OA12256A - Sodium-hydrogen exchanger type 1 inhibitor (NHE-1). - Google Patents

Abstract

NHE-1 inhibitor, methods of using the NHE-1 inhibitor and pharmaceutical compositions containing the NHE-1 inhibitor. The NHE-1 inhibitor is useful for the reduction of tissue damage resulting from tissue ischemia.

Description

012256 -1- • SODIUM-HYDROGEN EX CHANGER TYPE 1 INHIBITOR (NHE-1) 1

BACKGROUND OF INVENTION

This invention relates to a sodium-hydrogen exchanger type 1 (NHE-1)inhibitor. 5 Myocardial ischémie injury can occur in out-patient as well as in perioperative settings and can lead to the development of sudden death,myocardial infarction or congestive heart failure. There is an unmet medical needto prevent or minimize myocardial ischémie injury, particularly perioperativemyocardial infarction. Such a therapy is anticipated to be life-saving and can 10 reduce hospitalizations, enhance quality of life and reduce overall health care costsof high risk patients.

Pharmacological cardioprotection would reduce the incidence andprogression of myocardial infarction and dysfunction occurring in these surgicalsettings (perioperatively). In addition to reducing myocardial damage and 15 improving post-ischemic myocardial function in patients with ischémie heart disease, cardioprotection would also decrease the incidence of cardiac morbidityand mortality due to myocardial infarction and dysfunction in patients “at risk” (suchas greater than 65 years, exercise intolérant, coronary artery disease, diabètesmellitus, hypertension) that require non-cardiac surgery. 20 The mechanism(s) responsible for the myocardial injury observed after ischemia and reperfusion is not fully understood. A variety of publications hâve disclosed the use of guanidine dérivatives asuseful for the treatment of, for example, arrhythmias.

A recent published patent application, PCT/IB99/00206 published as WO 25 99/43663 on September 2, 1999, the disclosure of which is hereby incorporated by reference, discloses a variety of NHE-1 inhibitors including [5-cyclopropy,-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine. The publication further States * that “preferred salts of the immediately preceding compound are the mono- or di-mesylate salts." One group of preferred compounds, which include 30 hydroxyquinoline compounds, is described in claim 102 of the published application. Of course, some of these hydroxyquinoline compounds can exist in several tautomeric forms such as the quinolone form as described in that patent application. In addition, commonly assigned U.S. provisional application serial no. 012256 -2- 60/162,374 was filed on October 29, 1999 and is directed to crystal forms of the » i above described NHE-1 inhibitor. I PCT/JP97/04650 application published on June 25, 1998 discloses N- [(substituted five-membered heteroaryl)]guanidine compounds to be useful as 5 inhibrtors of Na+/H+ exchange and consequently effective for the treatment ofvarious diseases such as hypertension, arrhythmia, angina pectoris, myocardialinfarct, arteriosclerosis, and complications of diabètes.

Thus, there is cleariy a need and a continuing search in this field of art forcompounds for the treatment of perioperative myocardial ischemia.

10 SUMMARY OF THE INVENTION

This invention is directed to a compound of Formula I

15 a prodrug thereof, or a pharmaceutically acceptable sait of said compound or ofsaid prodrug, with the proviso that [5-cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine is not inciuded.

As used herein the phrase “a prodrug thereof, or a pharmaceuticallyacceptable sait of said compound or of said prodrug" includes the proviso that [5- 20 cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine is not inciuded.

This invention is also directed to substantially pure [5-cyclopropyl-1-(2- quinolon-5-yl}-1H-pyrazole-4-carbonyl]guanidine, or a pharmaceutically acceptable sait of said compound. 01 2256 -3-

Altematively, the above compound is named [5-cyclopropyl-1-(2-quinolon-5-yl)-1H-pyrazole-4-carbonyl]guanidine. A preferred sait is the hydrochloride sait, andmost preferably, the monohydrochloride sait.

Another aspect of this invention is a method of treating a mammal (e.g., 5 human) having a disease or condition mediated by NHE-1 by administering a therapeutically effective amount of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug to themammal.

Another aspect of this invention is directed to a method of reducing tissue10 damage (e.g., substantially preventing tissue damage, inducing tissue protection)resulting from ischemia comprising administering to a mammal (e.g., a female or male human) in need of such treatment a therapeutically effective amount of theFormula I compound, a prodrug thereof, or a pharmaceutically acceptable sait ofsaid compound or of said prodrug. 15 Preferred ischémie tissues taken individually or as a group are cardiac, brain, liver, kidney, lung, gut, skeletal muscle, spleen, pancréas, nerve, spinal cord,retina tissue, the vasculature, or intestinal tissue.

An especially preferred ischémie tissue is cardiac tissue.

It is especially preferred that the Formula I compound, a prodrug thereof, or 20 a pharmaceutically acceptable sait of said compound or of said prodrug isadministered to prevent perioperative myocardial ischémie injury.

Preferably, the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug isadministered prophylactically. 25 The ischémie damage may occur during organ transplantation either to the organ or the patient.

Preferably, the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug isadministered prior to, during and/or shortly after cardiac surgery or non-cardiac 30 surgery.

In one aspect of this invention the Formula I compound, a prodrug thereof,or a pharmaceutically acceptable sait of said compound or of said prodrug isadministered locally. 012256 -4- A preferred dosage is about 0.01 to 100 mg/kg/day of the Formula Icompound, a prodrug thereof, or a pharmaceutically acceptable sait of saidcompound or of said prodrug. An especially preferred dosage is about 0.01 to 50mg/kg/day of the Formula I compound, a prodrug thereof, or a pharmaceuticallyacceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method of reducingmyocardial tissue damage (e.g., substantially preventing tissue damage, inducingtissue protection) during surgery (e.g., coronary artery bypass grafting (CABG)surgeries, vascular surgeries, percutaneous transluminal coronary angioplasty(PTCA), organ transplantation, or other non-cardiac surgeries) comprisingadministering to a mammal (e.g., a female or male human) a therapeuticallyeffective amount of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method of reducingmyocardial tissue damage (e.g., substantially preventing tissue damage, inducingtissue protection) in patients presenting with ongoing cardiac (acute coronarysyndromes, e.g. myocardial infarction or unstable angina) or cérébral ischémieevents (e.g., stroke) comprising administering to a mammal (e.g., a femaie or malehuman) a therapeutically effective amount of the Formula I compound, a prodrugthereof, or a pharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a chronic method of reducingmyocardial tissue damage (e.g., substantially preventing tissue damage, inducingtissue protection) in a patient with diagnosed coronary heart disease (e.g., previousmyocardial infarction or unstable angina) or patients who are at high risk formyocardial infarction (e.g., âge > 65 and two or more risk factors for coronary heartdisease) comprising administering to a mammal (e.g., a female or male human) atherapeutically effective amount of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method of preventingischémie damage comprising the chronic oral administration to a mammal inneed of such treatment a therapeutically effective amount of the Formula Icompound, a prodrug thereof, or a pharmaceutically acceptable sait of saidcompound or of said prodrug. 012256 -5-

Another aspect of this invention is directed to a method for treatingcardiovascular diseases comprising administering to a mammal (e.g., a female ormale human) a therapeutically effective amount of the Formula I compound, aprodrug thereof, or a pharmaceutically acceptable sait of said compound or of saidprodrug.

Another aspect of this invention is directed to a method for treatingarteriosclerosis comprising administering to a mammal (e.g., a female or malehuman) a therapeutically effective amount of the Formula I compound, a prodrugthereof, or a pharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treatinghypertension comprising administering to a mammal (e.g., a female or malehuman) a therapeutically effective amount of the Formula I compound, a prodrugthereof, or a pharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treatingarrhythmia comprising administering to a mammal (e.g., a female or male human) atherapeutically effective amount of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treating anginapectoris comprising administering to a mammal (e.g., a female or male human) atherapeutically effective amount of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treating cardiachypertrophy comprising administering to a mammal (e.g., a female or male human)a therapeutically effective amount of the Formula I compound, a prodrug thereof, ora pharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treating rénaldiseases comprising administering to a mammal (e.g., a female or male human) atherapeutically effective amount of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treating diabeticcomplications comprising administering to a mammal (e.g., a female or malehuman) a therapeutically effective amount of the Formula I compound, a prodrugthereof, or a pharmaceutically acceptable sait of said compound or of said prodrug. 012256 -6-

Another aspect of this invention is directed to a method for treatingrestenosis comprising administering to a mammal (e.g., a female or male human) atherapeutically effective amount of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treating diseasesof cell prolifération comprising administering to a mammal (e.g., a female or malehuman) a therapeutically effective amount of the Formula I compound, a prodrugthereof, or a pharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treatingcancerous diseases comprising administering to a mammal (e.g., a female or malehuman) a therapeutically effective amount of the Formula I compound, a prodrugthereof, or a pharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treating fibroticdiseases comprising administering to a mammal (e.g., a female or male human) atherapeutically effective amount of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treatingglomerular nephrosclerosis comprising administering to a mammal (e.g., a femaleor male human) a therapeutically effective amount of the Formula I compound, aprodrug thereof, or a pharmaceutically acceptable sait of said compound or of saidprodrug.

Another aspect of this invention is directed to a method for treatingpulmonary fibrosis comprising administering to a mammal (e.g., a female or malehuman) a therapeutically effective amount of the Formula I compound, a prodrugthereof, or a pharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treating cerebroischémie disorders comprising administering to a mammal (e.g., a female or malehuman) a therapeutically effective amount of the Formula I compound, a prodrugthereof, or a pharmaceutically acceptable sait of said compound or of said prodrug.

Another aspect of this invention is directed to a method for treatingmyocardial stunning comprising administering to a mammal (e.g., a female or malehuman) a therapeutically effective amount of a the Formula I compound, a prodrugthereof, or a pharmaceutically acceptable sait of said compound or of said prodrug. 012256 -7-

Another aspect of this invention is directed to a method for treatingmyocardial dysfunction comprising administering to a mammal (e.g., a female ormale human) a therapeutically effective amount of the Formula I compound, aprodrug thereof, or a pharmaceutically acceptable sait of said compound or of said 5 prodrug.

Another aspect of this invention is directed to a method for treatingcerebrovascular diseases comprising administering to a mammal (e.g., a female ormale human) a therapeutically effective amount of the Formula I compound, aprodrug thereof, or a pharmaceutically acceptable sait of said compound or of said 10 prodrug.

Another aspect of this invention is directed to a method for treating organhypertrophies or hyperplasias comprising administering to a mammal (e.g., afemale or male human) a therapeutically effective amount of the Formula Icompound, a prodrug thereof, or a pharmaceutically acceptable sait of said 15 compound or of said prodrug.

This invention is also directed to pharmaceutical compositions whichcomprise an amount of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug and apharmaceutically acceptable carrier, vehicle or diluent. 20 This invention is also directed to pharmaceutical compositions for the réduction of tissue damage resulting from ischemia which comprise atherapeutically effective amount of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug and apharmaceutically acceptable carrier, vehicle or diluent. 25 The term “réduction” is intended to include partial prévention or prévention which, although greater than that which would resuit from taking no compound orfrom taking a placebo, is less than 100%, in addition to substantially totalprévention.

The term “damage resulting from ischemia" as employed herein refers to 30 conditions directly associated with reduced blood flow to tissue, for example due toa clôt or obstruction of blood vessels which supply blood to the subject tissue andwhich resuit, inter alia. in lowered oxygen transport to such tissue, impaired tissueperformance, tissue dysfunction and/or necrosis. Alternatively, where blood flow ororgan perfusion may be quantitatively adéquate, the oxygen carrying capacity of 012256 -8- the blood or organ perfusion medium may be reduced, e.g., in hypoxie environment, such that oxygen supply to the tissue is lowered, and impaired tissueperformance, tissue dysfunction, and/or tissue necrosis ensues.

The term "treating", "treat" or "treatment" as used herein includespreventative (e.g., prophylactic) and palliative treatment.

The expression "pharmaceutically-acceptable sait" refers to nontoxic anionicsalts containing anions such as (but not limited to) chloride, bromide, iodide, sulfate,bisulfate, phosphate, acetate, maleate, fumarate, oxalate, lactate, tartrate, citrate,gluconate, methanesulfonate (mesylate) and 4-toluene-sulfonate. Since more thanone basic moiety exists the expression may include multiple salts (e.g., di-salt). Theexpression also refers to nontoxic cationic salts such as (but not limited to) sodium,potassium, calcium, magnésium, ammonium or protonated benzathine (N,N-dibenzylethylenediamine), choline, ethanolamine, diethanolamine, ethylenediamine,meglamine (N-methyl-glucamine), benethamine (N-benzylphenethylamine),piperazine or tromethamine (2-amino-2-hydroxymethyl-1,3-propanediol).

The expression "prodrug" refers to compounds that are drug precursors whichfollowing administration, release the drug in vivo via some Chemical or physiologicalprocess (e.g., a prodrug on being brought to the physiological pH or through enzymeaction is converted to the desired drug form). Again, there is a proviso that [5-cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine is not included.

By "pharmaceutically acceptable" it is meant the carrier, diluent, excipients,and/or sait must be compatible with the other ingrédients of the formulation, andnot deleterious to the récipient thereof.

As used herein, the expressions "reaction-inert solvent” and "inert solvent"refers to a solvent or mixture of solvents which does not interact with startingmaterials, reagents, intermediates or products in a manner which adversely affectsthe yield of the desired product.

It will be recognized that the compound of this invention can exist inradiolabelled form, i.e., the compound may contain one or more atoms containingan atomic mass or mass number different from the atomic mass or mass numberordinarily found in nature. Radioisotopes of hydrogen and carbon include 2H, 3H,and 14C respectively. A compound of this invention, which contains thoseradioisotopes and/or other radioisotopes of other atoms are within the scope of thisinvention. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, radioisotopes are particularly 012256 -9- preferred for their ease of préparation and detectability. A radiolabelled Formula Icompound can generally be prepared by methods well known to those skified in theart. Conveniently, such radiolabelled compounds can be prepared by carrying outthe procedures disclosed in the Schemes and/or in the Examples below bysubstituting a readily available radiolabelled reagent for a non-radiolabelledreagent.

Other features and advantages will be apparent from the spécification anddaims which describe the invention.

DETAILED DESCRIPTION OF THE INVENTION

The compound of Formula I is a human métabolite of 5-cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine. Itwas identified from samples ofhuman plasma obtained after intravenous administration of 5-cydopropyl-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine to human subjects. The structureof this compound was independently verified by Chemical synthesis. In anotheraspect of this invention [5-cyclopropyl-1 -(2-quinolon-5-yl)-1 H-pyrazole-4-carbonyl]guanidine may be prepared by administering [5-cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine to a human and isoiating the desiredmétabolite from the plasma. Altematively, the métabolite need not be isolated fromthe human since it is produced in vivo.

In general the compound of this invention can be made by processes whichinclude processes analogous to those known in the Chemical arts, particularly inlight of the description contained herein. Certain processes for the manufacture ofthe compound of this invention are provided as further features of the invention andare illustrated by the following réaction scheme. Other processes are described inthe experimental section. A detailed description of the synthetic aspect of the invention follows: 012256 -10-

According ίο the scheme above, the formula IA compound (prepared asdescribed in Capps, J. D.; Hamilton, C. S. J. Am. Chem. Soc. 1938, 60,2104) is 5 dissolved in a protic solvent such as éthanol and treated with an appropriatereducing agent such as stannous chloride dihydrate. The resulting mixture isstirred at a température of about 0 °C to about 115 °C for about 30 minutes toabout 24 hours. The resulting mixture is cooled to about 23 °C and filtered. Theresulting solid material is purified in a suitable way, such as by triturating with 10 hydrochloric acid (for example, 1 M aqueous) at about 100 °C for about 1 hour,cooling to about 23 °C and filtering to provide the formula II compound as itshydrochloride sait. Alternatively, the formula II compound can be isolated from thereaction mixture as the free base by basifying with an inorganic base, andextracting with an appropriate organic solvent. The formula II compound can also 15 be isolated as other salts. Other réduction methods that will perform this réduction °’2256 -11- are known to those skilled in the art, such as for example catalytic hydrogénation.

The formula II compound is diazotized in hydrochloric acid and water using sodium nitrite at about 0 °C for about 15 minutes to about 2 hours. The resulting diazonium sait solution is reduced with an appropriate reducing agent, such as* stannous chloride dihydrate in hydrochloric acid and water at about 0 °C to about23 °C for about 30 minutes to about 6 hours. The resulting solid is collected byfiltration and purified in a suitable manner, such as by trituration with hydrochloricacid (for example, 1 M aqueous) at about 23 °C for about 30 minutes. Thecompound of formula III is collected by filtration as its hydrochloride sait.Altematively, the formula III compound can be isolated from the réaction mixture asthe free base by basifying, with an inorganic base, and extracting with anappropriate organic solvent. The formula III compound can also be isolated asother salts. Other reducing agents that will perform this transformation are knownto those skilled in the art.

The formula IV compound is prepared by methods known to those skilled inthe art, such as by the reaction of an Ν,Ν-dialkylformamide dialkylacetal, forexample, Ν,Ν-dimethylformamide dimethylacetal with an ester of 3-cyclopropyl-3-oxopropanoic acid at a température of about 23 °C to about 115 °C for about 1hour to about 4 hours with or without an acid catalyst. The group R1 and R2 areconveniently any alkyl, cycloalkyl, cycloalkylalkyl, or arylalkyl group. Furthermore,the two R2 groups may be tethered together to form a cyclic entity.

The formula IV compound is reacted with the formula III compound in analcoholic solvent such as éthanol at a température of about 23 °C to about 115 °Cfor about 15 minutes to about 12 hours. When the formula III compound is used asits hydrochloride sait, it is advantageous to carry out the reaction in the presence ofan excess of a non-nucleophilic base, such as triethylamine. The formula Vcompound is collected by filtration. Altematively, the formula V compound may beisolated by other methods such as concentration followed by addition of water andextraction with a suitable organic solvent. Altematively, the formula V compoundmay be prepared using the formula III compound and other compounds in place ofthe formula IV compound, such as a compound in which the (R2)2N group isreplaced by a R2O group.

The formula V compound is hydrolyzed with a base such as sodium orlithium hydroxide in a solvent such as water, and/or methanol, and/or THFconveniently at about 23 °C or at an elevated température such as at reflux 012256 -12- y température for about 30 minutes to about 12 hours. The formula VI acid is then isolated by, for example, removing the organic solvents, acidifying and filtering. e Alternative^, the formula VI compound can be isolated from the reaction mixture by removing the organic solvents, acidifying and extracting with an appropriate organic 5 solvent.

The Formula VI acid is coüpled with guanidine in the presence of a suitablecoupling agent. A suitable coupling agent is one which transforms a carboxylic acidinto a reactive species which forms an amide linkage on reaction with an amine.

The coupling agent may be a reagent which effects this condensation in a10 one pot process when mixed together with the carboxylic acid and guanidine.

Exemplary coupling reagents are 1-(3-dirnethylaminopropyl)-3-ethylcarbodiimidehydrochloride-hydroxybenzotriazole (EDC/HBT), dicyclohexylcarbodiimide(DCC)/hydroxybenzotriazole(HBT), 2-ethoxy-1 -ethoxycarbonyl-1,2-dihydroquinoline(EEDQ), and diethylphosphorylcyanide. The coupling is performed in an inert 15 solvent, preferably an aprotic solvent at a température of about -20°C to about50°C for about 1 to about 48 hours, in the presence of excess guanidine as base.Exemplary solvents include acetonitrile, dichloromethane, dimethylsulfoxide,dimethylformamide, chloroform and mixtures thereof.

The coupling agent may also be that agent which converts the carboxylic 20 acid to an activated intermediate which is isolated and/or formed in a first step andallowed to react with guanidine in à second step. Examples of such couplingagents and activated intermediates are thionyl chloride or oxalyl chloride to formthe acid chloride, cyanuric fluoride to form an acid fluoride or an alkyl chloroformatesuch as isobutyl or isopropenyl chloroformate or propanephosphonic anhydride 25 (propanephosphonic acid anhydride, PPA) with a tertiary amine base to form amixed anhydride of the carboxylic acid, or carbonyldiimidazole to form anacylimidazole. If the coupling agent is oxaiyl chloride, it is advantageous to employa small amount of dimethylformamide as cosolvent with another solvent (such asdichloromethane) to catalyze the formation of the acid chloride. This activated acid 30 dérivative may be coupled by mixing with the intermediate in an appropriate solventtogether with an appropriate base. Appropriate solvent/base combinations are forexample, dichloromethane, dimethylformamide or acetonitrile or mixtures thereof inthe presence of excess guanidine as base. Other appropriate solvent/basecombinations include water or a ((CrCs)alcohol) or a mixture thereof together with 35 a cosolvent such as dichloromethane, tetrahydrofuran or dioxane and a base such °'2256 -13- as sodium, potassium or lithium hydroxide in sufficient quantity to consume the acidliberated in the reaction. Use of these coupling agents and appropriate sélection ofsolvents and températures are known to those skilled in the art or can be readilydetermined from the literature in light of this disclosure. These and other 5 exemplary conditions useful for coupling carboxylic acids are described in Houben-Weyl, Vol XV, part II, E. Wunsch, Ed., G. Theime Verlag, 1974, Stuttgart; M.Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984; and ThePeptides, Analysis, Synthesis and Biology (ed. E. Gross and J. Meienhdfer), vols 1-5 (Academie Press, NY 1979-1983). 10 In one embodiment, the formula VI acid is activated with excess thionyl chloride at a température of about reflux température for about 15 minutes to about3 hours and the excess thionyl chloride is removed by concentration. The resultingacid chloride is combined with excess guanidine hydrochloride and an inorganicbase, such as sodium hydroxide in tetrahydrofuran and water. The reaction is 15 stirred conveniently at 23 °C or at an elevated température, such as reflux température, for about 30 minutes to about 6 hours. The formula I compound isisolated form this reaction mixture in a variety of ways. For example, the reactionmixture is concentrated to remove the THF; the aqueous layer is acidified to pH 9,and the solid is collected by filtration. Altematively, the formula I compound may be 20 isolated by extraction with an organic solvent. The formula I compound may betransformed to the corresponding hydrochloride sait by treating a solution of it inmethanol with hydrogen chloride in ether, and collection of the resulting solid by filtration or concentration. Other salts may be prepared by analogous methods.

Those skilled in the art will recognize that it is also possible to transform the 25 formula V compound directly to the formula I compound by treating the formula Vcompound with excess guanidine in an inert solvent, such as an alcoholic solvent,for example éthanol, or in the absence of a solvent, at about 60 °C to about 150 °C.

Those skilled in the art will recognize that the Formula I compound can existin several tautomeric forms. Ail such tautomeric forms are considered as part of 30 this invention. For example, ail of the tautomeric forms of the carbonylguanidinemoiety of the Formula I compound are included in this invention. Ail tautomericforms of the 2-quinolone moiety of the Formula I compound, such as the 2-hydroxyquinoline form, are also included in this invention.

The starting materials and reagents for the above described compounds, 35 are also readily available or can be easily synthesized by those skilled in the art θ’2256 -14- using conventional methods of organic synthesis.

In addition, when the compound of this invention forms métabolites,hydrates or solvatés they are also within the scope of the invention.

Some of the compounds (e.g., prodrugs) of this invention are acidic and theyform a sait with a pharmaceutically acceptable cation. Most of the compounds of thisinvention are basic and they form a sait with a pharmaceutically acceptable anion. Ailsuch salts, including di- salts are within the scope of this invention and they can beprepared by conventional methods. For example, they can be prepared simply bycontacting the acidic and basic entities, in either an aqueous, non-aqueous orpartially aqueous medium. The salts are recovered either by filtration, by précipitationwith a solvent followed by filtration, by évaporation of the solvent, or, in the case ofaqueous solutions, by lyophilization, as appropriate.

The Formula I compound, a prodrug thereof, or a pharmaceuticallyacceptable sait of said compound or of said prodrug inhibits the sodium/proton(Na+/H+) exchange transport System and hence is useful as a therapeutic orprophylactic agent for diseases caused by the accélération of the sodium/proton(Na+/H+) exchange transport System, for example, cardiovascular diseases [e.g.,arteriosclerosis, hypertension, arrhythmia (e.g., ischémie arrhythmia, arrhythmiadue to myocardial infarction, arrhythmia after PTCA or after thrombolysis, etc.),angina pectoris, cardiac hypertrophy, myocardial infarction, heart failure (e.g.,congestive heart failure, acute heart failure, cardiac hypertrophy, etc.), restenosisafter PTCA, shock (e.g. hémorrhagie shock, endotoxin shock, etc.)], rénal diseases(e.g., diabètes mellitus, diabetic nephropathy, ischémie acute rénal failure, etc.)organ disorders associated with ischemia or ischémie reperfusion [(e.g., heartmuscle ischémie reperfusion associated disorders, acute rénal failure, or disordersinduced by surgical treatment such as coronary artery bypass grafting (CABG)surgeries, vascular surgeries, organ transplantation, non-cardtac surgeries orpercutaneous transluminal coronary angioplasty (PTCA)], cerebrovasculardiseases (e.g., ischémie stroke, hémorrhagie stroke, etc.), cerebro ischémiedisorders (e.g., disorders associated with cérébral infarction, disorders caused aftercérébral apoplexy as sequelae, or cérébral edema). The Formula I compound, aprodrug thereof, or a pharmaceutically acceptable sait of said compound or of saidprodrug can also be used as an agent for myocardial protection during coronaryartery bypass grafting (CABG) surgeries, vascular surgeries, percutaneous 012256 -15- transluminal coronary angioplasty (PTCA), organ transplantation, or non-cardiacsurgeries.

Preferably, the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug can be usedas agents for myocardial protection before, during, or after coronary artery bypassgrafting (CABG) surgeries, vascular surgeries, percutaneous transluminal coronaryangioplasty (PTCA), organ transplantation, or non-cardiac surgeries.

Preferably, the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug can be usedas agents for myocardial protection in patients presenting with ongoing cardiac(acute coronary syndromes, e.g., myocardial infarction or unstable angina) orcérébral ischémie events (e.g:, stroke).

Preferably, the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug can be usedas agents for chronic myocardial protection in patients with diagnosed coronaryheart disease (e.g., previous myocardial infarction or unstable angina) or patientswho are at high risk for myocardial infarction (e.g., âge greater than 65 and two ormore risk factors for coronary heart disease).

In addition to this, the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug are notablefor their strong inhibitory effect on the prolifération of cells, for example theprolifération of fibroblast cells and the prolifération of the smooth muscle cells ofthe blood vessels. For this reason, the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug are valuabletherapeutic agents for use in diseases in which cell prolifération représente aprimary or secondary cause and may, therefore, be used as antiatheroscleroticagents, and as agents against diabetic late complications, cancerous diseases,fibrotic diseases such as pulmonary fibrosis, hepatic fibrosis or rénal fibrosis,glomerular nephrosclerosis, organ hypertrophies or hyperplasias, in particularhyperplasia or hypertrophy of the prostate, pulmonary fibrosis, diabeticcomplications or récurrent stricture after PTCA, or diseases caused by endothélialcell injury.

The utility of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug as medical 012256 -16- agents in the treatment of diseases, such as are detailed herein in mammals (e.g.,humans) for example, myocardial protection during surgery or mycardial protectionin patients presenting with ongoing cardiac or cérébral ischémie events or chroniccardioprotection in patients with diagnosed coronary heart disease, isdemonstrated by the activity of the Formula I compound, or a pharmaceuticallyacceptable sait of said compound in conventional in vitro and other preclinicalcardioprotection assays [see the in vivo assay in Klein, H. et al., Circulation 92:912-917 (1995); the isolated heart assay in Scholz, W. et al., Cardiovascular Research29:260-268 (1995); the antiarrhythmic assay in Yasutake M. et al., Am. J. Physiol.,36.Ή2430-Η2440 (1994); the NMR assay in Kolke et al., J. Thorac. Cardiovasc.

Surg. 112: 765-775 (1996)J. Such assays also provide a means whereby theactivities of the Formula I compound, or a pharmaceutically acceptable sait of saidcompound or of said prodrug can be compared with the activities of other knowncompounds. The results of these compensons are useful for determining dosagelevels in mammals, including humans, for the treatment of such diseases.

Measurement of Human NHE-1 Inhibitory ActivityMéthodologies for measurement of human NHE-1 activity and inhibitor potency are based on those published by Watson et al., Am. J. Physiol., 24:G229-G238,1991), where NHE-mediated recovery of intracellular pH is measured followingintracellular acidification. Thus, fibroblasts stably expressing human NHE-1(Couniilon, L. et al., Mol. Pharmacol., 44:1041-1045 (1993) are plated onto collagencoated 96 well plates (50,000/well) and grown to confluence in growth media (DMEMhigh glucose, 10% fêtai bovine sérum, 50 u/ml penicillin and streptomycin).

Confluent plates are incubated for 30 min at 37°C with the pH sensitive fluorescentprobe BCECF (5 μΜ; Molecular Probes, Eugene, OR). BCECF loaded cells areincubated for 30 min at 37°C in acid loading media (70 mM choüne chloride, 50 mMNHCU, 5 mM KCI, 1 mM MgCI2,1.8 mM CaCI2, 5 mM glucose, 10 mM HEPES, pH7.5), and then placed in a Fluorescent Imaging Plate Reader (Molecular Devices, CA). BCECF fluorescence is monitored using excitation and émission wavelengthsof 485 nM and 525 nM, respectively. Intracellular acidification is initiated via rapidreplacement of acid loading media with recovery media (120 mM NaCI, 5 mM KCI, 1mM MgCI2,1.8 mM CaCI2, 5 mM glucose, 10 mM HEPES, pH 7.5) ± test compound,and NHE-mediated recovery of intracellular pH is monitored as the subséquent time-dependent increase BCECF fluorescence. The potency of human NHE-1 inhibitors is °’2256 -17- calculated as the concentration that reduces recovery of intracellular pH by 50% (IC50). Under these conditions reference NHE inhibitors amiloride and HOE-642 hadIC50 values for human NHE-1 of 50 μΜ and 0.5 μΜ, respectively. The compound ofFormula I demonstrated an IC50 value of 200nM in the above assay. 5 It has been reported that brief periods of myocardial ischemia followed by coronary artery reperfusion protects the heart from subséquent severe myocardialischemia (Murry et al., Circulation 74:1124-1136,1986). This phenomenon is knownas ischémie preconditioning.

The therapeutic effects of the compounds of this invention in preventing heart 10 tissue damage resulting from an ischémie insult can be demonstrated in vitro alonglines presented in Liu et al. (Cardiovasc. Res., 28:1057-1061,1994), as describedspecifically herein. Cardioprotection, as indicated by a réduction in infarctedmyocardium, can be induced pharmacoiogicaliy using adenosine receptor agonists inisolàted, retrogradeiy perfused rabbit hearts as an in vitro model of myocardial 15 ischémie preconditioning (Liu et ai., Cardiovasc. Res., 28:1057-1061,1994). The invitro test described below demonstrates that an active test compound can alsopharmacoiogicaliy induce cardioprotection, i.e., reduce myocardial infarct size, whenadministered to a rabbit isolàted heart. The effects of the test compound arecompared to ischémie preconditioning and the A1/A3 adenosine agonist, APNEA (N6- 20 I2-(4-aminophenyl)ethyi]adenosine), that has been shown to pharmacoiogicaliyinduce cardioprotection in the rabbit isolàted heart (Liu et al., Cardiovasc. Res.,28:1057-1061,1994). The exact methodology is described below.

The protocol used for these experiments closely follows that described by Liuet al., Cardiovasc. Res., 28:1057-1061,1994. Male New Zealand White rabbits (3-4 25 kg) are anesthetized with sodium pentobarbital (30 mg/kg, i.v.). After deep anesthésia is achieved (determined by the absence of an ocular blink reflex) theanimal is intubated and ventilated with 100% O2 using a positive pressure ventilator.A left thoracotomy is performed, the heart exposed, and a snare (2-0 silk) is placedloosely around a prominent branch of the left coronary artery, approximately 2/3 of 30 the distance towards the apex of the heart The heart is removed from the chest and rapidly (<30 sec) mounted on a Langendorff apparatus. The heart is retrogradeiy perfused in a non-recirculating manner with a modified Krebs solution (NaC1118.5 mM, KCI 4.7 mM, Mg SO4 1.2 mM, KH2PO4 1.2 mM, NaKCO3 24.8 mM, CaCI2 2.5 mM, and glucose 10 mM), at a constant pressure of 80 mmHg and a température of 012256 -18- 37°C. Perfusate pH is maintained at 7.4-7.5 by bubbling with 95% O2/5% CO2. Hearttempérature is tightly controlled by using heated réservoirs for the physiologicalsolution and water jacketing around both the perfusion tubing and the isolated heart.Heart rate and Ieft ventricular pressures are determined via a latex balloon which isinserted in the Ieft ventricle and connected by stainless Steel tubing to a pressuretransducer. The intraventricuiar balloon is inflated to provide a systolic pressure of80-100 mmHg, and a diastolic pressure £10 mmHg. Total coronary flow is alsocontinuously monitored using an in-line flow probe and normalized for heart weight.

The heart is allowed to equilibrate for 30 min, over which time the heart mustshow stable Ieft ventricular pressures within the parameters outlined above. If theheart rate falls below 180 bpm at any time prior to the 30 min period of régionalischemia, the heart is paced at about 200 bpm for the remainder of the experiment.Ischémie preconditioning is induced by total cessation of cardiac perfusion (globalischemia) for 5 min, followed by reperfusion for 10 min. The régional ischemia isprovided by tightening the snare around the coronary artery branch. Following the 30min régional ischemia, the snare is released and the heart reperfused for anadditional 120 min.

Pharmacological cardioprotection is induced by infusing the test compound atpredetermined concentrations, starting 30 min prior to the 30 min régional ischemia,and continuing until the end of the 120 min reperfusion period. Hearts which receivetest compound do not undergo the period of ischémie preconditioning. The référencécompound, APNEA (500 nM) is perfused through hearts (which do not receive thetest compound) for a 5 min period which ends 10 min before the 30 min régionalischemia.

At the end of the 120 min reperfusion period, the coronary artery snare istightened, and a 0.5% suspension of fluorescent zinc cadmium sulfate particles (1-10pM) Duke Scientific Corp.(Palo Alto, CA) is perfused through the heart; this stains aliof the myocardium, except that area-at-risk for infarct development (area-at-risk).

The heart is removed from the Langendorff apparatus, blotted dry, wrapped inaluminum foii and stored ovemight at -20°C. The next day, the heart is sliced into 2mm transverse sections from the apex to the top of the ventricles. The slices arestained with 1% triphenyl tétrazolium chloride (TTC) in phosphate-buffered saline for20 min at 37’C. Since TTC reacts with living tissue (containing NAD-dependentdehydrogenases), this stain differentiates between living (red stained) tissue, and 012256 -19- dead tissue (unstained infarcted tissue). The infarcted area (no stain) and the area-at-risk (no fluorescent particles) are calculated for each slice of left ventricle using aprecalibrated image analyzer. To normalize the ischémie injury for différences in thearea-at-risk between hearts, the data is expressed as the ratio of infarct area vs.area-at-risk (%IA/AAR). Ali data are expressed as mean ± SE and comparedstatistically using a Mann-Whitney non-parametric test with a Bonferroni correction formultiple compensons. Significance is considered as p < 0.05.

The results from the above in vitro test can be used to demonstrate thatcompounds of this invention induce significant cardioprotection relative to the controlgroup.

Administration of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug can be viaany method which delivers the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug preferentiallyto the desired tissue (e.g., liver and/or cardiac tissues). These methods includeoral routes, parentéral, intraduodenal routes, etc. Generally, the Formula Icompound, a prodrug thereof, or a pharmaceutically acceptable sait of saidcompound or of said prodrug is administered in single (e.g., once daily) or multipledoses or via constant infusion in, for example, an isotonie saline solution.

The Formula I compound, a prodrug thereof, or a pharmaceuticallyacceptable sait of said compound or of said prodrug is useful, for example, inreducing or minimizing damage effected directly to any tissue that may besusceptible to ischemia/reperfusion injury (e.g., heart, brain, lung, kidney, liver, gut,skeletal muscle, retina), through for example médiation by NHE-1, as the resuit ofan ischémie event (e.g., myocardial infarction). The active compound is thereforeusefully employed prophylactically to prevent, (i.e., prospectively or prophylactically)to blunt or stem, tissue damage (e.g., myocardial tissue) in patients who are at riskfor ischemia (e.g., myocardial ischemia).

Generally, the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug of thisinvention is administered orally, or parenterally (e.g., intravenous, intramuscular,subeutaneous or intramedullary). Topical administration may also be indîcated, forexample, where the patient is suffering from gastrointestinal disorders or whenever θ’ 2256 -20- the médication is best applied to the surface of a tissue or organ as determined bythe attending physicien.

The amount and timing of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug administeredwill, of course, be dépendent on the subject being treated, on the severity of theaffliction, on the manner of administration and on the judgement of the prescribingphysician. Thus, because of patient to patient variability, the dosages given beloware a guideline and the physician may titrate doses of the drug to achieve thetreatment that the physician considère appropriate for the patient. In consideringthe degree of treatment desired, the physician must balance a variety of factorssuch as âge of the patient, presence of preexisting disease, as well as presence ofother diseases (e.g., cardiovascular disease).

Typically an amount of the Formula I compound, a prodrug thereof, or apharmaceutically acceptable sait of said compound or of said prodrug is used thatis effective for ischémie protection. A preferred dosage is about 0.01 to 100mg/kg/day of the Formula l compound, a prodrug thereof, or a pharmaceuticallyacceptable sait of said compound or of said prodrug of this invention. An especiallypreferred dosage is about 0.01 to 50 mg/kg/day of the Formula I compound, aprodrug thereof, or a pharmaceutically acceptable sait of said compound or of saidprodrug of this invention.

In one mode of administration the Formula I compound, a prodrug thereof,or a pharmaceutically acceptable sait of said compound or of said prodrug may beadministered just prior to cardiac surgery (e.g., within twenty-four hours beforesurgery), during or subséquent to cardiac surgery (e.g., within twenty-four hoursafter surgery) where there is risk of myocardial ischemia. In an especially preferredmode an infusion is administered with a loading dose of about 1 mg to about 300mg for about one minute to about one hour prior to surgery followed by a constantinfusion of about 1 mg/kg/day to about 100 mg/kg/day for the remainingpresurgery, surgery and post surgery période, including for example about 2 toabout 7 days post surgical treatment. The compound of this invention may also beadministered in a chronic daily mode.

The Formula I compound, a prodrug thereof, or a pharmaceutically acceptable sait of said compound or of said prodrug is generally administered in the form of a pharmaceutical composition comprising the Formula I compound, a 07 225 6 -21- prodrüg thereof, or a pharmaceutically acceptable sait of said compound or of saidprodrug together with a pharmaceutically acceptable vehicle, carrier or diluent.

Thus, the Formula I compound, a prodrug thereof, or a pharmaceuticallyacceptable sait of said compound or of said prodrug can be administered in anyconventiona, oral, parentéral, rectal or transdermal dosage form.

For oral administration a pharmaceutical composition can take the form ofsolutions, suspensions, tablets, pills, capsules, powders, and the like. Tabletscontaining various excipients such as sodium citrate, calcium carbonate andcalcium phosphate are employed along with various désintégrants such as starchand preferably potato or tapioca starch and certain complex silicates, together withbinding agents such as polyvinylpyrroiidone, sucrose, gelatin and acacia.

Additionally, lubricating agents such as magnésium stéarate, sodium lauryl sulfateand talc are often very useful for tabletting purposes. Solid compositions of asimilar type are aiso employed as fillers in soft and hard-filled gelatin capsules;preferred materials in this connection also include lactose or milk sugar as well ashigh molecular weight polyethylene glycols. When aqueous suspensions and/orélixirs are desired for oral administration, the compounds of this invention can becombined with various sweetening agents, flavoring agents, coloring agents,emulsifying agents and/or suspending agents, as well as such diiuents as water,éthanol, propylene glycol, glycerin and various like combinations thereof.

For purposes of parentéral administration, solutions, for example, insesame or peanut oil or in aqueous propylene glycol can be employed, as well asstérile aqueous solutions of the corresponding water-soluble salts. Such aqueoussolutions may be suitably buffered, if necessary, and the liquid diluent first renderedisotonie with sufficient saline or glucose. These aqueous solutions are especiallysuitable for intravenous, intramuscular, subeutaneous and intraperitoneal injectionpurposes. In this connection, the stérile aqueous media employed are ail readilyobtainable by standard techniques well-known to those skilled in the art.

For purposes of transdermal (e.g.,topical) administration, dilute stérile,aqueous or partially aqueous solutions (usually in about 0.1% to 5% concentration),otherwise similar to the above parentéral solutions, are prepared.

Methods of preparing various pharmaceutical compositions with a certain amount of active ingrédient are known, or will be apparent in light of this disclosure, to thosé skilled in this art. For examples of methods of preparing pharmaceutical 012256 -22- compositions, see Reminqton's Pharmaceutical Sciences, Mack PublishingCompany, Easter, Pa., 15th Edition (1975).

Pharmaceutical compositions according to the invention may contain forexampie 0.0001 %-95% of the Formula I compound, a prodrug thereof, or a 5 pharmaceutically acceptable sait of said compound or of said prodrug. In anyevent, the composition or formulation to be administered will contain an amounteffective to treat the disease/condition of the subject being treated.

The Formula I compound, a prodrug thereof, or a pharmaceuticallyacceptable sait of said compound or of said prodrug generally will be administered 10 in a convenient formulation. The following formulation examples are illustrative onlyand are not intended to limit the scope of the présent invention.

In the formulations which follow, "active ingrédient" means the Formula Icompound, a prodrug thereof, or a pharmaceutically acceptable sait of saidcompound or of said prodrug. 15 Formulation 1: Gelatin Capsules

Hard gelatin capsules are prepared using the following:

Ingrédient Quantity (mg/capsule) Active ingrédient 0.25-100 Starch, NF 0-650 Starch fiowable powder 0-50 Silicone fluid 350 centistokes 0-15 A tablet formulation is prepared using the ingrédients below: Formulation 2: Tablets Ingrédient Quantity (mg/tablet) Active ingrédient 0.25-100 Cellulose, microcrystalline 200-650 Silicon dioxide, fumed 10-650 Stéarate acid 5-15 The components are blended and compressed to form tablets. Altematively, tablets each containing 0.25-100 mg of active ingrédients are made up as follows: 012256 -23-

Formulation 3: Tablets

Ingrédient Quantity (mg/tablet) Active ingrédient 0.25-100 Starch 45 Cellulose, microcrystalline 35 Polyvinylpyrrolidone (as 10% solution in water) 4 Sodium carboxymethyl cellulose 4.5 Magnésium stéarate 0.5 Talc 1 10

The active ingrédient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone ismixed with the résultant powders which are then passed through a No. 14 meshU.S. sieve. The granules so produced are dried at 50° - 60°C and passed through aNo. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnésium stéarate,and talc, previously passed through a No. 60 U.S. sieve, are then added to thegranules which, after mixing, are compressed on a tablet machine to yield tablets.

Suspensions each containing 0.25-100 mg of active ingrédient per 5 mldose are made as follows:

Formulation 4: Suspensions

Ingrédient

Active ingrédient

Sodium carboxymethyl celluloseSyrup

Benzoic acid solution

Flavor

Color

Purified Water to

Quantity (mg/5 ml_)0.25-100 mg50 mg1.25 mg 0.10 mLq.v.q.v.

5 mL

The active ingrédient is passed through a No. 45 mesh U.S. sieve andmixed with the sodium carboxymethyl cellulose and syrup to form smooth paste.The benzoic acid solution, flavor, and color are diluted with some of the water and 15 added, with stirring. Sufficient water is then added to produce the required volume.An aérosol solution is prepared containing the following ingrédients:

01 2256 -24- Formulation 5: Aérosol Ingrédient Quantity (% by weight) Active ingrédient Ethanol Propellant 22 (Chlorodifluoromethane) 0.25 25.75 74.00 The active ingrédient is mixed with éthanol and the mixture added to aportion of the propellant 22, cooled to 30eC, and transferred to a filling device. Therequired amount is then fed to a stainless Steel container and diluted with the 5 remaining propellant. The valve units are then fitted to the container. Suppositories are prepared as follows: Formulation 6: Suppositories Ingrédient Quantity (mg/suppository) Active ingrédient Saturated fatty acid glycerides 250 2,000 The active ingrédient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted using the 10 minimal necessary heat. The mixture is then poured into a suppository mold ofnominal 2 g capacity and allowed to cool. An intravenous formulation is prepared as follows: Formulation 7: Intravenous Solution Ingrédient Quantity Active ingrédient Isotonie saline 25 mg 1,000 mL

The solution of the above ingrédients is intravenously administered to a 15 patient. GENERAL EXPERIMENTAL PROCEDURES300 and 400 MHz 1H NMR spectra were recorded on a Varian Unity+ 300 or 400 spectrometer (Varian Co., Palo Alto, CA) equipped with two RF channels,indirect détection, and pulsed-field gradients (Z-axis only). Spectra were generally 20 acquired near room température (21 °C), and standard auto-lock and auto-shimroutines were employed for shimming samples. Chemical shifts are expressed inparts per million downfield from trimethylsilane. The peak shapes are denoted as 012256 -25- follows: s, singlet; d, doublet; t, triplet, q, quartet; m, multiplet; bs, broad singlet.Résonances designated as exchangeable did not appear in a separate NMRexperiment where the sample was shaken with several drops of D2O in the samesolvent. Atmospheric pressure Chemical ionization mass spectra (APCIMS) were 5 obtained on a Fisons Platform II or a Micromass ZMD spectrometer (Microsmass,Manchester, U.K.). Where the intensity of chlorine or bromine-containing-ions aredescribed, the expected intensity ratio was observed (approximately 3:1 for3SCI/37CI-containing ions and 1:1 for ^Br/^Br-containing ions) and M is based on35CI and 7eBr. In some cases only représentative 1H NMR and APCIMS peaks are 10 given.

Column chromatography was performed with either Baker Silica Gel(40 pm) (J.T. Baker, Phillipsburg, N.J.) or Silica Gel 60 (EM Sciences, Gibbstown, N.J.) in glass columns or in Flash 12,40 or 75 (Biotage) (Charlottesville, VA)columns under low nitrogen pressure. Radial Chromatography was performed 15 using a Chromatotron, (Harrison Research, Palo Alto, CA.) Unless otherwisespecified, reagents were used as obtained from commercial sources.Dimethylformamide, 2-propanol, methanol, dimethylsulfoxide, 1,2-dichloroethane,tetrahydrofuran, toluene, dichloromethane and other réaction solvents were theanhydrous grade supplied by Aldrich Chemical Company (Milwaukee, Wisconsin). 20 Microanalyses were performed by Schwarzkopf Microanalytical Laboratory,

Woodside, NY. The terms "concentrated in vacuo" and “removed in vacuo" refer toremoval of solvent at reduced pressure on a rotary evaporator with a bathtempérature of less than 90°C. Reactions conducted at "0-20°C" or "0-25‘C” wereconducted with initiai cooling of the vessel in an insulated ice bath which was 25 allowed to warm to room température over several hours. The abbreviation "min"and "h" stand for "minutes" and "hours" respectively. EXAMPLE 1 5-Amino-2-quinolone hvdrochloride 30 A solution of the 5-nitro-2-quinolone (12.37 g, 65 mmol) (prepared as described in Capps, J. D.; Hamilton, C. S. J. Am. Chem. Soc. 1938, 60, 2104) inEtOH (176 mL) under a nitrogen atmosphère was treated with SnCI2«2H2O (73.4 g,325 mmol). The heterogeneous reaction mixture was allowed to stir at 23 °C for 2hours and heated at reflux for 2 hours. The resulting heterogeneous mixture was 35 cooled to 23 °C and filtered. The solid matériel was triturated in HCl (1 M aqueous, 01 2256 -26- 78 mL, 78 mmol) at reflux for 1 hour, cooled to 23 °C, and filtered to afford 10.08 gof the desired product (78 % yield). 1H NMR (400 MHz, DMSO-d6) 5 6.42 (d, J=10, 1H), 6.71 (d, J=7.6, 1H),6.81 (d, J=8,1H), 7.27 (d, J=8, 1H), 8.04 (d, J=10, 1H), 11.71 (bs, 1H). APCIMS 161 [M+1]+ EXAMPLE 2 5-Hvdrazino-2-quinolone hvdrochloride A solution of 5-amino-2-quinolone hydrochloride (10.08 g, 51.2 mmol) inHCl (concentrated, 42.7 mL) and H2O (18.2 mL) at 0 °C was treated dropwise witha solution of NaNO2 (3.53 g, 51.2 mmol) in H2O (26.4 mL) while maintaining thetempérature below 5 °C. The resulting red suspension was ailowed to stir at 0 °Cfor 1 hour.

In a separate three neck round bottom flask equipped with a mechanicalstirrer, a slurry of SnCI2*2H2O (25.41 g, 112.6 mmol) in HCl (concentrated, 34.2mL) and H2O (93.9 mL) was cooled to 0 °C. The suspension was treated dropwisewith the red diazonium sait suspension prepared above, while maintaining thetempérature below 5 °C. The reaction mixture was warmed to 23 °C and ailowed tostir for 3 hours. The resulting suspension was Filtered. The collected solid wastriturated with HCl (1 M aqueous, 61.4 mL, 61.4 mmol) at 23 °C for 30 minutes.

The solid was collected by filtration to afford 7.88 g of the desired product (73 %yield). 1H NMR (400 MHz, DMSO-efe) δ 6.43 (d, J= 10,1H), 6.63 (d, J=8, 1 H), 6.88 (d, J=8, 1H), 7.38 (t, J=8,1H), 7.98 (d, J=10, 1H), 8.97 (bs, 1H), 10.40 (bs,1H), 11.77 (bs, 1H). APCIMS 176 [M+1]+ EXAMPLE 3

Methvl 5-cvclopropyl-1 -(2-ouinolon-5-vl)-1 H-pyrazole-4-carboxvlate A solution of methyl 3-cyclopropyl-2-(dimethylaminomethylene)-3-oxopropanoate (7.85 g, 39.8 mmol) in EtOH (257 mL) was treated under a nitrogenatmosphère with triethylamine (7.77 mL, 55.8 mmol), followed by 5-hydrazino-2-quinolone hydrochloride (7.88 g, 37.2 mmol). The resulting heterogeneousreaction mixture was heated at reflux for 4 hours and cooled to 23 °C . A paleyellow solid precîpitated out and was collected by filtration to afford 5.06 g of the 012256 -27- desired product (44 % yield). 1H NMR (300 MHz, DMSO-d6) δ 0.59-0.73 (m, 4H), 1.87 (m, 1H), 3.79 (s,1H), 6.54 (d, J=10,1H), 7.17 (d, J=10, 1H), 7.34 (d, J= 7.5,1H), 7.49 (d, J=8, 1H),7.66 (t, J=8, 1H), 8.08 (s, 1H), 12.10 (bs, 1H). APCIMS 310 [M+1f EXAMPLE 4 5-Cvclopropvl-1 -(2-quinolon-5-yl)-1 H-pyrazole-4-carboxvlic acid A slurry of methyl 5-cyclopropyl-1-(2-quinolon-5-yl)-1/7-pyrazole-4-carboxylate (5.06 g, 16.4 mmol) in THF (81.8 mL) and MeOH (40.9 mL) wastreated with LiOH (1 M aqueous, 81.8 mL, 81.8 mmol) and heated at reflux for 2.5hours. The resulting heterogeneous mixture was cooled to 23 °C. The MeOH andTHF were removed in vacuo. The resulting slurry was cooled to 0 °C and acidifiedto pH 3-4 using HCl (concentrated). The mixture was filtered. The solid was driedto afford 4.94 g of desired product (100% yield). 1H NMR (400 MHz, DMSO-d6) δ 0.58 (d, J=4.4, 2H), 0.64 (d, J=8, 2H), 1.82(m, 1H), 6.50 (d, J=10, 1H), 7.12 (d, J=10,1H), 7.27 (d, J=7.6,1H), 7.44 (d, J=8.4,1H), 7.60 (t, J=8,1H). 7.97 (s, 1H). 12.03 (bs, 1H), 12.38 (bs, 1H). APCIMS 296 [M+1]* EXAMPLE 5 f5-Cvclopropvl-1 -(2-quinolon-5-vh-1 H-pvrazole-4-carbonvnquanidine hvdrochloride A solution of 5-cyclopropyl-1-(2-quinolôn-5-yl)-1H-pyrazole-4-carboxylic acid(13.27 g, 45.0 mmol) in SOCI2 (60 mL) was heated at reflux for 1 hour. The excessSOCI2 was removed in vacuo. The residue was treated with dry toluene andconcentrated in vacuo. A solution of the solid residue in THF (50 mL) was treatedwith a solution of guanidine hÿdrochloride (15.47 g, 162 mmol) in NaOH (2 Maqueous, 162 mL, 324 mmol) under a nitrogen atmosphère. The resulting mixturewas stirred at 23 °C for 2 hours. The reaction mixture was then concentrated invacuo to remove the THF. The aqueous layer was cooled to 0 °C and acidified topH 9. A pale yellow solid precipitated and was collected to afford 11.41 g of thefree base of the desired compound. A solution of the solid in MeOH (700 mL) wastreated with HCl (1 M in ether, 84.9 mL, 84.9 mmol). The solvent volume wasreduced in vacuo. The resulting pale brown precipitate was collected to afford 8.49g (51% yield). (Additional product as the free base may be obtained by extraction 012256 -28- of the aqueous layer using EtOAc-THF (1:1)). 1H NMR (400 MHz, DMSO-d6) δ 0.51 (m, 2H), 0.70 (dd, J=2, 8.4, 2H), 1.92 (m, 1H), 6.53 (dd, J=1.6,10,1H), 7.17 (d, J=10,1H), 7.32 (d, J=7,1H), 7.48 (d,J=8.4, 1H), 7.64 (t, J=8.2,1H), 8.34 (bs, 2H), 8.60 (bs, 3H), 11.64 (s, 1H), 12.10 (s, 5 1H). APCIMS 337 [M+1]* EXAMPLE 6

Identification of f5-cvclopropvl-1 -(2-quinolon-5-vl)-1 H-pvrazole-4-carbonvHguanidine 10 in human plasma samples bv liquid chromatoqraphv (LCVmass spectroscopv

(MSVMS and LC/NMR

Human subjects were dosed intravenously with 5-cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine monohydrochloride monohydrate. Humanplasma samples were pooled together and proteins were precipitated by twice 15 volume of acetonitrile. After centrifugation, the supematant was concentràted in awater bath with N2 at 40°C.

An aliquot (100 μΐ or 50 pL) of the concentràted sample was injected ontoan LC/MS/MS instrument equipped with a Zorbax Rx-C8 4.6 x 150 mm column,analyzed on PE Sciex API 2000 or Finnigan LCQ mass spectrometer and

20 monitored by UV detector at 254 nm. The eluent from the column was split so that5% of eluent was diverted to the mass spectrometer and the rest was passed to UVdetector. The analyses were performed using a mobile phase flow rate of 1ml/minute, where the mobile phase consisted of 5 mM ammonium formate (pH 3.0)as solvent A and acetonitrile as solvent B. A gradient included 5/95 to 40/60 of B/A 25 over a 20 minute period after an initial hold for 3 minutes. Under these LCconditions a métabolite was detected at a rétention time of 11.8 minutes ([5-cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine was detected at 13.7 minutes). The 16 mass units différence between 5-cyclopropyl-1-(quino,in-5-yl)-1H-pyrazole-4-carbonyi]guanidine (m/z 321) and métabolite (m/z 337) in human 30 plasma suggested that it is formed through oxidation. LC/MS/MS product ionspectrum of protonated métabolite at m/z 337 ([M+H]*) showed fragment ions atm/z 278, 260, 250, 236, 223 and 208.

The salts were removed from the above concentràted sample by solid phase extraction. The sample was concentràted to contain approximately 5-10 pg 35 of the métabolite. 50 pL of this sample was injected onto an LC/NMR instrument 012256 -29- equipped with a Zorbax Rx-C8 4.6 x 150 mm column, analyzed on a Bruker 500MHz NMR spectrometer and monitored by UV detector at 235 nm. The analyseswere performed using a mobile phase flow rate of 1 ml/minute, where the mobile ' phase consisted of 15 mM deuterated aqueous formic acid adjusted to pH 3.3 with5 deuterated ammonium hydroxide in D2O as solvent C and acetonitrile-d3 as solventD. A gradient included 5/95 to 20/80 of D/C over a 17 minute period after an initial hold for 3 minutes. The desired métabolite was captured in the NMR probe usingthe stop flow technique on the peak at 13.6 minutes. Proton NMR spectra andCOSY spectra of the human métabolite contained two doublets (6.64 and 7.41 10 ppm) which were coupled to each other but not to any other peaks. The absenceof the downfield peak at 8.92 ppm that is présent in [5-cyclopropyl-1-(quinolin-5-yl)-1/7-pyrazole-4-carbonyl]guanidine suggested that the 2-position on the quinolinering was the site of oxidation. On the basis of this data the structure of themétabolite was assigned as [5-cyclopropyl-1-(2-quinolon-5-yl)-1/7-pyrazole-4- 15 carbonyljguanidine. Comparison of the 1H LC/NMR spectrum of the métabolite tothat of a synthetic standard of [5-cyclopropyl-1-(2-quinolon-5-yl)-1/7-pyrazole-4-carbonyljguanidine hydrochloride, prepared as described in Example 5, confirmedthe structural assignment.

Claims (28)

1. 012256 -30- CLAIMS

   1. A compound having the Formula I

   a prodrug thereof or a pharmaceutically acceptable sait of said compoundor of said prodrug with the proviso that [5-cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine is not included. 10 2. [5-Cyclopropyl-1-(2-quinolon-5-yl)-1W-pyrazole-4-carbonyllguanidine, or a pharmaceutically acceptable sait thereof.
2. 3. Use of a compound of Claim 1, a prodrug thereof, or a pharmaceuticallyacceptable sait of said compound or of said prodrug, in the manufacture of a 1 g médicament for reducing tissue damage resulting from ischemia or hypoxia.
3. 4. Use as recited in claim 3 wherein the tissue is cardiac, brain, liver,kidney, lung, gut, skeletal muscle, spleen, pancréas, nerve, spinal cord, retinatissue, the vasculature, or intestinal tissue.
4. 5. Use as recited in claim 3 wherein the amount of the compound of 20 claim 1, a prodrug thereof or a pharmaceutically acceptable sait of said compoundor of said prodrug is about 0.01 mg/kg/day to about 50 mg/kg/day.
5. 6. Use as recited in claim 5 wherein the mammal is a female or malehuman. 012256 -31- 7 Use as recited in claim 6 wherein said tissue is heart tissue.
6. 8. Use as recited in claim 6 wherein said tissue is brain tissue. 9 Use as recited in claim 6 wherein said tissue is liver tissue.
7. 10. Use as recited in claim 6 wherein said tissue is kidney tissue.
8. 11. Use as recited in claim 6 wherein said tissue is lung tissue.
9. 12. Use as recited in claim 6 wherein said tissue is gut tissue.
10. 13. Use as recited in claim 6 wherein said tissue is skeletal muscle tissue.
11. 14. Use as recited in claim 6 wherein said tissue is spleen tissue.
12. 15. Use as recited in claim 6 wherein said tissue is pancréas tissue.
13. 16. Use as recited in claim 6 wherein said tissue is retina tissue.
14. 17. Use as recited in claim 6 wherein the compound is administeredprophylactically.
15. 18. Use as recited in claim 6 wherein the compound is administered priorto surgery.
16. 19. Use as recited in claim 6 wherein the compound is administered prior to cardiac surgery.
17. 20. Use as recited in claim 6 wherein the compound is administeredduring surgery.
18. 21. Use as recited in claim 6 wherein the compound is administered 20 during cardiac surgery.
19. 22. Use as recited in claim 6 wherein the compound is administered withintwenty-four hours after surgery.
20. 23. Use as recited in claim 6 wherein the compound is administered withintwenty four hours after cardiac surgery.
21. 24. Use as recited in claim 6 wherein the tissue damage resulting from ischemia is ischémie damage and is incurred during orgàn transplantation.
22. 25. Use as recited in claim 6 wherein the compound is administered toprevent perioperative myocardial ischémie injury.
23. 26. A pharmaceutical composition which comprises an amount of the 30 compound of claim 1, a prodrug thereof or a pharmaceutically acceptable sait ofsaid compound or of said prodrug and a pharmaceutically acceptable carrier,vehicie or diluent.
24. 27. A pharmaceutical composition for the réduction of tissue damage resultingfrom ischemia or hypoxia which comprises a therapeutically effective amount of the 01 2256 -32- compound of claim 1, a prodrug thereof or a pharmaceutically acceptable sait ofsaid compound or of said prodrug and a pharmaceutically acceptable carrier,vehicle or diluent.
25. 28. Use as recited in claim 6 wherein the compound is administered prior5 to, during and after surgery.
26. 29. Use as recited in claim 6 wherein thé compound is administered priorto, during and after cardiac surgery.
27. 30. Use of [5-cyclopropyl-l-(quinolin-5-yl)-lH-pyrazole-4-carbonyl]guanidine in the manufacture of [5-cyclopropyl-l-(2-quinolon-5-yl)-lH- 10 pyrazole-4-carbonyl]guanidine in a human.
28. 31. Substantially pure (5-cyclopropyl-1-(2-quinolon-5-yl)-1 H-pyrazole-4-carbonyl]guanidine, or a pharmaceutically acceptable sait of said compound.