WO2012174013A2

WO2012174013A2 - Treatment of cardiovascular disease, stroke, and inflammatory conditions

Info

Publication number: WO2012174013A2
Application number: PCT/US2012/042092
Authority: WO
Inventors: Karen J. Brunke; Toshio Uemura; Fujiko Takamura
Original assignee: Cardeus Pharmaceuticals, Inc.; Astellas Pharma, Inc.
Priority date: 2011-06-14
Filing date: 2012-06-12
Publication date: 2012-12-20
Also published as: WO2012174013A3

Abstract

Reversible P2Y12 receptor antagonists are administered for the treatment of cardiovascular disease, stroke, and other inflammatory diseases and conditions.

Description

TREATMENT OF CARDIOVASCULAR DISEASE, STROKE, AND

INFLAMMATORY CONDITIONS BACKGROUND OF THE INVENTION

[0001] This application claims the benefit of U.S. Patent Application No.

61/496,905 filed June 14, 2011, which is hereby incorporated by reference.

Field of the Invention

[0002] The present invention provides new treatments and pharmaceutical formulations and unit dose forms for treatment of cardiovascular disease and other inflammatory conditions and related diseases and so relates to the fields of biology, chemistry, medicinal chemistry, medicine, molecular biology, and pharmacology.

Description of Related Disclosures

[0003] P2Y12 receptor antagonists have been developed as therapeutic products based on their platelet aggregation inhibitory activity. They are useful as a preventive and/or therapeutic agent for circulatory organ system disease involving thrombus formation by platelet aggregation, such as unstable angina; acute myocardial infarction and its secondary prevention; re-obstruction and re-stricture after coronary artery bypass surgery, percutaneous transluminal coronary angioplasty (PTCA operation), or stent indwelling operation; coronary artery thrombolysis acceleration and re-obstruction prevention and similar ischemic diseases; transient cerebral ischemic attack (TLA), cerebral infarction, subarachnoid hemorrhage (vasospasm), and cerebrovascular accidents; chronic arterial occlusive disease and similar peripheral arterial diseases; and as an auxiliary agent at the time of cardiac surgical operation or vascular surgical operation. P2Y12 receptor antagonists constitute the largest segment of the antithrombotic market, and clopidogrel bisulfate (PLAVIX, marketed by Bristol Myers Squibb and Sanofi- Aventis) is the dominant drug in this category.

[0004] Clopidogrel has significant drawbacks, however, including variable patient response, delayed onset action, hepatic damage, and irreversible inhibition of the P2Y12 receptor. Clopidogrel carries a black box warning for poor metabolizers of the drug. In addition, irreversible inhibition requires discontinuation of irreversible P2Y12 receptor antagonist administration at least five days prior to most surgeries. Other P2Y12 receptor antagonists, such as prasugrel (EFFIENT, marketed by Eli Lilly and Company), ticagrelor (BRILINTA, marketed by AstraZeneca), and elinogrel (in development by Portola Pharmaceuticals), have similar or other drawbacks. For example, prasugrel is an irreversible inhibitor that has a black box warning for bleeding risk. Both ticagrelor and elinogrel have a variety of undesired side effects. Ticagrelor has an increased bleeding risk, dypsnea, slowed heart rate and renal impairment as examples of undesired side effects in comparison with clopidogrel, which may lead to contraindication of ticagrelor for patients with asthma or COPD. Ticagrelor has drug-drug interaction risks with drugs metabolized via CYP3 A4 or utilizing P-glycoprotein, especially statins and digoxin, and has a very active metabolite. Clopidogrel and prasugrel are prodrugs that require cleavage to produce the active drug.

[0005] A promising new class of P2Y12 receptor antagonists is described in U.S. patent number (USP) 8,133,882, incorporated herein by reference, but this class of compounds has not been tested in humans, and safe and efficacious dosing regimens are not known. Compounds in this class are not nucleic acid analogues like ticagrelor or thienopyridines like clopidogrel and prasugrel.

[0006] Due to their side effects, drug-drug interactions (DDIs), and black box warnings for side effects such as bleeding risk, P2Y12 receptor antagonists have not found application outside the treatment of high-risk conditions such as cardiovascular disease and stroke. Moreover, P2Y12 receptor antagonists act by inhibiting thrombus formation by platelet aggregation, and thrombus formation has not been established as a causative factor in diseases other than cardiovascular diseases, so there has been no significant development of P2Y12 receptor antagonists to treat other inflammatory conditions and diseases resulting therefrom. There could be significant benefit in developing safer treatments, dose regimens and unit dose forms for P2Y12 receptor antagonists for use in cardiovascular disease, and in expanding their safe and efficacious use to other inflammatory conditions. The present invention provides such benefits.

SUMMARY OF THE INVENTION

[0007] In a first aspect, the invention provides pharmaceutical formulations and unit dose forms of reversible P2Y12 inhibitors for use in methods for treating and/or preventing cardiovascular disease, stroke, or another inflammatory disease or condition in a subject in need of treatment by administering a therapeutically effective dose of a compound of Formula 1 ,

wherein

R¹ is cycloalkyl or lower alkylene-cycloalkyl, wherein the cycloalkyl in R¹ is optionally substituted;

hydrogen or halogen;

R⁴ is lower alkyl, lower alkylene-cycloalkyl, or cycloalkyl, wherein the cycloalkyl is optionally substituted;

R⁵ is lower alkylene-R⁶ or lower alkenylene- R⁶, wherein the lower alkylene or lower alkenylene is optionally substituted;

R⁶ is -C0₂R⁷, -C(0)NHOH, -C(0)N(R⁷) ₂, or -C0₂-lower alkylene-aryl; and R⁷ is hydrogen or lower alkyl,

or an active metabolite, prodrug, ester, or salt form of such a compound, and methods for their use.

[0008] In the methods of the invention, the therapeutically effective dose is a dose no higher than the dose that results in 100% inhibition of platelet aggregation (also referred to herein as "platelet inhibition"), and typically, the therapeutically effective dose is a dose that results in 90% platelet inhibition or less. Generally, the therapeutically effective dose will be a daily dose in the range of 5 mg/day to 200 mg/day, depending on the patient and indication to be treated, and the daily dose will be administered once per day (qd) or twice per day (bid).

[0009] In various embodiments of the formulations, unit dose forms, and methods of the invention, the reversible P2Y12 inhibitor compound is ASP 1645 (or one of its active metabolites, prodrugs, esters, or salt forms), which has the following structure and can be named, using ChemDraw Ultra, as (S)-2-(7-(cyclohexylamino)-l-cyclopentyl-6- fluoro-4-oxo-l ,4-dihydroquinolin-3-yloxy)propanoic acid.

[0010] In one embodiment, the present invention provides pharmaceutical formulations and unit dose forms of a compound of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, for use in the method of the invention for treating cardiovascular disease in a patient undergoing percutaneous coronary intervention (PCI), including but not limited to balloon angioplasty and PTCA or stent indwelling operation. In this embodiment of the invention, ASP 1645 or another reversible P2Y12 inhibitor of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, is administered at a dose that provides at least 50% inhibition of platelet aggregation (also referred to herein as platelet inhibition), typically greater than 75% platelet inhibition, and sometimes 90% platelet inhibition or greater, i.e., up to and generally no more than the amount that results in 100% platelet inhibition. Suitable doses for adult humans therefore typically include a daily dose equal to or less than 200 mg of a compound of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, including but not limited to ASP 1645, including 150 mg qd; 75 mg qd or bid; 50 mg qd or bid; and 25 mg qd or bid.

[0011] In another embodiment, the present invention provides a method for treating acute coronary syndrome (ACS), which includes unstable angina and acute myocardial infarction and its secondary prevention. In this embodiment of the invention, a compound of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, including but not limited to ASP 1645 is administered at a dose that provides at least 50% platelet inhibition, typically greater than 75% platelet inhibition, and sometimes 90% platelet inhibition or greater, i.e., up to and generally no more than the amount that results in 100% platelet inhibition. Suitable doses for adult humans therefore typically include a daily dose equal to or less than 200 mg of ASP 1645 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, including but not limited to 150 mg qd; 75 mg qd or bid; 50 mg qd or bid; and 25 mg qd or bid.

[0012] Other cardiovascular diseases amenable to prevention and/or treatment in accordance with the methods described herein include but are not limited to re-obstruction and re-stricture after coronary artery bypass surgery, coronary artery thrombolysis acceleration, and re-obstruction prevention and similar ischemic diseases; transient cerebral ischemic attack (TIA), cerebral infarction, subarachnoid hemorrhage

(vasospasm), and cerebrovascular accidents; chronic arterial occlusive disease and similar peripheral arterial diseases; stroke treatment and/or prevention; and as an auxiliary agent at the time of and/or immediately following surgery, including but not limited to major surgical operations, such as a cardiac surgical operation or a vascular surgical operation. In these cardiovascular diseases, the reversible P2Y12 inhibitor is administered as described above for the treatment of PCI and ACS.

[0013] In other embodiments of the methods of the invention, the inflammatory disease or condition treated or prevented is an inflammatory disease or condition other than cardiovascular disease or stroke. Such conditions amenable to treatment in accordance with the methods herein include joint and/or muscle inflammation, including but not limited to arthritis; skin inflammation; eye inflammation; lung and/or airway inflammation; GI tract inflammation; cancer; neurological inflammation; and/or neurological impairment. More specifically, such other diseases amenable to prevention and/or treatment in accordance with the methods described herein include but are not limited to rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, juvenile arthritis, scapulohumeral periarthritis, cervical syndrome, lumbago, eczema, dermatitis, conjunctivitis, asthma (all types, include severe and chronic asthma), bronchitis, pigeon fancier's disease, farmer's lung, COPD, aphthous ulcer, Crohn's disease, inflammatory bowel disease, ulcerative colitis, atopic gastritis, gastritis varioloforme, celiac disease, irritable bowel syndrome, regional ileitis, cystic fibrosis, gingivitis, menorrhalgia, systemic lupus erythematosus, scleroderma, polymyositis, tendinitis, bursitis, periarteritis nodose, rheumatic fever, Sjogren's syndrome, Behcet disease, thyroiditis, type I diabetes, metabolic syndrome, nephrotic syndrome, aplastic anemia, myasthenia gravis, uveitis contact dermatitis, psoriasis, Kawasaki disease, sarcoidosis, Hodgkin's disease, Alzheimer's disease, and non-asthma pulmonary diseases with inflammatory components, such as cystic fibrosis, acute lung injury, endotoxin- induced lung injury and adult respiratory distress syndrome.

[0014] In the treatment methods of the invention for treating inflammatory diseases and conditions other than cardiovascular disease and stroke, a reversible P2Y12 inhibitor of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, is administered at a dose that provides at least 25% or greater platelet inhibition, including greater than 50% platelet inhibition, and 75% platelet inhibition or greater, but typically less than 90% platelet inhibition. Suitable doses for adult humans in these embodiments therefore typically include a daily dose equal to or less than 100 mg of ASP 1645 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, qd, including but not limited to 75 mg qd, 50 mg qd or bid; 25 mg qd or bid; 10 mg qd or bid; and 5 mg qd or bid.

[0015] The treatment methods of the invention offer special benefit to certain patient populations. For example and without limitation, ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, are not metabolized by CYP3A4 and/or other cytochrome P450s and/or P-glycoprotein and so can be used to treat patients concurrently taking drugs that are metabolized by CYP3 A4 and/or other cytochrome P450s and/or P-glycoprotein, such as, for example and without limitation, statins and digoxin. In addition, certain anti-thrombotic agents currently marketed have a side effect of inducing dyspnea and so are not suitable for administration to patients suffering from dyspnea, including but not limited to patients experiencing shortness of breath, chronic obstructive pulmonary disease (COPD), or asthma, whereas ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, do not induce dyspnea and so can be used to treat such patients. In addition, patients with one or more inactivating mutations in CYP2C19 (about 25% of patients of Asian ancestry have such mutations) cannot be treated with clopidogrel, whereas ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, can be used to treat such patients. In addition, patients concurrently taking bradycardia drugs cannot be treated with ticagrelor, whereas

ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, can be used to treat such patients.

[0016] The pharmaceutical formulations and unit dose forms provided by the invention are useful in the methods described herein and include pharmaceutical formulations suitable for intravenous administration as well as pharmaceutical formulations suitable for oral administration. The present invention also provides unit dose forms of such pharmaceutical formulations. The unit dose forms of the invention contain a dose of a reversible P2Y12 receptor antagonist of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, that is equal to or lower than the dose that results in 100% inhibition of platelet aggregation. In various embodiments, this dose is a dose that results in 90%> or less platelet aggregation inhibition; 75% or less platelet aggregation inhibition; 50% or less platelet aggregation inhibition; and 25% or more platelet aggregation inhibition.

[0017] In various embodiments of the methods of the invention, the dose of the reversible P2Y12 inhibitor of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, such as ASP1645, is a 150 mg/day or lower dose, such as 150 mg dosed qd. Other suitable doses include, without limitation, 125 mg qd; 100 mg qd; 75 mg qd or bid; 50 mg qd or bid; 25 mg qd or bid; 10 mg qd or bid; 5 mg qd or bid. More generally, practice of the methods of the invention involves administering a dose of a P2Y12 inhibitor in an amount of from about 5 to about 150 mg/day.

[0018] In various embodiments, therefore, the pharmaceutical formulation contains a compound of Formula 1 or one of its active metabolites, prodrugs, esters, or salt forms. In one embodiment, the compound is ASP 1645 or one of its active

metabolites, prodrugs, esters, or salt forms. In various embodiments, the unit dose form contains 150 mg or less of ASP 1645 or another compound of Formula 1 or one of its active metabolites, prodrugs, esters, or salt forms. In various embodiments, the unit dose forms contain 150 mg, 125 mg, 100 mg, 75 mg, 50 mg, 25 mg, 20 mg, 10 mg, or 5 mg of a compound of Formula 1 or one of its active metabolites, prodrugs, esters, or salt forms, including but not limited to unit dose forms containing ASP 1645 or one of its active metabolites, prodrugs, esters, or salt forms.

[0019] These and other aspects and embodiments of the invention are described in more detail below.

BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1 shows the results of a study, described in Example 1 , of the effect of platelet washing on the inhibitory effects of ASP 1645 and clopidogrel on adenosine diphosphate (ADP)-induced platelet aggregation after oral administration to guinea pigs. In the figure, ** indicates a p value of <0.01 for the comparison of the post- wash inhibition versus the pre -wash inhibition.

[0021] Figure 2 shows the results of a study, described in Example 2, of the potent ADP-dependent platelet aggregation inhibition effect of ASP 1645 at various doses (in mg/kg). Increasing the ADP in the assay changed the percent inhibition of platelet aggregation by ASP 1645 in a concentration dependent manner. In the figure, ** indicates a p value <0.01 compared with the vehicle group (Dunnett's multiple comparison test). [0022] Figure 3 shows the results of a study, described in Example 4, of the effect of ASP 1645 on arterial pinch-injury-induced thrombosis in a guinea pig model. Circles represent the time to occlusion of each animal. Bars indicate the median values in each test group. Statistical analyses were performed using Steel's test. * indicates a p value of < 0.05 compared with the vehicle group.

[0023] Figure 4 shows the results of a study, described in Example 4, of the effect of clopidogrel on arterial pinch-injury-induced thrombosis in a guinea pig model. Circles, bars, statistical analysis, and * are as defined for Figure 3.

[0024] Figure 5 shows the results of a study, described in Example 4, of the effect of ASP 1645 on nail cuticle bleeding time in a guinea pig model. Circles represent the bleeding time of each animal. Bars indicate the median values in each experimental group. Statistical analyses were performed using Steel's test. * indicates a p value of < 0.05 compared with the vehicle group.

[0025] Figure 6 shows the results of a study, described in Example 4, of the effect of clopidogrel on nail cuticle bleeding time in a guinea pig model. Circles, bars, statistical analysis, and * are as defined for Figure 5.

[0026] Figure 7 shows the results of a study, described in Example 7, of the effect of ASP 1645 on P-selectin expression on ADP-stimulated human platelets in comparison with that of abciximab. Data represent the mean +SEM of 5 separate experiments. ** indicates a p value <0.01 compared with the ADP-stimulated platelets (Dunnett's multiple comparison test), "ns" is not statistically significant.

[0027] Figure 8 shows the results of a study, described in Example 7, of the effect of ASP 1645 on the percentage of monocytes with bound platelets in comparison with that of abciximab. Data represent the mean +SEM of 5 separate experiments. ** indicates a p value <0.01 compared with non-stimulated platelets (Paired t-test). "ns" is not statistically significant.

[0028] Figure 9 shows the results of a study, described in Example 7, of the effect of ASP 1645 on the percentage of granulocytes with bound platelets in comparison with that of abciximab. Data represent the mean +SEM of 5 separate experiments. ** indicates a p value <0.01 compared with non-stimulated platelets (Paired t-test).

[0029] Figure 10 shows the results of a study, described in Examples 8 and 9, of the effect of ASP 1645 or ticagrelor on the inhibition of ex vivo ADP-induced platelet aggregation at 12, 24, and 48 hours after cynomolgus monkeys were given one of three dose levels of either ASP 1645 or ticagrelor. Data represent the mean +SEM of 3 separate experiments.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides methods for treating cardiovascular disease, stroke, and other inflammatory diseases and conditions by administering a reversible P2Y12 inhibitor of Formula 1 to a patient in need of treatment.

Formula 1

wherein

Pv¹ is cycloalkyl or lower alkylene-cycloalkyl, wherein the cycloalkyl in R¹ is optionally substituted;

R is hydrogen or halogen;

R⁶ is ~C0₂R⁷, ~C(0)NHOH, -C(0)N(R⁷) ₂, or -C0₂-lower alkylene-aryl; and R⁷ is hydrogen or lower alkyl

or an active metabolite, prodrug, ester, or salt thereof.

[0031] The present invention also provides pharmaceutical formulations and unit dose forms useful in these methods.

[0032] In some embodiments, R¹ is cycloalkyl. In some embodiments, R¹ is cyclohexyl.

[0033] In some embodiments, R¹ is lower alkylene-cycloalkyl. In some embodiments, R¹ is lower alkylene-cyclopropyl.

2 2

[0034] In some embodiments, R is hydrogen. In some embodiments, R is halogen. In some embodiments, R is fluoro. 3 3

[0035] In some embodiments, R is hydrogen. In some embodiments, R is halogen. In some embodiments, R is fluoro.

2 3

[0036] In some embodiments, one of R or R is hydrogen and the other is fluoro.

[0037] In some embodiments, R⁴ is lower alkyl or cycloalkyl wherein the cycloalkyl is optionally substituted.

[0038] In some embodiments, R⁴ is lower alkyl.

[0039] In some embodiments, R⁴ is cycloalkyl wherein the cycloalkyl is optionally substituted. In some embodiments, R⁴ is cyclopentyl optionally substituted with one or two groups independently chosen from lower alkyl. In some embodiments, R⁴ is cyclopentyl.

[0040] In some embodiments, R⁵ is lower alkylene-R⁶ wherein the lower alkylene is optionally substituted. In some embodiments, R⁵ is -CH(CH₃)-R⁶.

[0041] In some embodiments, R⁶ is ~C0₂R⁷.

[0042] In some embodiments, R⁷ is hydrogen. In some embodiments, R⁷ is lower alkyl.

[0043] In some embodiments, the compound of Formula 1 is chosen from

2-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3- yloxy)pentanoic acid,

4-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3-yloxy)-3- hydroxybutanoic acid,

4-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3-yloxy)-2- hydroxybutanoic acid,

4-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3-yloxy)-2,2- dimethylbutanoic acid,

4-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3- yloxy)butanoic acid,

2-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3- yloxy)butanoic acid,

(S)-2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)butanoic acid,

2-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3-yloxy)acetic acid, 2- (7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3-yloxy)-2- methylpropanoic acid,

3- (7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3- yloxy)propanoic acid,

2-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3-yloxy)-3- methylbutanoic acid,

(S)-2-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3-yloxy)-

3-methylbutanoic acid,

2- { [7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3 - yl]oxy}propanoic acid,

(2S)-2- { [7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yl]oxy}propanoic acid,

2- {([7-(cyclohexylamino)-6-fluoro- 1 -isopropyl-4-oxo- 1 ,4-dihydroquinolin-3-yl]oxy} - propanoic acid, and

(2S)-2- {([7-(cyclohexylamino)-6-fluoro- 1 -isopropyl-4-oxo- 1 ,4-dihydroquinolin-3- yl]oxy} -propanoic acid,

or an active metabolite, prodrug, ester, or salt thereof.

[0044] In some embodiments, the compound of Formula 1 is chosen from the following esters:

methyl 2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)butanoate,

ethyl 2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)butanoate,

methyl (S)-2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)butanoate,

ethyl (S)-2-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3- yloxy)butanoate,

methyl 2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)-3 -methylbutanoate,

ethyl 2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)-3 -methylbutanoate,

methyl (S)-2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)-3 -methylbutanoate, ethyl (S)-2-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3- yloxy)-3 -methylbutanoate,

methyl 2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)acetate,

ethyl 2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)acetate,

methyl 2-(7-(cyclohexylamino)-6-fluoro- 1 -isopropyl-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)acetate,

ethyl {[7-(cyclohexylamino)-6-fluoro-l-isopropyl-4-oxo-l,4-dihydroquinolin-3- yl]oxy} acetate,

methyl 2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)propanoate,

ethyl 2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)propanoate,

methyl (S)-2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)propanoate,

ethyl (S)-2-(7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3- yloxy)propanoate,

methyl 2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)-2-methylpropanoate, and

ethyl 2-(7-(cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3- yloxy)-2-methylpropanoate.

[0045] In some embodiments, the compound of Formula 1 is chosen from (2S)-2- {[7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3- yl]oxy} propanoic acid, and (2S)-2-{([7-(cyclohexylamino)-6-fluoro-l-isopropyl-4-oxo- l,4-dihydroquinolin-3-yl]oxy} -propanoic acid, or an active metabolite, prodrug, ester, or salt thereof.

[0046] In some embodiments, the compound of Formula 1 is (S)-2-(7- (cyclohexylamino)- 1 -cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3-yloxy)propanoic acid or an active metabolite, prodrug, ester, or salt thereof. Definitions

[0047] As used in the present specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0048] In this description, the "lower alkyl", "lower alkylene" and "lower alkenylene" respectively mean hydrocarbon chains having from 1 to 6 carbon atoms which maybe in the straight chain or branched chain form, unless otherwise noted.

[0049] Accordingly, the "lower alkyl" means a Ci_₆ alkyl, and illustrative examples thereof include methyl, ethyl, propyl, butyl, pentyl or hexyl, or structures isomers thereof such as isopropyl, tert-butyl or the like, preferably a Ci_₅ alkyl, more preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl or 3-pentyl.

[0050] The "lower alkylene" means a divalent group in which one hydrogen is removed from an optional position of the "lower alkyl", and is illustratively methylene, methylmethylene, ethylene, propylene, butylene or the like, preferably a Ci_₄ alkylene, more preferably methylene, methylmethylene, ethylene or propylene.

[0051] The "lower alkenylene" means a divalent group in which one hydrogen is removed from an optional position of the "lower alkenyl", and is illustratively vinylene, propenylene, butenylene or the like, preferably a C₂_₃ alkenylene, more preferably vinylene, propenylene.

[0052] The "halogen" means a monovalent group of halogen atom, and fluoro, chloro, bromo, iodo or the like may be cited illustratively, of which fluoro or chloro is preferred.

[0053] The "cycloalkyl" means a C3_io non-aromatic hydrocarbon ring, and it may form a bridged ring or a spiro ring, partially have an unsaturated bond or be condensed with benzene ring. However, when benzene ring is condensed, the linking hand is present on the non-aromatic ring. Illustrative examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, cyclohexenyl, cyclooctdieneyl, adamantly, norbornyl, indanyl having a linking hand at from the 1- to 3-position and the like. Preferred is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl and more preferred is cyclopentyl or cyclohexyl.

[0054] The term "aryl" means a monocyclic to tricyclic C_6-14 aromatic hydrocarbon ring. Aryl groups may be monocyclic or polycyclic (i.e.., bicyclic, tricyclic). In some instances, both rings of a polycyclic aryl group are aromatic (e.g., naphthyl). In other instances, polycyclic aryl groups may include a non-aromatic ring (e.g., cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl) fused to an aromatic ring, provided the polycyclic aryl group is bound to the parent structure via an atom in the aromatic ring. Thus, a l,2,3,4-tetrahydronaphthalen-5-yl group (wherein the moiety is bound to the parent structure via an aromatic carbon atom) is considered an aryl group, while 1,2,3,4- tetrahydronaphthalen-l-yl (wherein the moiety is bound to the parent structure via a non- aromatic carbon atom) is not considered an aryl group

[0055] The term "optionally substituted" means "not substituted" or "substituted with 1 to 5 of the same or different substituent groups".

[0056] The substituents acceptable as those for the phrase "optionally substituted" satisfactorily include those for routine use in the art as substituents for the individual groups. In addition, when two or more groups are present, respective groups may be the same or different from each other.

[0057] In some embodiments, substituents for "lower alkylene" and "lower alkenylene" are independently selected from halogen, -OR⁰, -C0₂R° , -C0₂-lower alkylene-aryl and aryl where R° is -H or a lower alkyl.

[0058] In some embodiments, substituents for "cycloalkyl" (such as "cyclopentyl") are independently selected from halogen, lower alkyl, -OR⁰, -C0₂R° and -C(0)-aryl where R° is -H or a lower alkyl.

[0059] A "salt" may be prepared for any compound having a functionality capable of forming a salt, for example, an acid or base functionality. Salts may be derived from organic or inorganic acids and bases. Compounds that contain one or more basic functional groups, e.g., amino or alkylamino, are capable of forming salts with

pharmaceutically acceptable organic and inorganic acids. These salts can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting a purified compound in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate,

heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2- napthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, and tosylate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their acid addition salts. See, e.g., Berge et al. "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66: 1-19.

[0060] Compounds that contain one or more acidic functional groups are capable of forming salts with pharmaceutically acceptable bases. The term "salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds described herein. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Illustrative examples of some of the bases that can be used include sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate, N⁺(Ci_₄ alkyl)₄ hydroxide, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine,

ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. Also provided are salts wherein one or more basic nitrogen-containing groups are quarternized. Water or oil-soluble or dispersible products may be obtained by such quaternization. See, for example, Berge et al, supra.

[0061] The term "prodrug" refers to a substance administered in an inactive or less active form that is then transformed (e.g., by metabolic processing of the prodrug in the body) into an active compound. The rationale behind administering a prodrug is to optimize absorption, distribution, metabolism, and/or excretion of the drug. Prodrugs may be obtained by making a derivative of an active compound that will undergo a transformation under the conditions of use (e.g., within the body) to form the active compound. The transformation of the prodrug to the active compound may proceed spontaneously (e.g., by way of a hydrolysis reaction) or it can be catalyzed or induced by another agent (e.g., an enzyme, light, acid or base, and/or temperature). The agent may be endogenous to the conditions of use (e.g., an enzyme present in the cells to which the prodrug is administered, or the acidic conditions of the stomach) or the agent may be supplied exogenously. Prodrugs can be obtained by converting one or more functional groups in the active compound into another functional group, which is then converted back to the original functional group when administered to the body. For example, a hydroxyl functional group can be converted to a sulfonate, phosphate, ester or carbonate group, which in turn can be hydrolyzed in vivo back to the hydroxyl group. Similarly, an amino functional group can be converted, for example, into an amide, carbamate, imine, urea, phosphenyl, phosphoryl or sulfenyl functional group, which can be hydrolyzed in vivo back to the amino group. A carboxyl functional group can be converted, for example, into an ester (including silyl esters and thioesters), amide or hydrazide functional group, which can be hydrolyzed in vivo back to the carboxyl group.

[0062] The term "ester" refers to a compound formally derived from a carboxylic acid and an alcohol, phenol, heteroarenol, or enol by linking with formal loss of water from an acidic hydroxy group of the former and a hydroxy group of the latter.

[0063] Terms such as "active metabolite" and the like refer to a derivative of the reversible P2Y12 receptor antagonist that retains a detectable level, e.g., at least about 10%, at least about 20%>, at least about 30%> or at least about 50%>, of at least one desired activity of the parent compound. Determination of a desired activity may be accomplished as described herein. Such metabolites can be generated in the gastrointestinal tract, in blood or in one or more subject tissues. Such metabolites are detected using standard analytical methods, e.g., GC-MS analysis of an optionally radiolabeled parent compound and its metabolites, in blood, urine or other biological samples after it is administered to a subject by one or more routes as disclosed herein.

[0064] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. Pharmaceutically acceptable carriers include excipients and diluents.

[0065] The reversible P2Y12 receptor antagonists described herein can be enriched isotopic forms, e.g., enriched in the content of ²H, ³H, ⁿC, ¹³C and/or ¹⁴C. In one embodiment, the compound contains at least one deuterium atom. Such deuterated forms can be made, for example, by the procedure described in U.S. Patent Nos.

5,846,514 and 6,334,997. Such deuterated compounds may improve the efficacy and increase the duration of action of compounds disclosed and/or described herein.

Deuterium substituted compounds can be synthesized using various methods, such as those described in: Dean, D., Recent Advances in the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery and Development, Curr. Pharm. Des. , 2000; 6(10); Kabalka, G. et al, The Synthesis of Radiolabeled Compounds via

Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21; and Evans, E., Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64(1-2), 9-32.

[0066] The reversible P2Y12 receptor antagonists described herein are intended to include polymorphs, isomers, and tautomers.

[0067] "Isomers" are different compounds that have the same molecular formula. "Stereoisomers" are isomers that differ only in the way the atoms are arranged in space. "Enantiomers" are stereoisomers that are non-superimposable mirror images of each other. A 1 : 1 mixture of a pair of enantiomers is a "racemic" mixture. The symbol "(±)" may be used to designate a racemic mixture where appropriate. "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn-Ingold- Prelog R-S system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S. Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. Unless otherwise indicated, compounds described herein include all such possible enantiomers, diastereomers, and other stereoisomeric forms, including racemic mixtures, optically pure forms and intermediate mixtures. Optically active (R) and (S) isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.

[0068] "Polymorph," is meant to include all crystalline and amorphous forms of the compound, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms, as well as mixtures thereof, unless a particular crystalline or amorphous form is referred to.

[0069] "Tautomers" are structurally distinct isomers that interconvert by tautomerization. Tautomerization is a form of isomerization and includes prototropic or proton-shift tautomerization, which is considered a subset of acid-base chemistry.

Prototropic tautomerization or proton-shift tautomerization involves the migration of a proton accompanied by changes in bond order, often the interchange of a single bond with an adjacent double bond. Where tautomerization is possible (e.g. in solution), a chemical equilibrium of tautomers can be reached. An example of tautomerization is keto-enol tautomerization. A specific example of keto-enol tautomerization is the interconverision of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridin-4(lH)-one tautomers. When the compounds described herein contain moieties capable of tautomerization, and unless specified otherwise, it is intended that the compounds include all possible tautomers.

Treatment of Cardiovascular Disease and Stroke

[0070] The present invention provides methods for treating and/or preventing cardiovascular disease and stroke in a subject in need of treatment by administering a compound selected from the group consisting of reversible P2Y12 receptor antagonists of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, at a dose equal to or lower than the dose that results in 100% inhibition of platelet aggregation. In various embodiments, the compound used in the method is ASP 1645 (or one of its active metabolites, prodrugs, esters, or salt forms).

[0071] In one embodiment, the patient treated in accordance with the methods of the invention is a patient with cardiovascular disease who is suitable for or has undergone or is undergoing percutaneous coronary intervention (PCI). PCI is the most commonly performed invasive therapeutic cardiac procedure and plays an important role in the treatment of ischemic heart disease. The goals of pharmacotherapy during PCI are twofold: to mitigate the sequelae of iatrogenic plaque rupture from balloon angioplasty or stenting; and to reduce the risk of thrombus formation on intravascular PCI equipment. Platelet activation and subsequent platelet aggregation and thrombus formation are induced by multiple factors including thrombin, produced locally by tissue factor, and adenosine diphosphate (ADP) and thromboxane A2 (TXA₂), which are released from activated platelets. The transduction of the ADP signal involves platelet receptors, including the P2Y12 receptor, which is a target for certain antithrombotic agents that are P2Y12 receptor antagonists; compounds of Formula 1 are P2Y12 receptor antagonists. Importantly, the use of anticoagulation agents must balance reduction in thrombotic complications (periprocedural myocardial infarction [MI] and catheter thrombus) with the risk of periprocedural bleeding. Hemorrhagic complications in patients with ischemic heart disease are associated with death, recurrent MI, stent thrombosis, and stroke. Many patient characteristics associated with increased risk for bleeding are also independent predictors of ischemic outcomes, underscoring the importance of appropriate dosing of antithrombotic therapy to minimize both ischemic and hemorrhagic complications after PCI. PCI requires either oral and/or intravenous treatment with a platelet aggregation inhibitor to prevent thrombus formation during and following the procedure. Oral administration typically continues for up to one year following PCI.

[0072] Selection of the platelet aggregation inhibitor for use in PCI is based on its ability to rapidly inhibit platelet aggregation, its lack of drug-drug interactions with other drugs taken in this patient population or for this procedure, and its bleeding profile. A drug that performs well in all three areas is ideal for the PCI setting. Compounds of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, exhibit such performance within the dose ranges of the methods of the present invention, as demonstrated in the examples below. Prolonged bleeding is expected in a patient in which platelet inhibition is 100%. Example 8, below, demonstrates that 100% platelet inhibition is achieved in monkeys by dosing ASP 1645 at 3 mg/kg/day, which translates into a dose in excess of 200 mg/day in humans. The dose range provided by the methods of the invention also avoids the phototoxicity associated with ASP 1645 and other compounds of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, as demonstrated in Example 5, below. Example 1 , below, demonstrates the full reversibility of the platelet inhibition of ASP 1645. Example 2, below, demonstrates that potent inhibition of platelet aggregation inhibition can be achieved with ASP 1645, and Example 4, below, demonstrates potent reduction in thrombus formation with that compound. Example 3, below, demonstrates a lack of drug-drug interactions with ASP 1645, as no significant interactions with the cytochrome P450s were observed. Other animal metabolism studies demonstrate the majority of ASP 1645 is excreted as whole drug. Example 7, below, demonstrates inhibition of P-selectin expression with ASP 1645 consistent with inhibition of activation of new platelets. In addition, as patients with PCI devices frequently return for other coronary procedures within the year, a rapid rate of offset of a P2Y12 inhibitor is critical to the success of its use in this setting, and ASP 1645 and other compounds of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, exhibit such rapid offset rates, as demonstrated in Example 1. The compounds of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, are therefore ideal for use in the PCI setting.

[0073] Thus, in one embodiment, the present invention provides a method for treating cardiovascular disease by reducing clot formation in a patient undergoing percutaneous coronary intervention (PCI), including but not limited to balloon

angioplasty and PTCA or stent indwelling operation. In this embodiment of the invention, ASP 1645 or another reversible P2Y12 inhibitor of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, is administered at a dose that provides at least 50% platelet inhibition, typically greater than 75% platelet inhibition, and sometimes 90% platelet inhibition or greater, i.e., up to but generally no more than 100% platelet inhibition (physicians may employ a "loading" dose upon initiation of treatment that equals or exceeds the amount of drug that provides 100% platelet inhibition). Suitable doses for adult humans therefore typically include a daily dose equal to or less than 200 mg of ASP 1645 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, including 150 mg qd (once per day); 75 mg qd or bid (twice per day); 50 mg qd or bid; and 25 mg qd or bid, i.e., a dose range of 25 mg to 200 mg per day for an adult human. The present invention arose in part from the discoveries that administration of doses higher than the highest dose in this range results in unacceptable bleeding risk and phototoxicity and that administration of doses lower than the lowest dose in this range is not efficacious in the treatment of cardiovascular disease and stroke.

[0074] Treatment in accordance with the methods of the invention can be initiated at any time prior to PCI, concurrently with PCI, or shortly after PCI. Typically, if treatment hasn't been initiated prior to PCI, then treatment is initiated concurrently with PCI. Treatment typically is continued for at least one year after PCI. The initial treatment can be administered intravenously or orally; thereafter, for the convenience of the patient, treatments are typically administered orally, using one of the unit dose forms provided by the present invention.

[0075] Another cardiovascular disease suitable for treatment in accordance with the invention is acute coronary syndrome (ACS). ACS includes life-threatening clinical conditions ranging from unstable angina to non-Q-wave myocardial infarction and Q- wave myocardial infarction, which are a major cause of emergency medical care and hospitalization in the United States. The underlying cause is in large part due to coronary artery disease. Acute coronary syndrome requires treatment with platelet aggregation inhibitors at a dose that adequately inhibits platelets without undue side effects.

Ticagrelor, a reversible and direct-acting oral antagonist of the ADP receptor P2Y12, provides faster, greater, and more consistent P2Y12 inhibition than clopidogrel. In a dose- guiding trial, there was no significant difference in the rate of bleeding with the use of ticagrelor at a dose of 90 mg or 180 mg twice daily and the rate with the use of clopidogrel at a dose of 75 mg daily. However, dose-related episodes of dyspnea and ventricular pauses on Holier monitoring, which occurred more frequently with ticagrelor, led to the selection of the dose of 90 mg twice daily, which is now the approved dose. However, even at this lower dose, dyspnea was more common in the ticagrelor group than in the clopidogrel group (in 13.8% of patients vs. 7.8%). The rate of stroke did not differ significantly between ticagrelor and clopidogrel in studies, although there were more hemorrhagic strokes with ticagrelor than with clopidogrel.

[0076] The methods of the present invention avoid the problems associated with ticagrelor and clopidogrel. ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, do not have the side effects of ticagrelor and clopidogrel and other agents of the P2Y12 receptor inhibitor class. Optimal dosing of ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, in accordance with the methods of the invention results in efficacious platelet aggregation without significant side effects as demonstrated in the examples below and discussed above. In addition, ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, do not induce dyspnea, as demonstrated in Example 6, below. Thus, in one embodiment, the present invention provides a method for treating acute coronary syndrome, including unstable angina and acute myocardial infarction and its secondary prevention. In this embodiment of the invention, ASP 1645 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, is administered at a dose that provides greater than 50% platelet inhibition, typically greater than 75% platelet inhibition, and sometimes 90% platelet inhibition or greater, i.e., but generally is provided in an amount that is equal to or less than the amount that provides 100% platelet inhibition. Suitable doses for adult humans therefore typically include a daily dose equal to or less than 200 mg of ASP 1645, including but not limited to 150 mg qd; 75 mg qd or bid; 50 mg qd or bid; and 25 mg qd or bid.

[0077] Other cardiovascular diseases amenable to prevention and/or treatment in accordance with the methods described herein include but are not limited to re-obstruction and re-stricture after coronary artery bypass surgery, coronary artery thrombolysis acceleration, and re-obstruction prevention and similar ischemic diseases; transient cerebral ischemic attack (TIA), cerebral infarction, subarachnoid hemorrhage

[0078] Most cardiology patients undergo multiple surgeries over their life span including surgery such as CABG (coronary artery bypass graft). ASP 1645 and other compounds of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, have a fast offset rate and so can be used up until two or three days prior to surgery. Support can be found in Example 1 for the complete reversibility of ASP 1645.

Clopidogrel and aspirin are currently stopped one week before most surgeries and ticagrelor is stopped five days before surgery. The ability to continue treatment in accordance with the methods of the present invention up until two to three days prior to surgery is a significant advantage for patients at risk for thrombotic events. Thus, in one embodiment, the methods of the invention are useful in treating cardiovascular disease or stroke in a patient expecting to undergo surgery within one week.

[0079] The treatment methods of the invention offer special benefit to certain patient populations unsuitable for treatment with other drugs, including other antithrombotic agents. For example and without limitation, ASP 1645 and other compounds of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, are not

metabolized by CYP3A4 and/or other cytochrome P450s and/or P-glycoprotein and so can be used to treat patients concurrently taking drugs that are metabolized by CYP3 A4 and/or other cytochrome P450s and/or P-glycoprotein, such as, for example and without limitation, statins and digoxin. In addition, certain anti-thrombotic agents currently marketed have a side effect of inducing dyspnea and so are not suitable for administration to patients suffering from dyspnea, including but not limited to patients experiencing shortness of breath, chronic obstructive pulmonary disease (COPD), or asthma, whereas ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, do not induce dyspnea and so can be used to treat such patients. In addition, patients with one or more inactivating mutations in CYP2C19 (about 25% of patients of Asian ancestry have such mutations) cannot be treated with clopidogrel, whereas ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, can be used to treat such patients. In addition, patients concurrently taking bradycardia drugs cannot be treated with ticagrelor, whereas

Treatment of Inflammatory Conditions other than Cardiovascular Disease and Stroke

[0080] The present invention provides methods for treating and/or preventing inflammatory diseases and conditions other than cardiovascular disease and stroke in a subject in need of treatment by administering a compound of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, at a dose equal to or lower than the dose that results in 25% or greater but typically less than 90% inhibition of platelet

aggregation. In various embodiments, the compound used in the method is ASP 1645 (or one of its active metabolites, prodrugs, esters, or salt forms). [0081] Platelets are an important, although largely unappreciated, part of the inflammatory cascade. Platelets also produce a large number of proinflammatory lipid mediators and cytokines and are involved in the recruitment of leukocytes into the inflamed tissues. Platelets can be viewed as inflammatory cells in that they can undergo chemotaxis, contain and release adhesive proteins, activate other inflammatory cells, release vasoactive substances, and have the capacity to express or release

proinflammatory mediators such as thromboxane A2 (TXA₂), prostaglandins, platelet activating factor (PAF), brain-derived neurotrophic factor (BDNF) and platelet factor 4 (PF4 or CXCL4), as well as a large number of other chemokines and chemokine receptors. Although platelets do not have nuclei, they do have mRNAs and are capable of de novo protein synthesis. Platelets provide free arachidonic acid (AA) to

polymorphonuclear cells (PMNs), which enhances production of PMN-derived leukotriene B4 (LTB4) and the cysteinyl leukotrienes LTC4, LTD4, and LTE4.

[0082] In asthmatic individuals, LTE4 causes accumulation of eosinophils, mast cells and basophils in the bronchial mucosa. And, particularly in aspirin-sensitive asthmatics, their airways are hyperresponsive to LTE4. While the side effect profiles of approved P2Y12 receptor antagonists make them unlikely to be useful for treating diseases such as asthma, the present invention arose in part from the discovery that the better safety profiles for reversible P2Y12 receptor antagonists such as ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, would not similarly restrict their use. Thus, in one aspect, the present invention relates to the discovery that reversible P2Y12 receptor antagonists are useful in preventing and/or treating the pulmonary inflammation associated with asthma. Thus, unlike the irreversible P2Y12 receptor antagonists, the reversible P2Y12 receptor antagonist class useful in the methods, pharmaceutical formulations, and unit dose forms of this invention has a safety profile acceptable for use by those patients suffering from asthma and other inflammatory diseases. In addition, while other P2Y12 receptor antagonists have significant rates of dyspnea likely based on interactions with other cysteinyl leukotrienes, ATP analog structure, and/or potential for binding adenosine receptors and increasing adenosine, ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, do not have these properties and do not induce dyspnea and so avoid the pulmonary complications associated with other P2Y12 inhibitors. Example 6, below, demonstrates that ticagrelor binds the adenosine receptor whereas ASP 1645 does not.

[0083] Bronchial asthma is a chronic inflammatory disease associated with leukocyte infiltration and airway epithelial damage. Platelets and platelet-derived P- selectin are found in the lungs of allergic asthmatics. Platelet depletion in mice has been shown to remodel airway architecture. In addition, P-selectin expression on the surface of platelets is a major requirement for pulmonary eosinophilia in asthma as well as other inflammatory diseases such as acute lung injury and endotoxin-induced injury. It is also known that platelet-derived P-selectin is important in postischemic renal failure.

Activated platelets have been detected in the blood of patients with inflammatory bowel disease. Increased expression of platelet P-selectin is correlated with inflammatory disease in humans. Inflammatory diseases where platelet activation is known to be important include cystic fibrosis, adult respiratory distress syndrome, atherosclerosis, asthma, inflammatory bowel disease and renal disease, to name a few. Inhibition of P- selectin correlates with inhibition of the inflammatory state. Example 7, below, demonstrates that ASP 1645 inhibits the expression of P-selectin and reduced adhesion of activated platelets to monocytes and granulocytes. As leukocytes interact with activated platelets to produce inflammatory mediators, inhibition of expression of P-selectin correlates with decreased inflammation. Thus, use of ASP 1645 and other compounds of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof, in inflammatory diseases at the therapeutically effective doses provided by the methods of the invention that reduce inflammation without prolonging bleeding provides significant therapeutic benefit.

[0084] Platelets are a linking element between hemostasis, inflammation and tissue repair. Platelets can be activated by multiple pathways (thrombin, ADP and TXA₂). The P2Y12 receptor found on platelets and to which ADP binds (one mechanism for platelet activation) is the target for a P2Y12 receptor antagonist. The reversible P2Y12 receptor antagonist compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, that are useful in the methods, pharmaceutical formulations, and unit dose forms described herein show, as demonstrated in animal models using ASP 1645 and described in the examples below, overall safety, lack of drug-drug interactions, and relatively low bleeding risk. Thus, in addition to the cardiology indications described above, the pharmaceutical formulations and unit dose forms of the invention are also useful in the treatment of inflammatory diseases other than cardiovascular diseases and stroke, including but not limited to chronic asthma, inflammatory asthma, systemic lupus erythematosus, cystic fibrosis, inflammatory bowel disease, chronic kidney disease, glomerulonephritis and other inflammatory diseases. In addition, and unlike other P2Y12 receptor antagonists that cause dyspnea, ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, can be used in patients with pulmonary diseases who have cardiovascular disease or stroke, as discussed above.

[0085] Thus, the methods, pharmaceutical formulations, and unit dose forms described herein are useful in treating any inflammatory condition and related diseases, in part because the doses provided are safe and efficacious. In this range, dosing inhibits platelet activation (and inhibits and reduces platelet aggregation) with considerably lower risk of bleeding. Such dose ranges are generally (regardless of disease or indication) in the range of 5 to 200 mg per day, although patients with cardiovascular disease are generally dosed at the higher end of this range, stroke patients in the mid to upper part of the range, and patients with other inflammatory diseases are generally dosed at the lower end of this range. The daily dose can be administered qd or bid (for example, the 150 mg per day dose can be administered qd or 75 mg bid). In general, the pharmaceutical formulations of the invention comprise one or more pharmaceutically acceptable carriers.

[0086] In some embodiments, the pharmaceutical formulation of the invention is suitable for intravenous, intraperitoneal, subcutaneous or oral administration.

[0087] In some embodiments, the pharmaceutical formulation of the invention comprises the active ingredient (a compound of Formula 1) dissolved in water and an alkaline solution such as 0.1 M NaOH, and made essentially isotonic by the addition of normal saline, phosphate-buffered saline or other agent such as 0.5% methyl cellulose. The pH typically is adjusted to approximately, neutral, for example, between pH 6 and pH 8. The pharmaceutical formulation can then be aseptically filtered and filled into vials or bags. In some embodiments, the pharmaceutical formulation is injected directly into the patient as a bolus administration. In some embodiments, the pharmaceutical formulation is administered to the patient intravenously over time. In some embodiments, the intravenous administration is performed directly and the dosing controlled using a drip or a pump. In some embodiments, the intravenous administration is performed via a Y- connector to a bag with an intravenous catheter and the dosing controlled using a drip or a pump. [0088] In some embodiments, the pharmaceutical formulation of the invention is formulated into tablets or other suitable unit dose forms such as capsules for oral administration. In some embodiments, a tablet of the invention comprises the active ingredient, a filler, a binder, a disintegrant, a lubricant, and a coating agent. In some embodiments, a tablet comprises the active ingredient, e.g., 10 mg ASP1645; lactose monohydrate (filler), 106.1 mg; hydroxypropylcellulose (binder), 4.05 mg; low

substituted hydroxypropylcellulose (disintegrant), 13.5 mg; magnesium stearate

(lubricant), 1.35 mg; and OPADRY03A42172 (coating agent), 4 mg.

[0089] In some embodiments, the tablet comprises ASP 1645 (active ingredient), 50 mg; lactose monohydrate (filler), 180.85 mg; hydroxypropylcellulose (binder), 8.1 mg; low substituted hydroxypropylcellulose (disintegrant), 27 mg; magnesium stearate (lubricant), 4.05 mg; and OPADRY03A42172 (coating agent), 8 mg.

[0090] In some embodiments, a process of the invention for producing the tablets is employed and involves several sequential steps, such as: 1. Pulverizing: Pulverize the active ingredient using an impact mill; 2. Dissolving: Dissolve hydroxypropylcellulose into purified water as binding solution; 3. Granulating: Blend pulverized active ingredient obtained in step 1 with lactose monohydrate, and granulate the blended powder with binding solution obtained in step 2 by a fluidized bed granulator; and then, dry the granule and sieve through a screen; 4. Blending: Blend the granule obtained in step 3 with low substituted hydroxypropylcellulose and magnesium stearate using a diffusion mixer; 5. Tableting: Tablet the final blend from step 4 into tablets using a tableting machine; 6. Dispersing: Disperse OPADRY03A42172 into purified water as film-coating suspension; and 7. Film Coating: Coat core tablets with a film-coating suspension obtained in step 6 by a pan-coating system.

[0091] Thus, in accordance with the methods of the invention, a patient suffering from an inflammatory disease or condition other than cardiovascular disease and stroke can be administered a dose of a reversible P2Y12 inhibitor of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, such as ASP 1645, in an amount of about 150 mg/day or lower, such as 150 mg dosed qd. Other suitable doses include, without limitation, 125 mg qd; 100 mg qd; 75 mg qd or bid; 50 mg qd or bid; 25 mg qd or bid; 10 mg qd or bid; 5 mg qd or bid.

[0092] In various embodiments of these methods, the inflammatory disease or condition treated or prevented is joint and/or muscle inflammation, including but not limited to arthritis; skin inflammation; eye inflammation; lung and/or airway

inflammation; GI tract inflammation; cancer; neurological inflammation; and/or neurological impairment. More specifically, such other diseases amenable to prevention and/or treatment in accordance with the methods described herein include but are not limited to rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, juvenile arthritis, scapulohumeral periarthritis, cervical syndrome, lumbago, eczema, dermatitis, conjunctivitis, asthma (all types, include severe and chronic asthma), bronchitis, pigeon fancier's disease, farmer's lung, COPD, aphthous ulcer, Crohn's disease, inflammatory bowel disease, ulcerative colitis, atopic gastritis, gastritis varioloforme, celiac disease, irritable bowel syndrome, regional ileitis, cystic fibrosis, gingivitis, menorrhalgia, systemic lupus erythematosus, scleroderma, polymyositis, tendinitis, bursitis, periarteritis nodose, rheumatic fever, Sjogren's syndrome, Behcet disease, thyroiditis, type I diabetes, metabolic syndrome, nephrotic syndrome, aplastic anemia, myasthenia gravis, uveitis contact dermatitis, psoriasis, Kawasaki disease, sarcoidosis, Hodgkin's disease, Alzheimer's disease, and non-asthma pulmonary diseases with inflammatory components, such as cystic fibrosis, acute lung injury, endotoxin- induced lung injury and adult respiratory distress syndrome.

[0093] As discussed above, the treatment methods of the invention offer special benefit to certain patient populations unsuitable for treatment with other drugs, including other anti-thrombotic agents, and such patient populations include those suffering from an inflammatory disease or condition other than cardiovascular disease or stroke. For example and without limitation, ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, are not metabolized by CYP3A4 and/or other cytochrome P450s and/or P-glycoprotein and so can be used to treat patients concurrently taking drugs that are metabolized by CYP3A4 and/or other cytochrome P450s and/or P-glycoprotein, such as, for example and without limitation, statins and digoxin. In addition, ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, do not induce dyspnea and so can be used to treat patients suffering from dyspnea, including but not limited to patients experiencing shortness of breath, chronic obstructive pulmonary disease (COPD), or asthma. In addition, patients concurrently taking bradycardia drugs cannot be treated with ticagrelor, whereas ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, can be used to treat such patients. Combination Therapies

[0094] The methods, pharmaceutical formulations, and unit dose forms described herein are also useful in combination with other drugs and therapies that are now or may later be approved for the treatment of cardiovascular disease, stroke, and other inflammatory diseases and conditions including but not limited to asthma (severe, chronic) and chronic kidney disease.

[0095] For example, ASP 1645 or another compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, is, in some embodiments, used in combination with aspirin for treatment of cardiovascular disease or stroke. In some embodiments, ASP 1645 or another compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, is used instead of aspirin in these indications.

[0096] As another example, ASP 1645 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, is, in some embodiments, used in combination with other drugs for treatment of acute coronary syndrome such as anticoagulants, statins, vasodilators, ACE (angiotensin converting enzyme) inhibitors, ARBs (angiotensin II receptor blockers), cardiac glycosides and beta blockers.

[0097] As another example, ASP 1645 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, is, in some embodiments, used in combination with other anti-coagulants for treatment prior to, during and after PCI. Such anti-coagulants may include factor Xa inhibitors (heparin, low molecular heparin and others), Vitamin K antagonists (warfarin and others), and direct thrombin inhibitors as well as anti-platelets such as GPIIbllla inhibitors, P2Y12 receptor antagonists, COX inhibitors (aspirin), prostaglandin analogues, thromboxane inhibitors and

phosphodiesterase inhibitors.

[0098] As another example, ASP 1645 or another compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, is, in some embodiments, used in combination with other agents currently used for treatment of inflammation in asthma. Such agents, and the classes of anti-inflammatory agents to which they belong include, but are not limited to: anti-IgE agents, chemokine receptor antagonists, cytokine antagonists, immunosuppressive agents, leukotriene modifiers, mast cell stabilizers, phospholipase A2 antagonists, protease inhibitors, selective phosphodiesterase inhibitors, and steroids. In the treatment of asthma, anti-inflammatories are used in combination with beta-agonist bronchodilators to help open the airways, and bronchodilators, including beta-agonist bronchodilators, can be used in combination with ASP 1645 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof. Other types of bronchodilators include, but are not limited to: anti-cholinergics, anti- muscarinic agents, magnesium sulfate, and theophylline.

General Synthesis of Compounds

[0099] The compounds of this invention can be prepared from readily available starting materials using, for example, the following general methods and procedures. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA), Emka-Chemce or Sigma (St. Louis, Missouri, USA). Others may be prepared by procedures, or obvious modifications thereof, described in standard reference texts such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley, and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5, and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley, and Sons, 5th Edition, 2001), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Additionally, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. For example, numerous protecting groups are described in T. W. Greene and G. M. Wuts (1999) Protecting Groups in Organic Synthesis, 3rd Edition, Wiley, New York, and references cited therein.

[00100] Furthermore, the methods of this invention may employ compounds which contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of this invention, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents, and the like.

[00101] The substituted quinolin-4-ones of this disclosure can be prepared by the synthetic protocols illustrated in Scheme 1, where R¹, R², R³, R⁴ and R⁵ are as defined herein and LG is a leaving group. Scheme 1

1 -1 I

[00102] In Scheme 1, compound 1-1 is reacted with compound 1-2, where LG is a leaving group such as halogen, alkylsulfonate, hydroxyl, and the like, under standard substitution reaction conditions well known in the art. In one embodiment, the reaction is conducted in the presence of a suitable base in tetrahydrofuran, or other suitable solvent (e.g. DMF or diethyl ether), at room temperature or under elevated reaction temperatures. The reaction is continued until substantially complete which typically occurs within about 1 to 72 hours. Alternatively, the reaction can be performed at elevated temperatures in a microwave oven. Upon reaction completion, compound I can be recovered by conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration and the like.

Scheme 2

1 -1

[00103] In some embodiments, the substitution reaction conditions are Mitsunobu reaction conditions. For example, compound 1-1 is reacted with compound 1-3 and triphenylphosphine in tetrahydrofuran or other suitable solvent (e.g. diethyl ether) at 0 °C, and an azodicarboxylate such as diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD) is slowly added. The reaction mixture is then stirred for at room temperature for several hours. Upon reaction completion, compound I can be recovered by conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration and the like. [00104] In Scheme 1, compounds 1-1, 1-2 and 1-3 are commercially available or may be prepared by procedures, or obvious modifications thereof, described in USP 8,133,882, which is incorporated herein in its entirety.

EXAMPLES

Example 1. ASP 1645 is a Reversible P2Y12 Inhibitor

[00105] Eight-week-old male Hartley guinea-pigs were used in this study. All animals were fasted for 14-18 hours before oral administration of the drugs. ASP 1645 was dissolved in a half volume of distilled water by adding 1 M NaOH, then adjusted to 0.5% methylcellulose (MC) solution by adding another half volume of 1% MC solution. Clopidogrel was dissolved in a 0.5%> MC solution prior to use.

[00106] One hour before blood sampling, the test drugs (ASP1645, 3 mg/kg;

clopidogrel, 100 mg/kg) were administered through a gastric tube to conscious guinea pigs. While the animals were under diethyl ether anesthesia, 6 mL of blood were collected from the vena cava in a plastic syringe containing 3.2% trisodium citrate solution (10% of the final volume). Platelet-rich plasma (PRP) was prepared by centrifugation at 140-160g. Platelet-poor plasma (PPP) was obtained by centrifuging the PRP for 10 min at 1800g. Platelets in PRP were suspended in PPP to adjust the platelet count to 3 x 10⁵/μΕ. Platelet aggregation in PRP was induced by adding 2 μΜ of ADP, and measured using an aggregometer by recording the increase in light transmission through a stirred suspension maintained at 37 °C for 5 min. The inhibitory rate was calculated by dividing the area under the curve (AUC) of platelet aggregation in the test drug-treated animal samples by the average AUC in the vehicle -treated group samples.

[00107] The remaining PRP was then adjusted to pH 6.7 with 10 mM citric acid and 50 mM EDTA, and centrifuged at 600g for 15 min to obtain washed platelets. The sedimented platelets were suspended once in Tyrode's-HEPES buffer (3.8 mM HEPES, 137 mM NaCl, 2.7 mM KC1, 2.9 mM NaH2P04, 5.6 mM dextrose, pH 6.7) containing 0.35% bovine serum albumin and 50 mM EDTA. After centrifugation at 600g for 15 min, sedimented platelets were resuspended in PPP containing no drug to adjust the platelet count to 3 x 10⁵/μΕ. Platelet aggregation after the washing procedure was induced by adding 2 μΜ of ADP and was measured as described above.

[00108] The percent inhibition was calculated by dividing the mean value of platelet aggregation in the samples from treated animals by the mean value of platelet aggregation in the samples from animals treated only with vehicle. Data represent the mean effective dose (ed) over that determined for the 4 animals in each treatment group. Statistical analysis was performed using the paired t-test (before wash vs. after wash) for each drug. A value of P < 0.05 was regarded as significant.

[00109] Figure 1 shows the results of the study and demonstrates that the inhibitory activity of ASP 1645 on ADP-induced platelet aggregation disappeared completely after washing the platelets. In contrast, the inhibitory effect of clopidogrel on ADP-induced platelet aggregation did not change after washing the platelets. Thus, the study demonstrated that ASP 1645 is a reversible platelet inhibitor. The reversible nature of platelet inhibition with ASP 1645 has numerous advantages over the irreversible clopidogrel. For example, ASP 1645 is preferred over clopidogrel for use in patients expected to undergo a surgical procedure. Currently, clopidogrel is stopped about 7 days prior to surgery, whereas ASP 1645 treatment (or treatment with another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt form thereof) can be stopped only 2-3 days prior to surgery.

Example 2. ASP 1645 is a Potent Platelet Aggregation Inhibitor

[00110] ASP1645 was administered to guinea pigs for one hour at various doses (3 animals per dose). Platelet rich samples were obtained from the guinea pigs and ADP was added at two different concentrations for induction of platelet aggregation. No platelet washing was done. The results are shown in Figure 2. Data are expressed as the mean + SEM (SEM=statistical error of the mean) of 3 animals.

[00111] Increasing the ADP in the assay changed the percent inhibition of platelet aggregation by ASP 1645 in a concentration dependent manner. This shows that ASP 1645 is a potent inhibitor of platelet aggregation under varying conditions. In addition, the study showed a dose dependent increase in platelet aggregation inhibition with ASP 1645 at both ADP concentrations added to samples in this study.

Example 3. ASP 1645 Does Not Inhibit Cytochrome P450 Isozymes

[00112] ASP1645 was studied to determine if it inhibits cytochrome P450 isozymes in human liver microsomes. Many drug-drug interactions of other P2Y12 receptor antagonists are attributed to their inhibition of metabolism via major cytochrome P450 isozymes.

[00113] Specific prototypical substrates of human CYP isozymes were incubated individually with human liver microsomes in the presence of ASP 1645, and the metabolic activities were determined. The isozyme-specific metabolic activities were assessed using an index of remaining activity or IC50 values for ASP 1645. Substances known to be specific inhibitors of CYP isozymes were used as positive controls.

[00114] Human liver microsomes were diluted with buffer on an ice bath in the reaction mixtures for each isozyme assay (CYPl A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). These microsomes are referred to as diluted microsomes. Control samples were prepared to investigate the effects of ASP 1645 on the quantification of the prototypical substrate metabolite. Another control sample was prepared to confirm the retention times and peak area of the prototypical substrate metabolite and internal standard. Using these samples, it was investigated whether peaks derived from ASP 1645 or its metabolites (if any) interfere with the detection of the peaks for each prototypical substrate metabolite or internal standard.

[00115] At the highest concentration of 100 μΜ, the remaining activities were 98.2% for CYP1A2, 93.1% for CYP2C9, 86.5 % for CYP2C19, 85.0% for CYP2D6, 84.9% for CYP3A4 (testosterone), and 67.0% for CYP3A4 (midazolam). Because these remaining activities were more than 50%, it was considered that the IC50 values were more than 100 μΜ for the activities of CYPl A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4.

[00116] This study demonstrates that ASP1645 has only a very weak inhibitory effect on any metabolic reactions of the human CYP isozymes examined. This makes ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, superior to other P2Y12 receptor antagonists on the market and currently in development with respect to its potential for drug-drug interactions. The absence of significant drug-drug interactions makes it possible to co-administer ASP 1645 with other pharmaceutical agents without losing the effect of these other agents or increasing the effect of these other agents to the point of toxicity.

Example 4. Effect of ASP 1645 on Time to Occlusion and Bleeding Time

[00117] Another attribute of many P2Y12 receptor antagonists is that they can cause excessive bleeding times, which has resulted in black box warnings and reluctance by the medical community even to study these drugs outside of serious medical situations such as thrombosis in cardiology and stroke. In this study, ASP 1645 was assessed for its effect on bleeding times as well as its anti-thrombotic ability in comparison with clopidogrel. [00118] The study was conducted using male Hartley guinea pigs weighing 270.3- 315.1 g. All animals were fasted overnight before the experiment in the carotid arterial pinch-injury-induced thrombosis model.

[00119] This method is based on a previously described model [S. Roux et al, Thromb Haemost (1994)71 : 252-256] with some modification. ASP1645 (0.01, 0.03, 0.1, 0.3 mg/kg) or clopidogrel (1, 3, 10, 30 mg/kg) was administered 30 min or 2 h before thrombus induction, respectively, through a gastric tube to conscious guinea pigs. The animals were anesthetized with sodium pentobarbital (30 mg/kg, i.p.) 15 min before thrombus induction. While the animals were under pentobarbital anesthesia, a 2-3 cm segment of the left carotid artery was isolated, and a doppler flow probe was placed around the vessel. Arterial injury was induced just distal to the flow probe by pinching the vessel with a surgical forceps for 10 s, and then releasing. The time to occlusive thrombus formation was defined as the time required for blood flow to stop completely. If blood flow continued for 20 min, 20 min was the value recorded for statistical analysis.

[00120] After the animals were used in the thrombosis model, they were immediately used in the bleeding model there. An incision was made 1 mm from the root of a nail on the left foreleg using surgical scissors. The blood was then blotted with filter paper every 30 sec. The amount of time that passed before no blood could be observed on clean filter paper pressed to the cut was regarded as the bleeding time. Five animals were in each group. Statistical analyses were performed using the Steel test (vehicle group vs. the ASP 1645 groups, vehicle group vs. the clopidogrel groups). A p value of less than 0.05 was considered significant.

[00121] Figures 3 and 4 show the effects of ASP 1645 and clopidogrel on the arterial thrombosis, respectively. These agents prolonged the time to occlusion dose- dependently. The effect was statistically significant at 0.03 mg/kg or higher for ASP1645 and at 3 mg/kg or higher for clopidogrel. Figures 5 and 6 show the effects of ASP 1645 and clopidogrel on the bleeding time, respectively. Oral administration of 0.3 mg/kg of ASP 1645 or 30 mg/kg of clopidogrel caused significant prolongation of the bleeding time.

[00122] In this study, ASP 1645 and clopidogrel showed anti-thrombotic activity against arterial thrombosis without bleeding time prolongation at 0.03 mg/kg and 3 mg/kg, respectively. The minimal effective dose of ASP 1645 against the arterial thrombosis was significantly lower than that of clopidogrel. This study showed that ASP 1645 is a potent anti-thrombotic and that bleeding times with ASP 1645 should be comparable with clopidogrel at therapeutically efficacious doses. The importance of comparable bleeding times is that this makes ASP 1645 useful in both cardiovascular disease and stroke as well as other inflammatory indications. These results also demonstrate that ASP 1645 is especially useful prior to surgery or during or immediately following a PCI procedure as described above.

Example 5. Phototoxicity of ASP 1645

[00123] Oral phototoxicity was evaluated using male guinea pigs. Each group of 10 animals received vehicle or 10, 30 or 100 mg/kg ASP1645, or a positive control for phototoxicity. After dosing, animals were irradiated with ultraviolet A at a dose of about 10J/cm2. The actual duration of exposure to reach this level of irradiation was 40 minutes and 30 seconds. The results demonstrated that no phototoxicity was observed at an ASP 1645 dose of 10 mg/kg. At a dose of 30 mg/kg, 3 of 10 animals showed positive reaction of phototoxicity. At a dose of 100 mg/kg, all 10 animals showed phototoxicity.

[00124] Phototoxicity limits the highest doses that can be routinely and safely used in humans. Given the chronic nature of dosing for acute coronary syndrome and percutaneous coronary intervention, phototoxicity could be deleterious to many patients. Given the need for both efficacy and safety, daily doses of ASP 1645 for platelet aggregation inhibition for cardiovascular disease are in the range of 12.5 mg bid (25 mg qd) to about 150 mg qd.

Example 6. ASP 1645 Does Not Induce Dyspnea

[00125] A common side effect seen with certain P2Y12 receptor antagonists is dyspnea. One explanation for dyspnea with these compounds is that they behave as leukotriene D4 receptor agonists. The leukotriene D4 receptor is known to be important in bronchoconstriction when leukotriene D4 binds this receptor. Leukotriene D4 antagonists have been developed to alleviate the bronchoconstriction caused by the agonist activity of leukotriene D4. ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, do not have this side effect. In vitro receptor binding assays demonstrated that ASP 1645 inhibits binding of 2MeS-ADP to the P2Y12 receptor with an IC50 of 4.2 nM. ASP 1645 had an IC50 in the micromolar range with respect to its binding to the leukotriene D4 receptor. Thus, the prinicipal effect of ASP 1645, inhibition of P2Y12 receptor, occurs at an IC50 that is 2400 times lower than that for leukotriene D4 receptor binding, one activity associated with the dyspnea side effect. In this study, ASP 1645 had no agonistic or antagonistic effect on human CysLTl or human CysLT2 (collectively, the leukotriene D4 receptors).

[00126] A second mechanism for the dyspnea side effect observed with certain P2Y12 inhibitors, especially ticagrelor, is the block of reuptake of adenosine by red blood cells. This results in increased adenosine, which causes dyspnea by activation of cell surface adenosine receptors. Safety pharmacology studies with ticagrelor showed a dose dependent increase in respiratory rates. ASP 1645 demonstrated no changes in respiratory rate with increasing doses in safety pharmacology studies in both monkeys and rats. In addition, ASP 1645 did not bind the adenosine receptor where ticagrelor bound this receptor. ASP 1645 is not an adenosine analog and does not exhibit properties of adenosine analogs. Thus, both from receptor binding studies and safety pharmacology studies, ASP 1645 demonstrates no potential for causing dyspnea. This property differentiates ASP 1645 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt form thereof, from other P2Y12 receptor antagonists, making it a safer drug for patients with asthma or COPD.

Example 7. ASP 1645 Reduces P-selectin Expression

[00127] Asthma and COPD are respiratory diseases caused by chronic

inflammation that affects more than 300 million patients worldwide. Both diseases are associated with reduced airflow due to accumulation of immune cells in tissues of the lung. Leukocyte adhesion to vascular endothelial cells and subsequent extravasation into the peribronchial space triggers the inflammatory response. The initial adhesion process, called "leukocyte rolling", is a cascade of adhesion events which is primarily controlled through the binding of carbohydrate moieties of transmembrane glycoproteins on the leukocytes to selectins present on the vascular endothelium. There are three distinct selectin proteins. E-selectin and P-selectin are found on endothelial cells, whereas L- selectin is found on granulocytes, monocytes, and many types of lymphocytes. The initial rolling step is mediated by the interaction of leukocyte glycoproteins containing active moieties such as sialyl Lewisx (sLex) with P-selectin expressed on endothelial cells. Inhibition of this interaction, including by inhibiting P-selectin expression, provides a treatment of inflammatory diseases such as asthma and arthritis.

[00128] This example describes a study of the effect of ASP 1645 on P-selectin expression on ADP-stimulated human platelets in comparison with that of abciximab (ReoPro^®). P-selectin is rapidly expressed on the surface of newly activated platelets following activation and is widely utilized as a marker of platelet activation. P-selectin has been demonstrated to play a pivotal role in vascular inflammation and injury, thereby acting as a link between inflammation and thrombosis. P-selectin expression was inhibited by ASP 1645 in a concentration-dependent manner (see Figure 7). The IC50 value of ASP 1645 was 0.11 μΜ. In contrast, abciximab had no effect on ADP-induced P- selectin expression at 1 μΜ. As activated platelets secrete a number of inflammatory substances into the local microenvironment, this result shows that ASP 1645 inhibits ADP-induced granule secretion by inhibiting P-selectin expression.

[00129] In addition, as mentioned above, activated platelets can interact with leukocytes. ASP 1645 more effectively reduced adhesion of activated platelets to monocytes (see Figure 8) and granulocytes (see Figure 9) compared with abciximab. The inhibition of leukocyte-platelet aggregates mitigates inflammatory processes and thereby the development of atherosclerosis, asthma, and other inflammatory diseases. Thus, this study demonstrates that ASP 1645 reduces P-selectin expression and is useful for the treatment of inflammatory diseases such as atherosclerosis, asthma, COPD, and psoriasis. Example 8. Therapeutically Effective QD Administration of ASP 1645

[00130] Once daily dosing is an advantage for any drug, and is especially advantageous for drugs used to treat patients with cardiovascular diseases or stroke.

Patient compliance is highly dependent on the number of daily doses. The more doses per day, the lower the patient compliance. Lack of patient compliance with respect to medications for platelet aggregation inhibition leads to thrombotic events. Ticagrelor dosing is twice daily. Figure 10 shows the results of a study involving a single

administration of ASP 1645 to demonstrate the efficacy of QD (once daily) administration of ASP 1645. In the study, ASP 1645 and ticagrelor were compared with respect to inhibition of ex vivo ADP-induced platelet aggregation at 12, 24, and 48 hours after cynomolgus monkeys were given one of three dose levels of either drug. Data represent the mean +SEM of 3 separate experiments. The 1 mg/kg dose for both ASP 1645 and ticagrelor demonstrated that the inhibition of platelet aggregation was about twice as long with ASP 1645 as with ticagrelor. This result demonstrates that ASP 1645 can be administered using once daily dosing versus the twice daily dosing required for ticagrelor. In addition, the highest level of ASP 1645 (3 mg/kg) tested in monkeys gave about 100% platelet aggregation inhibition. Translationally, this dose is in excess of a 200 mg dose in humans. Example 9. Rapid Onset Rate of ASP 1645

[00131] Rapid rate of onset is important for platelet aggregation inhibitors used in conjunction with PCI. Thrombotic events occur quickly following balloon angioplasty or stent insertion. In general, platelet aggregation inhibitors are given throughout the procedure and at relatively high loading doses prior to PCI to achieve maximal platelet aggregation inhibition. In studies of absorption, ASP 1645 was rapidly absorbed, and concentrations were observed to appear in the bloodstream in a dose-dependent manner when orally administered, only slightly delayed from the rate of increase in plasma concentration observed with intravenous dosing. Rapid platelet aggregation inhibition was also demonstrated at all doses, as shown in Figure 10. Thus, the data demonstrate a very rapid rate of onset for ASP 1645.

Example 10. Compound Synthesis

[00132] The compounds and pharmaceutically acceptable salts described herein can be prepared by methods known in the art, including those described in U.S. Patent No. 8,133,882, which is incorporated herein by reference. A representative synthesis is provided below.

Example 10(1)

[00133] 213 μΐ of diisopropyl azodicarboxylate was added to a 5.0 ml

dichloromethane solution of 263 mg of benzyl (2R)-2-hydroxy-3-phenylpropanoate and 270 mg of triphenylphosphine at 0°C, followed by stirring for 15 minutes. Then, 177 mg of 7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-3-hydroxyquinolin-4(lH)-one was added thereto, followed by stirring at room temperature for 4 hours. Water was added to the reaction mixture, followed by extraction with EtOAc and washing with aqueous saturated sodium chloride. After drying over anhydrous sodium sulfate and subsequent evaporation under a reduced pressure, the residue was purified by silica gel column chromatography to obtain 160 mg of benzyl (2S)-2-{[7-(cyclohexylamino)-l-cyclopentyl-6-fluoro-4-oxo- 1 ,4-dihydroquinolin-3-yl]oxy} -3-phenylpropanoate.

Example 10(2)

[00134] 606 mg of diisopropyl azodicarboxylate were added to a 10 ml

dichloromethane solution of 313 mg of methyl (2R)-2-hydroxypropanoate and 786 mg of triphenylphosphine at 0°C, followed by stirring for 15 minutes. Then, 344 mg of 7- (cyclohexylamino)-l-cyclopentyl-6-fluoro-3-hydroxyquinolin-4(lH)-one was added thereto, followed by stirring at room temperature overnight. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to obtain 387 mg of methyl (2S)-2-{[7-(cyclohexylamino)-l- cyclopentyl-6-fluoro-4-oxo-l,4-dihydroquinolin-3-yl]oxy}propanoate.

[00135] ASP 1645 can be prepared as shown in Scheme 3, where PG is a suitable protecting group such as lower alkyl (e.g., methyl), according to the above procedure using the appropriate starting materials. The reaction can be conducted as described hereinabove, followed by basic hydrolysis to remove the protecting group using, for example, aqueous sodium hydroxide to afford the corresponding carboxylic acid

ASP 1645.

Scheme 3

[00136] The invention, having been described in detail and illustrated by example, is claimed below.

Claims

1. A pharmaceutical formulation for use in treating and/or preventing cardiovascular disease, stroke, and/or other inflammatory diseases or conditions in a patient in need thereof comprising a therapeutically effective dose of a reversible P2Y12 receptor antagonist of Formula 1 :

Formula 1 wherein

R is hydrogen or halogen;

R⁶ is -C0₂R⁷, -C(0)NHOH, -C(0)N(R⁷) ₂, or -C0₂-lower alkylene-aryl; and R⁷ is hydrogen or lower alkyl

or an active metabolite, prodrug, ester, or salt thereof.

2. The pharmaceutical formulation of claim 1, wherein the dose is a dose in a range of 5 mg to 200 mg per day.

3. The pharmaceutical formulation of claim 2, wherein the dose is administered qd or bid.

4. The pharmaceutical formulation of claim 3, wherein the dose is in a range of 25 to 150 mg per day, and the patient has cardiovascular disease.

5. The pharmaceutical formulation of claim 3, wherein the dose is in a range of 25 to 150 mg per day, and the patient has had a stroke or is at risk of having a stroke.

6. The pharmaceutical formulation of claim 4, wherein the cardiovascular disease is acute coronary syndrome.

7. The pharmaceutical formulation of claim 4, wherein the patient has been diagnosed as needing percutaneous coronary intervention (PCI) or has had PCI.

8. The pharmaceutical formulation of claim 3, wherein the dose is in a range of 5 to 150 mg per day and the patient has an inflammatory disease or condition other than, or in addition to, cardiovascular disease or stroke.

9. The pharmaceutical formulation of claim 8, wherein the inflammatory disease or condition is selected from the group consisting of asthma and chronic kidney disease.

10. The pharmaceutical formulation of any of claims 1 to 9, wherein the compound of Formula 1 is ASP 1645.

11. A method for treating and/or preventing cardiovascular disease, stroke, and/or other inflammatory diseases or conditions in a patient in need thereof comprising administering to the patient a therapeutically effective dose of a reversible P2Y12 receptor antagonist of Formula 1 :

Formula 1 wherein

R is hydrogen or halogen;

or an active metabolite, prodrug, ester, or salt thereof.

12. The method of claim 11 , wherein the dose is a dose in a range of 5 mg to 200 mg per day.

13. The method of claim 12, wherein the dose is administered qd or bid.

14. The method of claim 13, wherein the dose is in a range of 25 to 150 mg per day, and the patient has cardiovascular disease.

15. The method of claim 13, wherein the dose is in a range of 25 to 150 mg per day, and the patient has had a stroke or is at risk of having a stroke.

16. The method of claim 14, wherein the cardiovascular disease is acute coronary syndrome.

17. The method of claim 14, wherein the patient has been diagnosed as needing percutaneous coronary intervention (PCI) or has had PCI.

18. The method of claim 13, wherein the dose is in a range of 5 to 150 mg per day and the patient has an inflammatory disease or condition other than, or in addition to, cardiovascular disease or stroke.

19. The method of claim 18, wherein the inflammatory disease or condition is selected from the group consisting of asthma and chronic kidney disease.

20. The method of any of claims 11 to 19, wherein the compound of Formula 1 is ASP 1645.