate product (0.260 g, 0.409 mmol) was dissolved in 3 mL of anhydrous N,N-dimethyl formamide, sodium azide (0.266 g, 4.09 mmol) was added, and the mixture ill was heated at 80 0 C in a closed vial for 7 h while being stirred. The mixture was diluted with 50 mL of dichloromethane and extracted with 5% sodium bicarbonate solution and brine. The organic layer was separated, dried over anhydrous sodium sulfate and solvent was removed in vacuo. The azide derivative was recovered (0.183 g, 87%) as a white solid. MW calculated for C 25 H 23 N 9 O 4 : 514.19 (MH + ), found 514.1.
  • thionyl chloride 0.3 g, 2.5 mmol was added at 0 0 C. After addition, the cold bath was removed followed by addition of two drops of N,N-dimethyl formamide. The reaction mixture was heated to 50 0 C for 30 minutes. The excess of thionyl chloride was removed under vacuum and the solid residue was washed with ethyl ether to give the acid chloride.
  • the acid chloride (61 mg, 0.125 mmol) was added to a vial containing L-proline methyl ester (23 mg, 0.138 mmol) and triethylamine (28 mg, 0.275 mmol) in 1 mL of dichloromethane at 0 0 C.
  • the reaction was gradually warmed to RT overnight.
  • ethyl acetate 75 ml
  • the residue was purified by elution from a silica column using 2 % methanol in dichloromethane to give purified prolylmethyl ester product (10 mg, 14 %).
  • Example 9 Enzymatic synthesis of a mixture of l-EthyI-3-[9-(6-hydroxymethyl-2- styryl-tetrahydro-furoP ⁇ -dlll ⁇ ldioxoM-yO ⁇ H-purin-o-yll-urea isomers from the corresponding isomeric mixture of 5'-AMP acetal/urea derivatives:
  • the reaction was worked up by adding more MeOH (20 mL), heating to 60 C to denature the enzyme, and filtering through a 0.22 uM filter.
  • the methanol was evaporated in vacuo, and a white, fine-particle solid precipitated from the remaining solvent. This mixture was cooled in an ice bath and filtered. Washing the material with water, followed by drying over P2O5 in a dessicator afforded the title product as a mixture of acetal diastereomers. Dry weight 440 mg (0.10 mmol, 71 % yield).
  • ACD acid/citrate/dextrose
  • the platelet pellet is resuspended in 40 mL of HEPES-buffered Tyrode's solution (137 mM NaCl, 2.7 mM KCl, 1 niM MgCl 2 , 2 mM CaCl 2 , 12 mM NaHCO 3 , 0.36 mM NaH 2 PO 4 , 5.5 mM glucose, 5 mM HEPES pH 7.4, 0.35% bovine serum albumin or 0.35% human serum albumin) containing l0U/mL heparin and 5 ⁇ M (final concentration) prostaglandin I2 (PGI 2 ).
  • HEPES-buffered Tyrode's solution 137 mM NaCl, 2.7 mM KCl, 1 niM MgCl 2 , 2 mM CaCl 2 , 12 mM NaHCO 3 , 0.36 mM NaH 2 PO 4 , 5.5 mM glucose, 5 mM HEPES pH 7.4,
  • the platelet suspension is incubated in a 37°C water bath for 10 minutes and then 5 ⁇ M (final cone.) PGI 2 is added just before centri&gation at 190Ox g for 8 minutes.
  • the resulting pellet is resuspended in 40 mL of HEPES-buffered Tyrode's solution containing 5 ⁇ M (final concentration) PGI 2 and then is incubated for 10 minutes in a 37°C water bath.
  • a small aliquot (500 ⁇ L) of the platelet suspension is removed for platelet counting.
  • 5 ⁇ M (final concentration) PGI 2 Prior to centrifugation 5 ⁇ M (final concentration) PGI 2 is added to the suspension and then the suspension is centrifuged at 190Ox g for 8 minutes.
  • the pellet is resuspended at a density of 5 x 10 s cells/mL in HEPES- buffered Tyrode's solution containing 0.05 U/mL apyrase.
  • ADP-induced platelet aggregation is determined by measuring the transmission of light through a 0.5 ml suspension of stirred (1000 rpm) washed platelets in a lumi-aggregometer at 37 0 C (Chrono-Log Corp. Havertown, PA). The baseline of the instrument is set using 0.5 ml of Hepes-buffered Tyrode's solution. Prior to aggregation measurements, the platelet suspension is supplemented with 1 mg/ml fibrinogen. Platelet aggregation is initiated by the addition of indicated concentrations of ADP or other agonists, and the light transmission is continuously recorded for at least 8 min.
  • IC50 values represent the concentration of antagonist necessary to inhibit by 50% the aggregation elicited by a given concentration of ADP.
  • Human blood is obtained from informed healthy adult volunteers. Blood is collected into syringes containing heparin, sodium citrate, PPACK or hirudin as anticoagulant. Blood is carefully transferred to a conical tube and maintained at room temperature. Assays are conducted within 60 min from the collection of the blood sample. ADP-induced platelet aggregation is performed using the impedance mode of an aggregometer (Chrono-Log Corp. Havertown, PA). Blood is gently mixed and an aliquot of 500 ⁇ L is transferred to a measurement cuvette, then, 450 ⁇ L of warm sterile saline is added to each cuvette and the sample is stirred at 1000 rpm. The impedance probe is introduced into the cuvette and the sample is allowed to warm for approx.
  • the basal impedance is recorded for 1 minute and then 50 ⁇ L of the appropriate concentrations of ADP are added to generate an ADP dose response curve.
  • blood samples are supplemented with 50 ⁇ L of the antagonist or vehicle and after 2 minutes, 50 ⁇ L of ADP (EC90; usually 5-10 ⁇ mol/L ADP) are added and the impedance is recorded for up to 8 minutes.
  • the potency of agonists and inhibitors of platelet aggregation is calculated from the impedance values obtained in each sample by fitting the data to a four-parameter logistic equation using the GraphPad software package (GraphPad Corp. San Diego, CA).
  • Surgical Preparation and Instrumentation Male Sprague-Dawley rats are anesthetized. Body temperature is maintained at 37 ⁇ 0.5 0 C with a heating lamp. Animals breathe spontaneously and a tracheotomy is performed to ensure a patent airway.
  • a cannula containing heparinized saline is introduced into the left femoral artery and connected to a transducer to record blood pressure and heart rate.
  • Cannulae containing non-heparinized saline are introduced into the left common carotid artery and left jugular vein for withdrawal of arterial blood samples and intravenous administration of compounds, respectively.
  • Rats (CD-I; male; approximately 350 grams; Charles River, Raleigh, NC), are anesthetized with sodium pentobarbital (70 mg/kg i.p.).
  • the abdomens are shaved and a 22 gauge intravenous catheter is inserted into a lateral tail vein.
  • a midline incision is made and the intestines are wrapped in saline-soaked gauze and positioned so the abdominal aorta is accessible.
  • the inferior vena cava and abdominal aorta are carefully isolated and a section (approximately 1 cm) of the abdominal aorta (distal to the renal arteries proximal to the bifurcation) is dissected.
  • All branches from the aorta in this section are ligated with 4-0 silk suture.
  • a 2.5 mm diameter flow probe connected to a Transonic flow meter is placed on the artery and a baseline (pre-stenosis) flow is recorded.
  • Two clips are placed around the artery decreasing the vessel diameter by approximately 80%.
  • a second baseline flow measurement is taken (post-stenosis) and the hyperemic response is tested.
  • Animals are then treated with either compound or saline intravenously via tail vein catheter.
  • Thrombosis is induced five minutes after treatment by repeated external compressions of the vessel with hemostatic forceps. Two minutes post-injury, the vessel compressions are repeated and a 10 minute period of flow monitoring is started.
  • Animals are monitored continuously for a minimum of the first ten minutes post-injury. After twenty minutes (post-injury), a flow measurement is repeated and the animals are euthanized. The section of the aorta that includes the injured section is harvested and placed in 10% formalin for possible histologic evaluation.
  • mice Male Sprague-Dawley rats are anesthetized using an inhaled anesthetic. A cannula containing heparinized saline is introduced into the jugular vein for withdrawal of venous blood samples. Animals are allowed a 48-hour recovery period prior to dose administration. Either compound or vehicle is administered to each animal as an oral gavage. Blood samples are taken immediately prior to compound administration, and at up to 12 time points ranging from 15 min to 24 hours following compound administration. HPLC-MS/MS is used to measure the amount of compound and/or metabolite in the blood samples.
  • Example 19 Inhibition of Thrombus Formation in Anesthetized Dogs: To evaluate the effect of the compounds of this invention on dynamic thrombus formation in vivo, the following experimental protocol, similar to the method of J. L. Romson et al. (Thromb. Res. 17:841-853, 1980), is performed.
  • Surgical Preparation and Instrumentation Briefly, purpose-bred dogs are anesthetized, intubated and ventilated with room air. The heart is exposed by a left thoracotomy in the fifth intercostal space and suspended in a pericardial cradle. A 2-3 cm segment of the left circumflex coronary artery (LCCA) is isolated by blunt dissection. The artery is instrumented from proximal to distal with a flow probe, a stimulation electrode, and a Goldblatt clamp. The flow probe monitors the mean and phasic LCCA blood flow velocities. The stimulation electrode and its placement in the LCCA and the methodology to induce an occlusive coronary thrombus have been described previously (J. K.
  • Example 20 IC 5 O values for representative compounds of the present invention.
  • Platelet IC50 data were determined using washed human platelets, according to the protocol of example 14.
  • Agonist challenge (ADP) typically in the range of 1-5 ⁇ M. Data are presented in ⁇ M and are from the average of two experiments or more.
  • Table 1 illustrates that a diverse set of compounds of the present invention show activity as antagonists of P2Yi 2 -mediated platelet aggregation.
  • compounds falling under Formula I (1, 14, 19, 24, 26, and 36) show P2Y 12 antagonist activity, despite having differing moieties at the 4' and 273' (acetal) positions of the ribose , and/or 6 (urea) position of the base.
  • compounds falling under the definition of Formula III demonstrate that conjugation of aromatic residues (with or without substituents) via a 2 or 3 atom ether linkage to the 4' carbon of the ribose ring leads to P2Yi 2 antagonists.
  • compounds falling under preferred Formula IV illustrate that diastereomerically-pure molecules containing a prolinamide residue at the 4' position, a phenyl or styryl acetal moiety at the 273' position, and an ethyl urea at the 6 position have improved potency relative to the less preferred compounds previously listed as falling under Formula I.
  • Table 1 also shows that replacing the oxygen of the benzyl ether moiety and reducing the linking group between the ribose ring 4' position and the terminal phenyl ring falling under the definition of X and Formula II to two total atoms instead of three (158) still can lead to active molecules.
  • preferred compounds falling under Formula VII show that P2Yi 2 antagonist activity can be obtained by modification of the ribose 4' position with simple, acyclic, non-aromatic moieties, in conjunction with the preferred styryl acetal and ethyl urea moieties previously described.
  • preferred compounds falling under Formula DC can show P2Yi 2 antagonist activity when one of the linking groups at the 4' position falling under the definition of A and B in Formula I is a carbamate moiety.
  • preferred compounds falling under Formula X (239) show potent activity when part of the linking group between the 4' position of the ribose and the phenyl ring falling under the definition of moiety X in Formula I and Formula II is an amide.
  • compounds falling under the definition of Formula XII (298) can be active when the moiety defined by X in Formula I and Formula II is a 5- membered heterocyclic ring.
  • Table 1 illustrates that a wide variety of molecules falling under the definitions of Formulae IV-XII can be useful as antagonists of P2Yi2 mediated platelet aggregation, and consequently potentially useful as therapeutics in diseases where inhibition of platelet aggregation would be beneficial.
  • Example 21 Coating of a Stent with a polymer incorporating a P2Yi 2 antagonist compound
  • a stent is coated with a P2Yi2 antagonist compound with procedures modified from that described in Example 4 of U.S. Patent No. 6,908,624 (Hossainy).
  • a stent is suspended in isopropanol and cleaned in an ultrasonic bath for 30 minutes.
  • the stent is dried and cleaned in a plasma chamber.
  • a poly(ethylene- vinyl alcohol) solution is made by dissolving one part poly(ethylene-vinyl alcohol) in seven parts dimethylsulfoxide, with stirring and shaking at 6O 0 C for 24 hours.
  • a P2Yi 2 antagonist compound (for example compound 41 ; typically in the range of 2-10% by weight of the total) is added to the poly(ethylene- vinyl alcohoiydimethyl sulfoxide solution and the solution is mixed, vortexed and placed in a tube.
  • the stent is attached to a mandrel wire and dipped into the solution.
  • the coated stent is briefly passed over a hotplate at 60 0 C, then is cured for 6 hours at ambient temperature, after which it is dried for 24 hours in a vacuum oven at 40-60 0 C. The above process is repeated two or three times to give two or three layers. Following final drying, the stent is optionally sterilized by electron beam radiation.

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Abstract

La présente invention concerne un procédé de prévention ou de traitement de maladies ou de conditions associées à l’agrégation de plaquettes. La présente invention porte également sur un stent pour élution de médicament, le stent étant revêtu d’un ou de plusieurs composés antagonistes récepteurs P2Y12 non nucléotides ou d’un sel, solvate, ou hydrate pharmaceutiquement acceptable de ceux-ci. Lorsque le stent est placé dans un vaisseau sanguin rétréci ou endommagé, une quantité thérapeutiquement efficace du composé antagoniste récepteur P2Y12 est éluée en continu entre le stent et l’environnement local du stent. Les stents pour élution de composé antagoniste récepteur P2Y12 servent à la prévention de la thrombose et de la resténose, et sont efficaces pour inhiber la formation de thrombus, inhiber la contraction de cellules musculaires lisses vasculaires, inhiber la prolifération cellulaire, et réduire l’inflammation.
EP06827504A 2005-11-04 2006-11-02 Stents pour élution de médicament avec composé antagoniste récepteur p2y12 non nucléotide Withdrawn EP1943261A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US11/267,941 US7749981B2 (en) 2003-10-21 2005-11-04 Drug-eluting stents coated with non-nucleotide P2Y12 receptor antagonist compound
PCT/US2006/017781 WO2006119507A2 (fr) 2005-05-05 2006-05-05 Composition non nucleotidique et technique d'inhibition d'agregation de plaquettes
PCT/US2006/043089 WO2007056217A2 (fr) 2005-11-04 2006-11-02 Stents pour élution de médicament avec composé antagoniste récepteur p2y12 non nucléotide

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